Explore Inchworm motion of a rationally designed DNA … Projects...0 Faculty of Science, Department of Physics Explore Inchworm motion of a rationally designed DNA nanomotor Thesis

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Faculty of Science, Department of Physics

Explore Inchworm motion of a rationally designed

DNA nanomotor

Thesis submitted in partial fulfilment of the requirements for the degree of

Bachelor of Science (Honours)

Leow Ke Yun

A0114542R

Supervisor: Associate Professor Wang Zhisong

3 April 2017

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Abstract

An autonomous inchworm DNA nanomotor that moves on the pure duplex track is designed

and experimented in this project. This autonomous motion, driven by a series of length change

in motor, is brought about by the introduction of DNA fuel strands and enzyme, Nt.BbvCI. The

binding between the motor leg and the duplex track forms triplex segment and is bound together

via Hoogsteen bond.

In this project, we tested two versions of the motor, which differs in the leg length, in two

different buffers. The triplex binding between the track and motor leg occurs in both buffers

under acidic condition and both the motors demonstrates forward binding bias during the

binding process. Results from the preferential dissociation experiment revealed that the

dissociation bias of the motor system has buffer dependence. When choline chloride-Mg2+

buffer is used, both the motors demonstrate preferential front leg dissociation upon fuel

addition, which is an inchworm behaviour.

In addition, experimental results have shown that the behaviour of the motor system is

dependent on the leg length. The motor with 12-nucleotide leg has a higher dissociation bias

as compared to its counterpart with 9-nucleotide leg, as such, it is a better candidate for the

motor system.

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Acknowledgements

First of all, I would like to express my most heartfelt gratitude to my Final Year Project (FYP)

supervisor, Associate Professor Wang Zhisong, for his patient guidance throughout the year.

This project will not be possible without his foresight, advice and encouragement. Beside the

academic supports that he rendered, the sharing of his beliefs and life experiences has spurred

a lot of new perspectives in me and will be of great value for lifetime.

I would also like to extent my gratitude Dr Loh Iong Ying for his training and supervision

throughout the year. He shared a lot of insightful advice and taught me a lot on the experimental

techniques and data analysis. This project would not have progress this far without his guidance

and kind assistance.

Also, I would like to thank Tee Shern Ren for providing me a different insight to my data.

Despite his busy schedule, he was very helpful and patient and was always very ready to help

me with anything I am unsure of.

In addition, I would like to thank Hu Xinpeng and Chiang Yi Herng for their assistance, support

and kind understanding over the past year. The feedbacks they gave have rendered me more

directions during both the motor designing and experimentation stage.

I would also like to thank my fellow peers, Goh Shi Ying and Liu Xiao Rui for their companion

and encouragement throughout our FYP. The insightful discussions with them has given me a

lot of new ideas to my project.

Finally, I would like to thank everyone in the molecular motor lab for their constant support

and understanding over the past year. I have learnt a lot from the sharing during the weekly

meeting and your advice has helped me progress in various ways.

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Table of Content

Abstract ................................................................................................................................................... 0

Acknowledgements ................................................................................................................................. ii

Table of Content .................................................................................................................................... iii

List of Figures ......................................................................................................................................... v

Chapter 1: Introduction ........................................................................................................................... 1

1.1 Significance of Nanomotors.................................................................................................... 1

1.2 Project Motivation .................................................................................................................. 2

1.3 Objective and Scope ............................................................................................................... 3

Chapter 2: Theory ................................................................................................................................... 4

2.1 Operation of DNA nanomotors ............................................................................................... 4

2.1.1 Hand-on-hand motor ....................................................................................................... 5

2.1.2 Inchworm motor .............................................................................................................. 6

2.2 Shearing and unzipping forces ................................................................................................ 8

2.3 DNA Duplex and Triplex ........................................................................................................ 9

2.4 Absorbance ........................................................................................................................... 11

2.5 Native polyacrylamide gel electrophoresis (PAGE) ............................................................. 12

2.6 Fluorescence experiment: the use of Dye and Quencher ...................................................... 13

2.7 Enzyme ................................................................................................................................. 14

2.8 Buffers................................................................................................................................... 15

Chapter 3: Motor System Design .......................................................................................................... 16

3.1 Motor design and operation mechanism ............................................................................... 16

3.1.1 Components in the motor system ......................................................................................... 16

3.1.2 Elaboration on the inchworm motion ................................................................................... 18

3.2 Length Selection ................................................................................................................... 20

3.3 Sequence Generation............................................................................................................. 23

3.4 Fuel and enzyme cycle .......................................................................................................... 25

Chapter 4: Materials and Methods ........................................................................................................ 27

4.1 Preparation of DNA single stranded stock (100m) ............................................................. 27

4.2 Annealing .............................................................................................................................. 28

4.3 Single stranded stock (5l) and fuel stock (10l) preparation .............................................. 29

4.4 Native polyacrylamide gel electrophoresis (Native PAGE) ................................................. 30

4.4.1 Gel preparation ..................................................................................................................... 30

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4.5 pH measurement ................................................................................................................... 33

4.6 Fluorescence experiment....................................................................................................... 34

4.6.1 Binding experiment .............................................................................................................. 34

4.6.2 Operation experiment ........................................................................................................... 35

4.7 Absorbance experiment......................................................................................................... 37

Chapter 5: Experimental results ............................................................................................................ 38

5.1 Native PAGE ........................................................................................................................ 38

5.1.1 Quality of annealed sample .................................................................................................. 38

5.1.2 Functionality of the newly designed single hairpin engine .................................................. 39

5.2 Fluorescence experiment....................................................................................................... 42

5.2.1 Binding of M12 under choline chloride-Mg2+ buffer ............................................................... 42

5.2.2 Binding of M9 under choline chloride-Mg2+ buffer ............................................................. 47

5.2.3 Operation of M12 under choline chloride-Mg2+ buffer ........................................................ 48

5.2.4 Operation of M9 under choline chloride-Mg2+ buffer .......................................................... 51

5.2.5 Summary of the motor systems in choline chloride-Mg2+ buffer......................................... 53

5.2.6 Binding of M12 under TAE-Mg2+ buffer ............................................................................. 54

5.2.7 Binding of M9 under TAE-Mg2+ buffer ............................................................................. 58

5.2.8 Operation of M12 under TAE-Mg2+ buffer .......................................................................... 59

5.2.9 Operation of M9 ................................................................................................................... 61

5.2.10 Summary of the motor systems in TAE-Mg2+ buffer and comparison with choline

chloride-Mg2+ buffer ..................................................................................................................... 63

5.3 Absorbance ........................................................................................................................... 64

Chapter 6: Discussion ........................................................................................................................... 66

6.1 Further discussion on experimental results ........................................................................... 66

6.1.1 Comment on dyes................................................................................................................. 66

6.1.2 Stability of DNA triplex under shearing and unzipping force ............................................. 67

6.2 Areas for improvements ........................................................................................................ 67

6.3 Future works ......................................................................................................................... 68

Chapter 7: Conclusion ........................................................................................................................... 69

References ............................................................................................................................................. 70

v

List of Figures Figure (1): A diagram to illustrate (a) forward binding bias and (b) backward binding bias ................. 5

Figure (2): Hand-on-hand motion of motor ............................................................................................ 5

Figure (3): Inchworm motion of motor ................................................................................................... 6

Figure (4): Components of the motor system. It consists of the motor, track, fuel and enzyme. The

arrows illustrate the direction of DNA by pointing to the 3’ end. The track in the illustration consist of

five binding sites, each represented by a pair of rectangular cell. For simplicity, the enzyme, which

acts as a fuel cutter, is illustrated by a scissors. .................................................................................... 16

Figure (5): One complete inchworm step starting from (a) to (g) ......................................................... 18

Figure (6): The relevant conformations and the labelling. A is the length of a repeating unit in the

track, x is the duplex length in the hairpin, w is the contour length of the single stranded loop in the

hairpin, y is the length of the motor bridge and z is the length of each linker that connects the motor

with its leg. Note that the length of the leg is not specified as it does not affect the motor size and is a

variable that will be changed in this experiment. .................................................................................. 20

Figure (7): DNA system illustration of M12 and M9 ........................................................................... 24

Figure (8): Fuel addition change the motor from a shortened state to a lengthened state..................... 25

Figure (9): Enzyme activity on lengthened motor to return it to the shortened state. In the process, the

fuel is cut into smaller remnants. The triangle symbol is used to represent the enzymes. .................... 25

Figure (10): The lengthened and shortened motor state ........................................................................ 26

Figure 11: Set up for gel solidification in electrophoresis .................................................................... 31

Figure (12): Gel electrophoresis to test the annealed sample. (a) annealing test for motor M9 and M12,

(b) annealing test for track. ................................................................................................................... 38

Figure (13): Gel electrophoresis performed for engine test .................................................................. 39

Figure (14): Raw binding data for M12 system with normal dye placement (choline chloride-Mg2+

buffer). .................................................................................................................................................. 43

Figure 15: Control experiment for binding experiment with normal dye placement (choline chloride-

Mg2+ buffer) ......................................................................................................................................... 43

Figure (16): Control calibrated binding data for M12 system with normal dye placement (choline

chloride-Mg2+ buffer) .......................................................................................................................... 44

Figure (17): Beta plot for M12 with normal dye placement (choline chloride-Mg2+ buffer) .............. 45

Figure (18): Control calibrated binding data for M12 system with switch dye placement (choline


Figure 19: Beta plot for M12 with switch dye placement (choline chloride-Mg2+ buffer) ................. 46





Figure 22: Raw operation data for M12 system under normal dye (choline chloride-Mg2+ buffer) ... 48

Figure (23): Control calibrated operation data for M12 system under normal dye (choline chloride-

Mg2+ buffer) ......................................................................................................................................... 49

Figure (24): Alpha plot for M12 system with normal dye placement (choline chloride-Mg2+ buffer) 50

Figure (25): Control calibrated operation data for M12 system with switch dye placement (choline


Figure (26): Alpha plot for M12 with switch dye placement (choline chloride-Mg2+ buffer) ............ 51

Figure 27: Control calibrated operation data for M9 system with normal dye placement (choline


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Figure 28: Control calibrated operation data for M9 system with switch dye placement (choline


Figure (29): Raw binding data for M12 system with normal dye placement (TAE-Mg2+ buffer) ...... 54

Figure (30): Control calibrated binding data for M12 system with normal dye placement (TAE-Mg2+

buffer) ................................................................................................................................................... 55

Figure (31): Beta plot for M12 with normal dye placement (TAE-Mg2+ buffer) ................................ 55

Figure (32): Raw binding data for M12 system with switched dye placement (TAE-Mg2+ buffer) ... 56

Figure (33): Control calibrated binding data for M12 system with switched dye placement (TAE-

Mg2+ buffer) ......................................................................................................................................... 56

Figure (34): Beta plot for M12 with switched dye placement (TAE-Mg2+ buffer) ............................. 57


buffer) ................................................................................................................................................... 58

Figure (36): Control calibrated binding data for M9 system with switched dye placement (TAE-Mg2+

buffer) ................................................................................................................................................... 58

Figure (37): Control calibrated operation data for M12 system under normal dye (TAE-Mg2+ buffer)

.............................................................................................................................................................. 60

Figure (38): Control calibrated operation data for M12 system under switched dye (TAE-Mg2+

buffer) ................................................................................................................................................... 60

Figure (39): Control calibrated operation data for M9 system under normal dye (TAE-Mg2+) .......... 61

Figure (40): Control calibrated operation data for M9 system under switched dye (TAE-Mg2+) ....... 61

Figure (41): Absorbance at 260nm for M12 in TAE- Mg2+ buffer ..................................................... 64

Figure (42): Absorbance at 260nm for M12 in choline chloride- Mg2+ buffer ................................... 65

Figure (43): Absorbance at 260nm for M9 in choline chloride- Mg2+ buffer ..................................... 65

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Chapter 1: Introduction

1.1 Significance of Nanomotors

The importance of nanomotors can be easily observed through the vital functions that natural

existing molecular motors in the human body are responsible for. Kinesin and dynein, which

are made up of proteins, are efficient and directional motors responsible for various cellular

transportation and play crucial roles in other cellular activities like cell division. Also, research

has found out kinesin is responsible for regulation brain functions and tumour suppression [10-

11].

These efficient and directional motors have inspired the creation of artificial nanomotors which

has the potential to support the advancement of nanotechnology because such machine, if

successfully developed, could perform a myriad of task such as transportation and fabrication

in nanoscale. Due to the small size, it can also perform with molecular precision. It is

envisioned to assist in targeted drug delivery which is especially useful to reduce the harmful

effect in chemotherapy. The importance of nanomotors has also been recognised by the

scientific committee for awarding the Nobel prize in Chemistry to the project on nanomachines.

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1.2 Project Motivation

The nanomachine that is awarded is a rotor that can spin directionally converting these motion

into useful work. Differing from these nanomachine which rotates, the nanomotors in the

project, as well as the published work by the research lead by Associate Professor Wang

Zhisong, has been working on motor systems made up of DNA which are designed to move

along a track and has the potential to make long range displacement or even make revolving

motion depending on the structure of the track. Several motors have been published and these

bipedal motors are able to walk in hand-on-hand manner either driven by light or autonomously

[1,2,12]. Although a variety of nanomachines have been developed, this area on the nanomotor

is still at its infancy as very few of these motors are robust enough to be exploited for real life

application.

Most of the motor systems developed currently all involve tracks with overhangs. Tracks with

overhangs are those with single stranded DNA suspending on top of the duplex backbone that

has sequences complementary to the motor leg. However, this might not be very desirable as

it might interact with other DNA present in the system. As the team further works to improve

the efficiency of the motor systems for real life application, the concern on overhang is one

area that should be reduced or eliminated.

To tackle this issue, an overhang free track has been designed several years back. Instead of

having the motor to bind to the overhangs, the single stranded binding sites act as ‘sticky end’

to attract the motor leg onto it. Although the motor operates on the track, the rigidity of the

track has been compromised due to the single stranded binding site, limiting the potential of

the motor system.

The pursuit to find an overhang free system, which can greatly simplify the system, and the

discovery that asymmetry exist between the sheering and unzipping force in the triplex

complex formed by LNA with DNA duplex [13], are motivated this project. The sheering and

unzipping asymmetry, which will be explained in greater details in the later chapters, is an

important element as it gives rise to ratchet effect, ensuring the motor’s motion. This finding

on the asymmetry inspired the creation of a motor that runs on a pure double stranded track

through the formation of triplex segments.

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1.3 Objective and Scope

The project aims to develop an autonomous inchworm nanomotor motor that moves on

overhang-free track. Specifically, the overhang free track in this system is made up of a pure

double stranded DNA which is rationally designed to have binding sites that forms triplex

structure with the DNA motor via Hoogsteen hydrogen bond. In addition, this project also

involves the designing of a new engine system that enables autonomous motion of the motor.

The engine adopts a single hairpin structure and interact with fuel and enzyme to create motion.

Upon designing, the subsequent experimentation on the motor system will allow us to verify

the hypothesis generated during the designing stage.

Due to the limitation of time, the project only covers experimentation of the motor on a two-

site track, testing the first round of hypothesis on the motor system. With the motor and two-

site track, we aim to investigate:

1) the functionalities of the new single hairpin engine,

2) DNA triplex formation and the binding preference, and

3) dissociation bias (shearing and unzipping effect) of the motor system upon fuel addition.

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Chapter 2: Theory

This chapter will discuss the relevant theory and information that serves to provide a better

understanding for the readers from various discipline.

2.1 Operation of DNA nanomotors

The motion of the DNA nanomotors are fundamentally driven by two types of biases, the

preferential dissociation bias and forward binding bias. Up till date, the published motors

engineered by members in the molecular motor lab are all designed to advance on the track in

the hand-on-hand manner [1,2]. In this project, we attempt to design a motor that moves in an

inchworm motion where a series of length extension and contraction of the motor length drive

the motion of the motor.

Just like walking, for a motor to make a successful advancement, the first key step will always

involve the dissociation of a leg. Preferential dissociation bias occurs when a particular leg of

the motor experiences a higher dissociation probability as compared to the other. This can bias

can be attained by inducing an asymmetry between the two leg-track binding of the motor

system.

Forward binding bias occurs when a motor, only bounded by a single leg, preferentially binds

to the binding site closer to the positive end of the track, where positive end is defined to be

the direction where the motor is advancing towards. The binding bias can be created by

adjusting the parameters of the motor design, such as altering leg and binding site distance, so

that the motor leg will be easily captured by the desired subsequent binding site upon

dissociation. It is important to note that these biases can be also induced by the intrinsic nature

of the motor leg-track binding. Hand-on-hand motor depends a lot on this factor for forward

binding bias.

The figure below illustrates one example of the forward and backward binding bias of a motor

bounded by a single leg. When the motor has a higher probability to bind to the binding site

closer to the positive end, as shown in figure 1(a), it will have a higher tendency flip over to

the positive end. On the other hand, if the motor has a higher probability to bind to the site

nearer to the negative end, it has a backward dissociation bias and will flip over to the negative

end more often. Since we want the motor to move from the positive to the negative end, the

forward binding bias is often more desired.

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Figure (1): A diagram to illustrate (a) forward binding bias and (b) backward binding bias

The following section will describe the mechanism behind both hand-on-hand and inchworm

motor.

2.1.1 Hand-on-hand motor

The hand-on-hand motor occupying two sites on the track can make a complete forward step

in two sub-steps: detaching the rear leg and binding of the rear leg to the third binding site. The

underlying criteria to the two steps are that we require the motor system to have a preferential

dissociation in the rear leg as well as a forward binding bias for the motor system.

Figure (2): Hand-on-hand motion of motor

The preferential rear leg dissociation will ensure that the rear leg has a higher probability of

detaching itself from the track as compared to the front leg. To achieved this, we must create

an asymmetry in the leg-track binding. Since the motors legs are designed to be identical, we

cannot depend on the length or sequence difference to alter the binding energy. However, we

can create this asymmetry by subjecting the two legs with forces which acts in different

direction. These forces can be brought about by the length changes of the motor, which in turn

induce tension in the motor legs [1]. An alternative way to create this leg-track binding

asymmetry is through track adjustment when give rise to a difference in the binding energy

between the two legs [2].

As seen from figure (2), upon rear leg dissociation, if the motor manages to flip and bind to the

binding site closer to the positive end, the motor will complete a forward hand-on-hand step.

However, it is also possible that the dissociated leg will bind back to the binding site which it

detaches from, causing a futile dissociation. Nonetheless, as long as the motor experiences

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forward binding bias, coupled with the rear leg dissociation bias, statistically, the motor will

advance towards the positive end.

2.1.2 Inchworm motor

The inchworm motor occupying two adjacent binding sites requires four sub-steps to complete

a forward step. The completion of a forward step in made possible by a series of motor length

contraction and extension. First, it requires the front leg to dissociate preferentially. This

dissociation can be induced by an extension of the motor length which in turn creates an

asymmetry in the leg-track binding. As the front leg dissociates, it immediately gets attracted

to the adjacent binding site, forming the conformation shown in figure 3(c). Following, a length

contraction in the motor will induce a rear leg dissociation shown in figure 3(d). The dissociated

leg will immediately bind and occupy the binding site where the front leg originally occupies

and this completes a full inchworm step.

Figure (3): Inchworm motion of motor

As seen above, the inchworm motion requires the front leg and rear leg dissociation bias in the

first and second dissociation stage respectively. The forward binding tendency of the inchworm

motor can be induced by designing the track such that the binding sites are either overlapping

or close together. By doing this, the dissociated leg can get attracted immediately to the

subsequent binding site upon dissociation of the correctly leg.

Even though the inchworm motor involves more steps to complete a full inchworm step, this

type of motor, if successfully designed, can better ensure directionality because the subsequent

binding site is always adjacent to the one it detaches from and thus the probability of attaching

to this adjacent binding site is much higher as compared to the others. For hand-on-hand system,

the motor must do a 180 flip to reach the subsequent binding site. Hence directionality can be

more easily attainable for inchworm motor.

Another advantage of the inchworm motor is that much more tolerant to any less desired

behaviour if the motor has an intrinsic forward binding bias. For example, when an undesired

rear leg dissociation happened instead of the front leg dissociation, having an intrinsic forward

binding bias gives the motor a hopeful chance of flipping over and attach to the binding site

7

closer to the positive end. This will recover the directionality of the motor. In addition, instead

of binding to the adjacent binding site in figure 3(d), the forward flipping tendency induced by

the forward binding bias, will speed up the forward motion of the motor. In this experiment,

we will be testing out such intrinsic forward binding bias even though it has little relevance in

the inchworm motion.

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2.2 Shearing and unzipping forces

As mentioned earlier, dissociation bias can be induced when there is an asymmetrical leg-track

binding and this can be attained by introducing asymmetrical force on the front and rear leg.

These asymmetrical forces take the form of a shearing and unzipping orientation. When each

of the force acts on the identical motor legs, the two legs will take a different orientation and

the stability of the leg-track binding will vary [1]. This asymmetry in the forces is not only

useful to the motor system, it is also useful in single molecular manipulation as the more stable

geometry is exploited to act as handle for the attachment of biomolecules [13,14].

It has been reported that under such asymmetry, the DNA duplex breaks apart into its individual

strands at different force magnitude when the force applied changes from shearing geometry

to unzipping geometry, indicating the difference in the stability as we change the geometry. In

shearing geometry, the base pairs in the duplex experience force together and the force acts in

the direction parallel to the single strands; in the unzipping geometry, the duplex unzips in a

base pair by base pair manner. The geometry induced force asymmetry in DNA has been

studied and it is found out that a larger force is required to separate a duplex when the force is

applied in the shearing manner as compared to the unzipping manner [14]. This finding has

been incorporated into a light-driven motor system [1] and positive results are obtained.

Other than DNA duplex, it is also found out that LNA-DNA triplex also has varied stability in

response to the direction of applied mechanical force. The LNA oligomer is two orders of

magnitude more stable under shearing force as compared to the unzipping force [13].

This finding has surfaced the possibility of creating a track that forms a triplex structure when

the motor leg binds onto it. However, in this project, instead of using the LNA-DNA triplex, a

pure DNA triplex is designed, using the same 15 nucleotide lambda genome sequence tested

in the LNA-DNA triplex.

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2.3 DNA Duplex and Triplex

DNA is a macromolecule built up by monomeric units called nucleotides. Each nucleotide is

made up of a five-carbon sugar (2’-deoxyribose), a phosphate group as well as a base. The

bases can be divided into two categories: purines and pyrimidines. In the case of DNA, there

are two purines, adenine (A) and guanine (G) and two pyrimidines, thymine (T) and cytosine

(C). These bases are responsible for the formation of hydrogen bond between or within DNA

molecules.

Majority of the naturally occurring DNA exist in the form of duplex where two antiparallel

strands of DNA oligonucleotides pair up by forming Watson-Crick hydrogen bond between

the bases. In the double stranded DNA, the adenine will pair with thymine, forming two

hydrogen bond between them and guanine will pair with cytosine, forming three hydrogen

bond between them. Due to the difference in the number of hydrogen bond, the GC pair is

slightly stronger than the AT pair.

The existence of triplex stranded DNA was first reported by Felsenfeld and Rich in 1957 [3].

The triplex structure of DNA, like its duplex counterpart, is sequence specific. It has been

discovered that there exist two types of triplex structure, the parallel and the antiparallel form.

This characterisation is determined by the composition and the orientation of the third DNA

strand [4].

The parallel structure takes the form of pyrimidine-purine-pyrimidine in the triplex. It is

defined as such because the third pyrimidine strand binds in a parallel way with respect to the

purine strand of the duplex. In another word, this parallel form of triplex involves the binding

of a pyrimidine-rich third strand with a duplex that consist of one purine rich and one

pyrimidine rich strand bounded by Watson-Crick hydrogen bond. The third strand will bind to

the purine strand of the double stranded DNA via Hoogsteen hydrogen bond. And the base

triplets binds in such a specific form C+*G:C and T*A:T (where * represents the Hoogsteen

hydrogen bond and : represent the Watson-Crick hydrogen bond). One condition to form

parallel structured triplex is that we require the cytosine in the third strand to be protonated and

thus slight acidic condition is required. It has also been reported that a small amount of MgCl2,

or more generally, divalent cations or high monovalent cation concentrations, in the solution

induces the formation of parallel triplexes [4] at room temperature [7].

The antiparallel form of triplex involves a purine third strand orientated in an anti-parallel

manner with respect to the purine strand of the Watson-Crick double stranded DNA, forming

10

base triplets of G*G:C and A*A:T [5]. The three strands forms purine-purine-pyrimidine

triplex. The bond involves in the antiparallel triplex is reverse Hoogsteen bond. It is reported

that unlike the parallel triplex, the antiparallel form of triplex can be formed at neutral pH in

the presence of magnesium ions. In addition, it has a binding energy of the third strand is about

twice as much as that in the parallel structure [5].

It is noticed that the binding of the third nucleotide or strand is always to the purine rich strand

of the double strand for both the parallel and antiparallel triplex structure. This observation can

be attributed to the fact that purines contain potential hydrogen bonds with incoming third

strand bases, as a result it can support the formation of the third strand through Hoogsteen bond

[6]. Structurally, Hoogsteen hydrogen bond and Watson-Crick hydrogen bond differs in the

orientation of the adenine and thymine base pairing. In the Hoogsteen base pairing, the

hydrogen bond is formed between the thymine and adenine is rotated by 180 as compared to

the one found by Watson and Crick [8]. Specifically, Hoogsteen hydrogen bonds is formed

between the N3 and O4 of thymine and N7 and N6 of adenine respectively [17]. In the reverse

Hoogsteen bond, the thymine is rotated by 180 as compared to the AT structure bound by

Hoogsteen bond [9].

11

2.4 Absorbance

Absorbance is a common method that is used to detect the formation and destruction of

secondary structures in DNA [4,15,16,17].

DNA molecules have characteristic absorbance at 260nm. This characteristic absorbance arises

due to the conjugated double bond and ring systems that is present in the purine and pyrimidine

nucleotides. Upon duplex or triplex formation, the absorbance of the DNA sample decreases

as the hydrogen bonds that is formed in the duplex and triplex limits the resonance behaviour

of the aromatic ring of the bases. The reduction in the UV absorbance is termed as

hypochromicity.

This absorbance property of the DNA is tested in this project and the results is used to as a

support for the formation of Hoogsteen bond and triplex.

12

2.5 Native polyacrylamide gel electrophoresis (PAGE)

Gel electrophoresis is a method that is used to separate macromolecules like DNA. Under an

applied voltage, the negatively charged DNA molecules will move to the positively charged

end. However, not all the molecules will move at the same speed. In general, the molecules

that have a lower molar mass will sieve through the dense polymer gel network more easily

and have a higher mobility in the gel as compared to those with a higher molar mass. As such,

the DNA molecules with a lower molar mass will be displaced a longer distance from the

starting point.

Native polyacrylamide gel electrophoresis, also known as non-denaturing gel electrophoresis,

is a type of electrophoresis which adopts a non-denaturing approach so that the DNA

macromolecules retain their structure during the electrophoresis run. As such, native gel

electrophoresis is not only useful to allow us to determine the molecular mass of sample and

the number of components in the sample but also gives us an idea of the secondary structures

formed within the sample. This is because the mobility of the DNA molecules in the dense

polymer network is also influenced by the different structures and shapes they take.

In this project, a series of electrophoresis experiments are carried out to test out the

effectiveness of DNA annealing and the new engine, specifically on the fuel and enzyme cycle.

Since we are interested to see the effect of the binding, the non-denaturing gel electrophoresis

is used so that we can retain and test the formation of the secondary structures. As the structures

of the DNA molecules have influence on the mobility, it is important to note that we will have

to analyse the gel by the number of bands and the relative position of each band. The ladder

lane will not be a good indication as the DNA molecules in the ladder lane has different

structures from the DNA samples, even though they have the same number of nucleotides, the

results will not be comparable due to the difference in the structure. The experimental methods

used to analyse the results will be discuss in detail in the later section.

13

2.6 Fluorescence experiment: the use of Dye and Quencher

In this project, fluorescence spectroscopy is the main method that used to test the statistical

behaviour of the motor system. For us to use this method to probe the system, specifically to

see the motor’s behaviour on the track, we artificially introduce dye and quencher into the DNA

molecules.

In the project, the dyes, fluorescein dT and TAMRA are added onto each end of the track and

two identical quenchers (3’ Iowa Black FQ) are incorporated onto each end of the motor leg.

fluorescein dT and TAMRA has an excitation/emission wavelength of 495nm/520nm and

559nm/583nm respectively and 3’ Iowa Black has an absorption spectrum ranging from 420 to

620 nm with peak absorbance at 531 nm. These dye and quencher, together with the DNA

oligomer are purchased from Integrated DNA Technologies Pte. Ltd.

When the dye is exposed to light that within its excitation spectrum, it will get promoted to a

higher energy state. As it de-excites back to the ground state, it will emit photons and this

photon can be detected. However, in the presence of quenchers, these emitted photons will be

absorbed and the intensity of the dye drops. The quencher can exist as both fluorescent or non-

fluorescent form [18].

In the case of a fluorescent quencher, this quencher will be called as an acceptor whereas the

dye will be labelled as donor. As the acceptor absorbs the photon emitted by the donor, the

acceptor will get excited and its de-excitation re-emits another photon with a higher wavelength

(a lower energy photon).

In this project, a non-fluorescence quencher is used. In the presence of such quencher, the

excited dye can return to the ground state by transferring the energy to the quencher. The

quencher will be promoted to its excited state and deexcite in the undetectable range or amount.

As compared to the fluorescent quencher, this non-fluorescence counterpart is preferred as we

will only be handling with one wavelength and the signal can be easily interpreted from the

change in the intensity of fluorescence [18].

The above mechanism works if the dye-quencher distance falls within the range of 20-100 Å

and these are due to the increase in the efficiency of fluorescence resonance energy transfer

(FRET). However, if the dye and quencher are brought even close than 20 Å separation, both

the intensity of the dye and fluorescence quencher will drop because most of the absorbed

energy will be dissipated as heat but not as light [18]. This phenomenon is termed as contact

14

quenching. Notice that at this distance, the effectiveness of dye and fluorescent quencher will

drop and the signal will not be an accurate indication of the dye-quencher distance. However,

the non-fluorescent quencher will still be useful in the contact quenching range as we do not

depend on the signal from the two dyes.

The dye and quencher interactions is a very useful experimental method to monitor the real

time behaviour of the motor systems. By attaching the dyes on the track and the quencher on

the motor, we will be able to track if binding between the track and motor leg occurs as binding

will results in an intensity drop in the emission by the dye on the track.

Studies have also revealed that strong bonds can be formed between some dye-quencher pairs

and these bonds differs between various dye-quencher pair [18]. Thus, even though the binding

affinity between the DNA strands is still the most significant factor that brings the quencher

and dye together, this introduced modification may give us biased signal because of the

additional attractive force between the dye and quencher.

This might be a potential problem to the motor system as the we adopted two different dye-

quencher pair. To investigate the significance of this problem, a reverse dye experiment is

conducted. These experiments will be elaborated in more details in the later chapters.

2.7 Enzyme

In this project, the restriction enzyme Nt.BbvCI is used, this enzyme is purchased from New

England BioLabs Inc. The purpose of the enzyme, together with the fuel, is to switch the motor

between contraction and extension mode, the mechanism behind this switching of mode will

be explained in detail in later chapters.

Nt.BbvCI is a nicking endonuclease that cleaves only one strand of the DNA on a double-

stranded DNA substrate. It recognises seven base pairs and makes a cleave at a on one strand

at a specific position as shown below:

15

2.8 Buffers

The buffer used is of critical importance as triplex structure requires a more stringent condition

to be formed as compared to its duplex counterpart. The section will summarise the findings

on the buffer.

Studies have shown that under choline dihydrogen phosphate (choline dhp) buffer, the DNA

triplexes are able to stabilise more significantly than in aqueous buffer at neutral pH. In addition,

the stability of Hoogsteen base pairs is found comparable to the Watson-Crick hydrogen bond

under this buffer [15]. In the study, NaCl buffer is also tested, and it is found out that triplex

does not form in a sequence with high G*C content. In addition, other than choline dhp, the

triplexes are also stable under choline chloride. However, studies have shown that choline dhp

stabilises the triplex structure more significantly than choline chloride. It is believed that

choline ions can surround the third strand of the triplex and strengthen its binding to the duplex,

thereby stabilising the G*C pair at neutral pH.

Several papers have reported that Mg2+ ions can stabilise triplex formation [4,7]. In fact,

triplexes will be stable in a buffer containing divalent cations or high monovalent-cation

concentrations at room temperature.

In this project, we explored several combinations of buffers, the key criteria that defines a

successful buffer one that supports triplex formation.

16

Chapter 3: Motor System Design

The motor system involve in this project is an entirely new system where its motion is

dependent on a newly designed single hairpin engine. As mentioned, the nanomotor in this

system is designed to advance on its own in the presence of fuel in an inchworm manner. One

key feature in this system is that the motor legs and binding sites on the track are designed to

bind via Hoogsteen bond forming short DNA triplex segments. This feature provides the

system with an overhang free track which can greatly simplify and reduce unnecessary

interaction between the track and other DNA strands in the system.

Generally, there is no fix rule on motor designing and we rely greatly on physical intuitions

and past experimental experiences to help us plan and come up with a design for the motor.

3.1 Motor design and operation mechanism

This section will provide a brief introduction on the components in the motor system and

elaborate on how the motor in the system makes one complete inchworm step.

3.1.1 Components in the motor system

The components of the motor system are the motor, duplex track, fuel, and enzyme. The

illustration of each component is shown in the figure below. The motor, track and fuel are all

constructed with DNA nucleotides whereas the enzyme used is called Nt.BbvCI.

Figure (4): Components of the motor system. It consists of the motor, track, fuel and enzyme. The

arrows illustrate the direction of DNA by pointing to the 3’ end. The track in the illustration consist of

five binding sites, each represented by a pair of rectangular cell. For simplicity, the enzyme, which acts

as a fuel cutter, is illustrated by a scissors.

The motor consists of two single stranded legs that are each connect to a short linker. The

purpose of the linkers is to provide flexibility for the system. The engine of the motor takes a

single hairpin structure; it consists of a duplex segment (in blue) as well as a loop of single

17

stranded DNA (in red). In addition, there is a motor bridge segment to provide certain degree

of rigidity to the motor.

The track is made up of two antiparallel DNA strands that are bound together by Watson-Crick

hydrogen bond. The track illustrated in figure (3) has five binding sites, however, the number

of binding sites used in the experiment can be varied depending on the purpose of the

experiment. In this project, we are investigating the behaviour of the motor using a track with

two binding sites. As the motor settles down onto the track, the leg on the motor will bind to

the track via Hoogsteen bond forming DNA triplex segments. As the motor takes on different

length configuration, the motor is expected to advance on the track moving from the positive

end to the negative end as labelled on the track. As this is an inchworm system, the track is

made up of repeating units of the binding sites with no spacer to ensure that the binding site is

as close as possible to promote the forward binding bias.

The length change in the motor is driven by the intrusion of fuel and the activity of the enzyme.

The fuel is made up of a single stranded DNA which serves to interact with the hairpin engine

on the motor. As it attached to the hairpin, specifically at the single stranded loop, it is

constructed to peel the hairpin structure, changing the motor to an elongated state (extension

mode). The fuel is specifically designed to have sequence that is complementary to the red

segment of the hairpin.

On the contrary to the fuel, the enzyme, Nt.BbvCI, promotes the motor to take a shortened state.

Both the elongated and shortened state are crucial for the advancement of the motor in the

motor system. The Nt.BbvCI enzyme recognises a specific sequence of double stranded DNA

and by constructing the fuel sequence to be the sequence recognised by the enzyme, we will

expect the enzyme to cut the fuel strand into shorter segments. As the short fuel remnants

dissociate away from the motor by thermal fluctuation, the hairpins form back, obtaining the

shorter state.

The above is a brief introduction of each components, more details will be elaborated in the

consecutive sections. In the next section, we see how all the components are expected and

designed to work together, allowing the motor to make a successful forward step.

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3.1.2 Elaboration on the inchworm motion

Figure (5): One complete inchworm step starting from (a) to (g)

To start off, we first consider that the motor legs manage to form Hoogsteen bond with the

binding site on the track and settle down on the first two binding sites as shown in the figure

5(a) below. The motor starts off being in a short state where the engine takes the hairpin

structure. For this configuration to be favourable, the motor size and the spacing between each

binding site should be set such that it will minimise any tension within the motor, creating a

state with a lowest free energy.

Notice that the motor leg and the track strand bind in a parallel manner taking on parallel triplex

structure. In another word, the 3’ end of DNA are both at the same direction. This orientation

hardly occurs in duplex structure, but it is a feasible in the triplex structure. In fact, this is a

deliberate design to ensure that the motor leg only forms triplex segments with the duplex track

but will not compete and form any duplex structures with any unbounded single stranded

complementary track strands that might be present in the system.

19

Next, fuel strands are introduced into the system as shown in figure 5(b). The fuel is designed

such that it can first interact with the motor by binding to the single stranded red loop and

subsequently peel off the hairpin (blue segment). After the fuel gets incorporated in to the

motor system, the motor takes an elongated state (extension mode). This increases the tension

in the motor system because of the size mismatch.

To start off an inchworm motion, it is desirable for the front leg to be preferentially dissociated

first. This can be done by fixing the configuration of the system such that we can induce

asymmetry in the leg-track binding. As mentioned, in the LNA-DNA triplex, LNA strand is

less stable when it takes an unzipping orientation with respect to an applied force, as compared

to shearing orientation. Presuming that pure DNA triplex also experiences such asymmetry, we

design the system such that the front leg and rear leg experience unzipping and shearing force

respectively as illustrated in figure 5(c). By doing this, the front leg will be less stable on the

track and is likely to be dissociated preferentially and forms a stable elongated motor state on

the third binding site which is in the proximity.

To make a complete forward advancement, the enzyme is added into the system as shown in

figure 5(e). The purpose of the enzyme is to nick the fuel strands that are attached to the motor

so that the motor can return to its short state. As the fuel remnants dissociate from the engine

of the motor, the hairpin re-forms with one leg on the first binding site and another on the third.

A tension is induced on motor leg and the rear leg again as illustrated in figure 5(f). Under this

configuration, the rear leg now experiences unzipping force which is relatively less stable and

will be preferentially dissociated. The rear leg will be attracted to the second binding site on

the track, completing an inchworm step as shown in figure 5(g).

In summary, length change of the motor coupled with leg-track binding asymmetry in the motor

system is sufficient to induce the motor’s motion. The forward binding bias in the system is

constructed by having a track with no spacer. The removal of spacer allows the dissociated

motor leg to get attracted to the subsequent binding site which is just in vicinity.

20

3.2 Length Selection

The length of motor and track is a crucial factor that ensures the motor’s forward advancement

on the track. The length of the motor and track are restricted by three constrains. Firstly, the

motor in the shortened state must be stable on when it binds to two adjacent binding sites on

the track, this is configuration is shown in figure 5(a) and 5(g). Secondly, as illustrated in figure

5(d), the elongated motor must be long enough to form a stable conformation across three

binding sites so that the first advancement step can take place. Thirdly, when the motor takes

the conformation in figure 5(c), the motor must still be able to barely fit into the adjacent

binding sites with the assistance of single stranded section (linkers and one segment of the

hairpin). This is to ensure that the expulsion mode can take place to induce the shearing and

unzipping force on rear and front leg respectively. From the three constrains, three governing

formula are derived intuitively below.

1) y + 6 = A

2) y + w + x = 2A

3) y + w + x - 2z (nt) - x (nt) > A

The constant 6 in the first equation is referring to the 2nm width of the double stranded DNA

which is about 6 base pairs long.

Figure (6): The relevant conformations and the labelling. A is the length of a repeating unit in the track,

x is the duplex length in the hairpin, w is the contour length of the single stranded loop in the hairpin,

y is the length of the motor bridge and z is the length of each linker that connects the motor with its leg.

Note that the length of the leg is not specified as it does not affect the motor size and is a variable that

will be changed in this experiment.

The linkers usually serve to provide some flexibility in the system and are not accounted for in

formula (1) and (2) to provide some freedom for the system. However, if necessary, we can

21

compromise some of these freedoms for a more stable system. If we were to forgo the freedoms

by accounting all the flexible linkers, we have the following three more generic formula:

1) y + 6 - 2z (nt) < A < y + 6 + 2z (nt)

2) y + w + x – x (nt) - 2z (nt) < 2A < y + w + x + x (nt) + 2z (nt)

3) y + w + x - 2z (nt) - x (nt) > A

The three equations above are in the unit of length and, for simplicity, will be expressed in

terms of number of base pairs. The last two terms in the left-hand side of equation (3) are

contributed by the single strands segments and for clarity are labelled with nt in parenthesis to

differentiate them from the other terms which are contributed by base pair length. In fact, the

nucleotide of single strands and base pairs of duplex vary slightly in terms of their length, as

summarised in the table (1) below. In the calculations, they are approximated to be the same

as they length differs only by a little.

The table below summaries the various length scales that are relevant to this nanomotor.

Length (nm) Length (base pairs)

Width of a double strand 2 Approximately 6

1 nucleotide (nt) 0.4 Approximately 1

1 base pair (bp) 0.34 1

Table (1): Various length scales relevant in the motor system

Considering the three formula as well as the stability of DNA duplex under thermal fluctuation,

below is the number of base pairs each segment will take:

• A = 15 bp

• y = 15 bp

• w = 10 bp

• x = 10 bp/nt

• z = 4 nt

To make comparison with the LNA-DNA triplex [13], the sequence of the track is also taken

from the lambda genome (4404-4418) and one repeating unit in the track is made up of 15 base

pairs. In addition, keeping in mind that any duplex with less than ten nucleotides will be easily

dissociated under thermal fluctuation, the segments that require for duplex binding are set to

take a length that is take more than 10 nucleotides long.

22

In this project, we will be testing two motor systems that differs in the length of the motor legs.

One system involves motor with 9 nucleotides long legs while the other has motor with 12

nucleotides long legs. The table summarises the acronym and length involved in the DNA

strands used in this project.

Component Description Acronym Number of

nucleotides

Track (2 binding sites) Track strand 1 T1 30

Track strand 2 T2 30

Motor (9nt leg) Motor strand 1 (9nt) MS1 (9nt) 58

Motor strand 2 (9nt) MS2 (9nt) 28

Motor (12nt leg) Motor strand 1 (12nt) MS1 (12nt) 61


Fuel Fuel Fuel 20

Table (2): DNA strands used in this experiment

23

3.3 Sequence Generation

The sequences are carefully designed to ensure that the various strands and segments bind

accordingly. The sequence of the track and the motor leg are adopted from the lambda genome

as these sequences are tested to show asymmetry with different force orientation in the case of

LNA. The fuel and the hairpin are made up of the sequence that is recognised by the enzyme.

For the other segments are generated using CANADA software to minimise any possible

unwanted intra-strand interactions. The secondary structures and thermodynamic calculations

of the sequence are then predicted using Mfold web server.

As mentioned, the track is made up of repeating units of a short segment in the lambda genome.

Unlike other motor designs [1,2], this track is deliberately designed to not consist of any spacers

so that the front leg can be attracted to the adjacent binding site without much delay upon

dissociation. A track with two binding sites will have two of such 15 nucleotides sequence.

The sequences of the duplex track are shown below:

T1: 5’-agaggaggagaagagagaggaggagaatag-3’

T2: 5’- ctattctcctcctctctcttctcctcctct-3’

To prevent the formation of an infinitely long track during the annealing of track, a point

mutation is introduced to the track, as indicated by the bold in the track strands.

The motor and fuel are designed carefully such that they contain sequences that can be

recognised by the enzyme. As mentioned, the enzyme recognises the following sequence and

nicks only on one strand as shown below:

The sequences are as shown below:

MS1 (9nt): 5’-accacattgtccggcgagctgagggctgaggcaccccctaagctcttttcctcctctt-3’

MS2 (9nt): 5’-gccggacaatgtggtttttcctcctctt-3’

MS1 (12nt): 5’-accacattgtccggcgagctgagggctgaggcaccccctaagctctttttctcctcctctt-3’

MS2 (12nt): 5’-gccggacaatgtggttttttctcctcctctt-3’

24

Fuel: 5’-ggtgcctcagccctcagctc-3’

For the fuel remnants to be easily dissociated by thermal fluctuation, two nicking sites are

introduced in the fuel as shown below.

Due to the hairpin structure, the motor will also consist of a segment that the enzyme recognises

and cuts. To prevent the motor to be nicked by the enzyme, a point mutation is done to in the

MS1 as indicated by the bold.

To measure the behaviour of the motor, two dyes (Fluorescein dT and TAMRA) and two

identical quenchers (3’ Iowa Black FQ) are attached on the track and motor legs respectively.

Fluorescein dT and TAMRA have an emission/excitation wavelength of 495nm/520nm and

559nm/583nm respectively. At proximity with the quencher, the intensity of the dye will

diminish allowing us to detect the leg-track binding.

Along with the quencher, the expected structure of the 2 sites track and motor takes

conformation as shown below.

Figure (7): DNA system illustration of M12 and M9

25

3.4 Fuel and enzyme cycle

With the DNA conformation shown in figure (7), we follow the how the fuel and enzyme work

together to create the shortened and lengthened state of the motor.

The fuel is made up of sequence that is complementary to the loop of the hair pin in figure (7).

As the fuel got incorporated into the motor, the hairpin is expected to break and the entire fuel

strand will bind with one segment of the hairpin while the other segment of the hairpin becomes

single stranded contributing to the flexibility of the motor.

Figure (8): Fuel addition change the motor from a shortened state to a lengthened state

As shown in figure (8), when the enzyme is added into the system, it recognises the fuel binding

on the motor and nicks it into shorter fuel remnants. The fuel is deliberately designed to take

the sequence of the strand which enzyme cuts while the strand on the motor takes on its

complementary strand. There are two nicking sites on the fuel and this is to ensure that the fuel

remnants can be dissociated by thermal fluctuation and the hairpin structure can be reformed.

From figure (8), we can also clearly see the need for a point mutation on the hair pin structure

a bolded adenine is introduced in place of a cytosine nucleotide. If the point mutation is not

introduced, the enzyme will recognise and nick any motor that is in the shortened state which

is not desirable for the system.

Figure (9): Enzyme activity on lengthened motor to return it to the shortened state. In the process, the

fuel is cut into smaller remnants. The triangle symbol is used to represent the enzymes.

26

If there are sufficient fuel strands, the motor will cycle between the lengthened and shortened state until

all the fuel strands are used up.

Figure (10): The lengthened and shortened motor state

27

Chapter 4: Materials and Methods

4.1 Preparation of DNA single stranded stock (100m)

The DNA used in this experiment are purchased from Integrated DNA Technologies Pte. Ltd

(IDT) and they appeared in the dry (lyophilised) form. To resuspend the oligonucleotide to a

stock concentration of 100m, Tris-EDTA buffer (10mM Tris-HCl; 1mM disodium EDTA at

pH8) into each single stranded tube. The following steps are taken when preparing the single

stranded stock.

1) Dried single stranded DNA are spun down prior to opening the tube to ensure that these

DNA strands will not be dislodged from the tube upon opening.

2) Various amount of TE buffer is added into each tube to create a stock of concentration

100m. This amount is specified in the specification sheet and they are summarised in

the appendix (1).

3) The 100m single stranded stock is then vortexed and centrifuged to ensure a

homogeneous stock.

4) The prepared stock is placed a -4C freezer for storage.

28

4.2 Annealing

This section describes the steps taken to anneal the single stranded DNA to form duplex

structures. In this project, the three duplex structures involved are namely, track (T), motor

with 12 nucleotides leg (M12) and motor with 9 nucleotides leg (M9). Upon annealing, each

double stranded stock will have the concentration of 5m. A solution consisting NaCl and TE

buffer act as the annealing buffer for duplex to form. The following steps are taken to obtain a

double stranded stock same of 5M.

1) 5.844g of NaCl salt crystals are added into 50ml of TE solution to produce a NaCl stock

of 2M.

2) 2l of the 2M NaCl is pipetted into Eppendorf tube and mixed with 1l of T1 from

100M stock, 1l of T2 from 100M stock and 16l of TE solution to create a 20l

solution with 0.2M NaCl. (For T)

3) Similarly, 2l of the 2M NaCl is pipetted into Eppendorf tube and mixed with 1l of

MS1 (12nt) from 100M stock, 1l of MS2 (12nt) from 100M stock and 16l of TE

solution to create a 20l solution with 0.2M NaCl. (For M12)

4) 2l of the 2M NaCl is pipetted into Eppendorf tube and mixed with 1l of MS1 (9nt)

from 100M stock, 1l of MS2 (9nt) from 100M stock and 16l of TE solution to

create a 20l solution with 0.2M NaCl. (For M9)

5) The three tubes (T tube, M12 tube and M9 tube) are then vortex to ensure a

homogeneous solution and centrifuged so that all the samples are spun down to the

bottom of the tube.

6) The annealing protocol is set as follow: The temperature of the annealing sample is

heated up to 95C for the first 20 mins. Very quickly, the temperature is lowered to

90C and subsequently lowered, step wise, at 0.1C every 25 seconds to 80C.

Subsequently, the temperature is further lowered to 80C, again step wise, and

maintained at 20C.

7) Upon annealing each tube consist of 5M of the double stranded DNA stock and are

all transferred into a screw cap tube.

8) The stocks are then kept in the freezer of -4 degree Celsius for storage.

29

4.3 Single stranded stock (5l) and fuel stock (10l) preparation

In some part of the experiment, we are required to use the single stranded oligonucleotide and

the following steps are taken to create single stranded stock that has a concentration of 5M.

These smaller concentrations are more convenient for experiment as most of the experiments

only require a small amount of DNA. The fuel, which also prepared in the single stranded form,

is prepared at a higher concentration of 10M.

To prepare a 10M fuel stock, the following steps are taken:

1) 2l of fuel from 100M stock is mixed with 18l of TE buffer in a screw cap tube.

2) The prepared 20l solution is vortexed to ensure a homogeneous solution and kept in

the freezer of -4 degree Celsius for storage.

There are eight single stranded stock to prepare and they are T1, T2, T1 (3 site), T2 (3 site),

MS1 (12nt), MS2 (12nt), MS1 (9nt) and MS2 (9nt). To prepare a 5M single stranded stock,

the following steps are taken:

1) 1l of each single strands from 100M stock is mixed with 19l of TE buffer in a screw

cap tube.

2) The prepared 20l solution is vortexed to ensure a homogeneous solution and kept in

the freezer of -4 degree Celsius for storage.

These strands are mainly used for in electrophoresis to test the quality of annealing.

30

4.4 Native polyacrylamide gel electrophoresis (Native PAGE)

As mentioned in the earlier chapter, this non-denaturing form of electrophoresis is used in this

experiment to test the annealing of the various strands as well as to test the fuel and enzyme

cycle.

It is noted that about 200ng of DNA macromolecules is required and sufficient to produce a

gel of appropriate brightness. Other than the DNA, gel loading dye are also added into the

sample. TE buffer is used to top up so that each lane can be loaded with 6l of sample. The

purpose of the loading dye is to allow the DNA to sink to the bottom of the well and for us to

visualise the DNA as they run down the gel.

Other than the DNA sample, one of the lane is loaded with DNA ladder instead of the DNA

macromolecules. The DNA ladder consist of a set of reference duplex so that we can compare

our DNA samples with these duplex standards. However, most of the samples that are loaded

into the wells do not take a pure duplex structure. In fact, DNA oligonucleotides that are single

stranded are added into some wells and these strands are expected to form clumps. The various

structures will result in a different mobility in PAGE as compared to the pure duplex of the

same molecular weight. Being so, the formation of motor and tracks are confirmed by making

comparison with the bands of that consist of single strands.

The composition of individual samples used in experiment to test for duplex and fuel-enzyme

cycle is included in the appendix (2) and appendix (3) respectively.

4.4.1 Gel preparation

The polyacrylamide gels are created from the following reagents:

1) 30% Acrylamide

2) DI water

3) 10x TBE

4) 10% APS

5) TEMED

Polyacrylamide gel is used rather than agarose gel in this experiment because the DNA

oligonucleotides used in the experiment are relatively short. The 30% polyacrylamide is made

up of a mixture of acrylamide and bisacrylamide in 29 to 1 ratio. The polyacrylamide gel is

formed by a free radical polymerisation of the acrylamide and bisacrylamide. Ammonium

31

persulfate (APS) provides the free radical required for this process to occur and

tetramethylethylenediamine (TEMED) is used as a catalyst to speed up the gel formation.

The following steps are taken to prepare the 10% gel:

1) 6.6ml of DI water, 1.2ml of 10x TBE and 200l of 10% APS are pipetted and mixed

into a tube. The sample are then brought to the fume hood for the addition of 4ml 30%

Acrylamide and 10l of TEMED.

2) The sample are then vortexed to get a homogeneously mixed sample.

3) The glass slides that are used to form the cast for the gels are cleaned thoroughly with

DI water followed by ethanol. This is to ensure that the gel can be more easily removed

after electrophoresis.

4) The prepared sample is poured into the ensembled cast as shown in the figure (11)

below and the comb is then gently inserted making sure that there are no air bubbles

created in the process.

Figure 11: Set up for gel solidification in electrophoresis

5) The set-up is then left untouched for about 1 hour until the gel solidifies. The tube

containing some remaining solution is turned upside down to help to gauge the extent

of solidification.

6) Upon solidification, gently remove the comb and release the cast from the solidification

set up. The gel together with the cast is then mounted on the holder for electrophoresis.

The entire set up is moved into the gel box, making sure that the positive and negative

terminal of the holder correspond to the one indicated on the gel box.

32

7) Next, the electrophoresis buffer (1x TBE) is then filled up to the labelled height. When

filling the buffer, fill from the gel holder first, flushing the small wells in the gel with

the buffer as it overflows into gel box.

8) To create the electrophoresis buffer (1x TBE), Add 5ml to 50ml of DI water.

9) Load the 6l of DNA samples into each well. It is advisable not to load the DNA

samples at the first and last well.

10) The voltage is set at 90V, which is about 5V/cm measured from the positive to negative

terminal. It is recommended that the voltage should not exceed 20V/cm.

11) It is also important to note that the current is proportionate to the number of gel samples

in the tub, the 4 gel samples should have a current of about 80mA. There should be air

bubbles appearing at the gel with electrophoresis is taking place.

12) The timing for the electrophoresis is set to 50 minutes. However, regular checks on the

gels are advised making sure that the dye do not move past the end of the gel.

13) To make the DNA visible under UV light, gel red is used to stain the DNA. The function

of gel red is the same as the well-known DNA fluorescence tag, ethidium bromide, but

it is a safer version as it is not a mutagen. Gel red is produced by mixing 50ml of DI

water with 5l of 10000x gel red.

14) The gel is carefully peel off from the cast and placed in a container containing the 50ml

gel red. The container is put onto a rocker for 25 minutes for the gel red to stain the

DNA on the gel.

15) Place the gel on the UV plate for band detection.

33

4.5 pH measurement

As the project involves triplex structure which is pH sensitive, it is required in the experiment

to measure the pH of buffers. In this experiment, the pH meter from SCHOTT Instruments is

used to measure the pH of the solution. However, the limitation of this pH meter is that it

requires at least 5ml of solution to give a measurement. In all the fluorescence experiment, the

volume of the sample used is approximately 700l (0.7ml) and this amount is insufficient for

the pH meter to give a reading as a result scaling up of the solution is required.

For example, to measure the pH of a 500l TAE-Mg2+ buffer with 62.5l of HCl, another

sample will be prepared by mixing 50ml of TAE-Mg2+ buffer with 6.25ml of HCl to have

obtain a solution with the same pH.

The following steps are taken when measuring the pH of the scaled-up sample:

1) The tip of the pH meter is cleaned with DI water thoroughly.

2) Immerse the tip of the pH meter in to the prepared sample ensuring that the solution is

filled up to the line indicated on the pH meter.

3) Wait for the readings on the pH meter to stabilise and record the value.

4) Wash the tip again with DI water again.

5) Fill the cap of the pH meter with 3M KCl and soak the tip of the pH meter into the KCl

solution.

34

4.6 Fluorescence experiment

The fluorescence experiment is carried out to detect the statistical motion of the motor. For us

to obtain fluorescence signal, we introduce two different dyes and two quenchers on the track

and motor respectively. The two dyes are Fluorescein dT and TAMRA where their

excitation/emission wavelength are 495nm/520nm and 559nm/583nm. The two quenchers

attached at the end of each motor leg are both Iowa Black FQ quencher which has an absorption

spectral ranging from 420nm to 620nm peaking at 531nm. At proximity, the quencher will

absorb the fluorescence emitted by the detector and causing the intensity of the intensity of the

emitted light to decrease. By making use of this dye-quencher mechanism, we will be able to

monitor the motor leg and track position by measuring the emission intensity in real time. Cary

Eclipse Fluorescence Spectrophotometer is used in the fluorescence experiment. This machine

is used to carry out both binding experiment and operation experiment.

4.6.1 Binding experiment

Binding experiment is carried out to test out to track the binding of motor on the track. In this

project, the motor and track are designed to bind via Hoogsteen bond, forming triplex motor

leg-track segments. The formation of triplex bond, which is relatively weaker, requires much

stringent condition to be formed. Hence this experiment is also, other than tracking the motor

binding process, is also at the same time used to detect triplex formation.

Below is the procedure taken when carrying out the binding experiment using fluorescence

method:

1) The fluorescence spectrophotometer is set to emit wavelengths of 495nm and 559nm

to excite Fluorescein dT and TAMRA dye in the track respectively. The detection

wavelength is set at 520nm and 583nm. The voltage is set to 800V and both the

emission and excitation slit width is set to 5nm.

2) The sample is prepared by mixing 2l of 5M annealed track into 696l of operation

buffer with desired pH. The sample is pipetted into a 3mm cuvette.

3) The cuvette is cleaned and dried on the exterior and placed into the fluorescence

spectrophotometer. The experiment is set to run to for 10 minutes to measure the track

only intensity.

4) After 10 minutes, 2l of 5M of annealed motor (M9/M12) is added into the cuvette.

The experiment is set to run again to measure the intensity of the motor-track system.

5) The experiment is carried for 2 hours.

35

6) Control for this experiment follows the same experimental procedure from step 1 to 5

with the exception that instead of adding motor (M9/M12) to the system in step 4, the

buffer in the motor is added. The motor buffer consists of TE solution and 20mM of

NaCl.

If triplex is formed, the intensity of the dye emission will drop as the quencher on the motor

leg binds and quenches the dye emission. Upon verifying the triplex formation with the

intensity drops, the motor and track complex is incubated overnight and is used for operation

experiment.

4.6.2 Operation experiment

The operation experiment is useful to allow us to investigate the dissociation bias in the motor

system. For the motor to progress in the inchworm manner, the front leg should dissociate more

than the rear leg, hence front leg dissociation bias is favoured. However, it has yet to be proven

that for DNA triplex shearing mode is stronger than unzipping mode, thus this experimental

result is opened to uncertainty.

The operation experiment on two site track involves the addition of fuel into the motor-track

incubated system. The following procedures are taken during the operation experiment.

Usually the sample used in this experiment is the same batch of sample used in the binding

experiment to reduce wastage.

1) The sample is prepared by mixing 2l of 5M annealed track into 696l of operation

buffer with desired pH. This sample is incubated for about 15 minutes before motor

(M9/M12) is added into the sample.

2) The sample is incubated overnight. To prevent any possibility of over-quenching of the

dye present in the sample, the solution of sample is wrapped in aluminium foil during

the incubation.

3) After overnight incubation, the sample is pipetted into the 3mm cuvette and placed into

the fluorescence spectrophotometer. The fluorescence spectrophotometer is set to emit

wavelengths of 495nm and 559nm to excite Fluorescein dT and TAMRA dye in the

track respectively. The detection wavelength is set at 520nm and 583nm. The voltage

is set to 800V and both the emission and excitation slit width is set to 5nm.

4) The cuvette is cleaned and dried on the exterior and placed into the fluorescence

spectrophotometer. The experiment is set to run to for 10 minutes to measure the motor-

track intensity.

36

5) After 10 minutes, 2l of 10M of fuel is added into the cuvette. The experiment is set

to run again to measure the intensity of the system.

6) The experiment is carried for 90 minutes.




NaCl.

37

4.7 Absorbance experiment

Absorbance experiment is carried out the verify the triplex formation. The characteristic

absorption wavelength for DNA nucleotides is at 260nm. The absorption at 260nm is tested to

test for the formation of triplex structure and monitor any decrease in the intensity signal as

fuel is added. This experiment serves as a verification of any bond formation and breakage and

the result is useful to support the experimental conclusion derive from the fluorescence

experiment. The Cary 50 Bio UV-Visible Spectrophotometer is used in the experiment to

measure the absorbance.

The following procedures are taken for the absorbance experiment:

1) The emission wavelength is set at 260nm

2) 696l of operation buffer is added into the 3mm cuvette. The cuvette is cleaned and

dried on the exterior and placed into the fluorescence spectrophotometer. The

experiment is set to run to for 10 minutes to measure the buffer only intensity.

3) 10l of the track is added into the cuvette; the experiment is set to run for another 10

minutes to measure the absorbance of the track at 260nm.

4) After another 10 minutes, 10l of 5M of annealed motor (M9/M12) is added into the

cuvette. The experiment is set to run again to measure the absorbance of the motor-

track system.

5) The experiment is carried for 1 hours.




NaCl.

38

Chapter 5: Experimental results

In this project, a series of experiments are carried out to examine the behaviour of the motor

and allow us to verify some of the hypothesis made during the motor designing stage. The

experiments can be categorised into three main section:

1) Electrophoresis

2) Fluorescence

3) Absorbance

In this chapter, the experimental results will be presented and analysed.

5.1 Native PAGE

Gel electrophoresis is performed to determine the quality of the annealed sample and to test

out the functionality of the newly designed single hairpin engine.

5.1.1 Quality of annealed sample

The figure below is the results obtained from the electrophoresis done on one batch of

annealing.

Figure (12): Gel electrophoresis to test the annealed sample. (a) annealing test for motor M9 and M12,

(b) annealing test for track.

The individual bands in each lane represent different components or structures that exist in the

sample. From figure (12), it can be seen that the annealing process is successful. In figure 12(a),

the single strands that form M12 and the annealed motor M12 are placed in lane 1, 2 and 3

respectively. The single band in lane 3 shows that the annealing for M12 of this batch is done

nicely. Notice that M12 having a larger mass as compared to the individual strands is displaced

39

a shorter distance from the well. This observation fits into the theory that a heavier DNA,

having a lower mobility, will make a shorter displacement.

Similarly, the single strands of M9 and the annealed M9 are placed in lane 4, 5 and 6

respectively. It is noticed that two bands appeared in lane 6. Closer analysis and comparison

with lane 4 allow us to conclude that the extra band corresponds to MS1(9nt). The presence of

this band indicates that an excess MS1(9nt) is added into the sample during the annealing

process.

As seen from figure 12(b), the annealing of the track is also done successfully. It is noticed that

the intensity of the band is much higher than in lane 1. This is because dyes are present in these

samples, causing an amplified intensity to the bands.

From these results, we can see that there is only a single prominent band in each annealed

sample and other than checking for the quality of the annealing, we can infer that the motor

and track bind very likely according to what we planned.

5.1.2 Functionality of the newly designed single hairpin engine

Figure (13) below shows the electrophoresis results that is performed to test the performance

of the single hairpin engine. In this experiment, the fuel and motor complex samples are

incubated for one hour and the samples with enzyme are incubated for 1.5 hours before the

enzyme is denatured by incubating the sample in 80C for 20 minutes.

Figure (13): Gel electrophoresis performed for engine test

40

The enzyme, Nt.BbvCI, is a nicking endonuclease that cleaves only one strand of the DNA on

a double-stranded DNA substrate. In the motor system, we design the system such that this

enzyme only recognises and cuts the fuel strand that is attached onto the motor. The enzyme

must not cut the motor hairpin and single stranded fuel for the system to work autonomously.

In this experiment, lane 1 consists of the strand that consist of the engine of M12 (MS1(12nt))

and lane 2 consist of MS1(12nt) and enzyme. The cutsmart (cs) buffer, that is necessary to

boost the activity of the enzyme, is added into each lane as well. As seen from lane 1 and 2,

even though enzyme is added into the system, the bands appear the same indicating that the

enzyme has no effect on the sequence on the engine. This also proves that the point mutation

done on the hairpin strand, as mentioned in section 3.3, is effective to prevent enzyme from

cutting it. In addition, this also shows that the enzyme activity is very sequence specific and a

single point mutation on a nucleotide is capable to dysfunction the enzyme.

A similar test is performed on the fuel strands on lane 3 and 4; Lane 3 consist of fuel only and

lane 4 consist of both fuel and the enzyme. As seen from figure (12), both the bands appear at

the same displacement from the well. This serves as an indication that the enzyme does not

have any activity on single strands.

Lane 5 and 6 are constructed to test the functionalities of the engine. In lane 5, the control lane,

the MS1(12nt) and fuel strands are added. In addition to what is added in lane 5, lane 6 consists

of the enzyme as well. In the two wells, the enzyme and fuel ratio is 1:3, an excess fuel is added

to test out the effectiveness of the enzyme and fuel cycle.

In lane 5, we can clearly see two distinct bands which correspond to the fuel and the motor-

fuel complex. We can conclude that the band nearer to the well corresponds to the motor-fuel

complex but not the motor because this band takes a displacement that is slightly smaller than

the MS1(12nt) strands in lane 1 and 2.

Analysing the results in lane 6, we can see that the two bands which appeared corresponds to

the motor-fuel complex and the MS1(12nt). Making comparison with the results lane 5, we

observe that fuel band disappeared and instead MS1(12nt) appeared with the addition of

enzyme. Since we tested that the enzyme will not cut the single stranded fuel that is in excess,

the possible reason for the disappearance for the fuel band is be attributed to the fact that the

enzyme is able to cut the fuel strands which got incorporated into the engine. This cycle repeats

41

itself until the fuel strands are all used up. The re-appearance of MS1 (12nt) tells us that the

fuel remnants can dissociate effectively from the motor just by thermal fluctuation.

From the analysis above, we can conclude that this engine works well and is able to support

the fuel-enzyme cycle. In addition, we can also conclude that the fuel strands can effectively

interact and bind to the hairpin structure forming the desired structure, which subsequently give

rise to a series of fuel-enzyme cycle until the fuel strands are exhausted.

As mentioned earlier, the enzyme is introduced for 1.5hours into the system before it is

denatured. This timing might be too short for the enzyme to exhaust all the fuel strands. It is

expected that if a longer enzyme incubation time is allowed for the system, only one band, the

MS1(12nt) will be present. It is also noted that the fuel remnant strands which are about six or

seven nucleotides long are not observed in the gel. One reason for this is that the mobility of

these remnants move so fast that they have made their way out of the gel by the end of the

electrophoresis experiment.

A similar experiment is also conducted under a normal TE buffer. It is found out from that

experiment that the enzyme does not work in that buffer, signifying that the enzyme activity is

buffer dependent. The results for this is in appendix (4).

42

5.2 Fluorescence experiment

The fluorescence experiment is carried out to test out the statistical behaviour of the motor

system in real time. As mentioned in section 2.6, the fluorescence signals are emitted by the

two dyes (TAMRA and Fluorescein dT) that are attached on each end of the two-site track. For

the two-site track, the signal on the positive end represents the behaviour of the front leg and

the signal on the negative end represents the behaviour of the rear leg. The precise dye and

quencher placement is shown in figure (7) above.

In this project, we tested two motors with differing leg length, one of the motor has a 12-

nucleotide leg (M12) and another has a 9-nucleotide leg (M9). Numerous buffer conditions are

tested and two of which shows positive binding results under acidic pH. The two buffers are

choline chloride-Mg2+ and Tris-Acetate-EDTA-Mg2+ (TAE-Mg2+) with an operation pH of 6.1

and 5.0 respectively.

The choline chloride-Mg2+ buffer [15] consists of 50mM Tris, 1mM Na2EDTA, 4M choline

chloride and 50mM magnesium acetate. HCl is added into the buffer system to adjust the pH

and the operating pH is set to be at pH6.1. The TAE-Mg2+ buffer [19] consists of 40mM Tris,

20mM acetic acid, 2mM EDTA and 12.5mM magnesium acetate. Similarly, the pH for TAE-

Mg2+ is adjusted with HCl and the operation pH is set at pH5. The constituents of the other

tested buffers are attached in the appendix (5).

Experiments are conducted under the two buffers mentioned above and the results are presented

in the following sections.

5.2.1 Binding of M12 under choline chloride-Mg2+ buffer

Figure (14) below shows the binding result of the M12 system. The experiment is first started

off with the track samples and at the 10th minute, M12 is added into the system. This experiment

is set to run for 2 hours measuring the binding occurrence between the motor leg and the track.

In this experiment, TAMRA is attached to the positive end of the track while fluorescein dT is

attached to the negative end of the track to detect the behaviour of the front and rear leg

respectively. If binding takes place, we will see a drop in the intensity upon motor addition.

This is because binding will bring the dye and quencher into proximity causing quenching.

As observed from figure (14), indeed, the signal drops with time, proving binding has taken

taking place. This binding is verified with absorbance test which will be discussed in section

5.3.

43

0 20 40 60 80 100 120

0

20

40

60

80

100

120

140

160

Inte

nsity (

a.u

.)

Time (min)

iFluorT (-)

TAMRA (+)


buffer).

A control experiment is done to ensure that the intensity drop represents a real effect from the

motor. In the control, instead of motor strands, buffer of the motor is added instead. The data

obtained from the control experiment is shown in figure (15) below. As observed in the control

experiment, the signal does not have any significant change in the intensity upon buffer

addition. This signifies that the signal in figure (14) comes from the motor binding.

Figure 15: Control experiment for binding experiment with normal dye placement (choline chloride-

Mg2+ buffer)

0 20 40 60 80 100 120

0

20

40

60

80

100

120

140

Inte

nsity (

a.u

.)

Time (min)

iFluorT (-)

TAMRA (+)

44

The control calibrated data is obtained by dividing the raw data by the control. Upon calibrating

the raw data with the control set, we obtain the control calibrated result shown in figure (16)

below.


chloride-Mg2+ buffer)

From figure (16), we can see that there is a slight difference between the binding behaviour of

the two legs. Nonetheless, the binding is present for M12 in the choline chloride-Mg2+ buffer.

Beside finding out the presence of binding, we can extract the binding bias information from

this binding curve as well. During the binding process, it is expected that the motor will form

a single leg binding structure by binding with the track first. The second leg will only proceed

to bind to the track after forming this single leg conformation. The binding bias can be

calculated by taking the ratio of the intensity change between the two dyes attached at the two

binding sites on the track, as shown in the formula below.

𝛽 =∆𝐼+

∆𝐼−

A value that is more than 1 indicates that there this system binds preferentially forward,

whereas a value less than 1 indicates a backward preferential binding. Using the average of

the first 10 minutes of the track only signal as the reference initial intensity, we obtain a plot

for the with respect to time, shown in figure (17) below. The system reaches a value slightly

more than 1 at equilibrium as observe in figure (17).

0 20 40 60 80 100 120

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Inte

nsity (

a.u

.)

Time (min)

iFluorT (-)

TAMRA (+)

45

Figure (17): Beta plot for M12 with normal dye placement (choline chloride-Mg2+ buffer)

To obtain a numerical approximate of this binding bias, we calculate the average value from

the last 100 minutes of the signal. The first 10 minutes is disregarded because during the first

10 minutes we expect the motor to be in the midst of forming the single leg states which can

be explained by the relatively big fluctuation in the beta plot. Since represents the binding

preference upon the formation of a single leg state, we only take the average for points at the

later timings.

Upon calculation, we obtain a beta value of 𝛽1 = 1.16 for this set of binding experiments.

The entire set of experiments is carried out again with track samples that have the dye switched

in position. By doing this we can identify and eliminate any dye effects that may be present in

the system with two different dyes. In this set of the experiment, TAMRA is attached at the

negative end of the track while fluorescein dT is attached to the positive end of the track. The

control calibrated plot and the beta plot is shown in figure (18) and figure (19) below. The raw

binding data for the switch dye experiment is included at the appendix (6) for reference.

0 20 40 60 80 100 120

-3.0

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Beta

Time (min)

M12

(normal dye)

46



Figure 19: Beta plot for M12 with switch dye placement (choline chloride-Mg2+ buffer)

Similar to the method done earlier, the beta value is calculated for last 100 minutes and we

obtain a value of 2

= 0.94. The difference in the beta value might indicate that there is a

possible dye effect in the system, this effect will be discused in chapter 6. To eliminate this dye

effect we use the following formula to get an resultant beta value for this system:

𝛽 = √𝛽1×𝛽2 = √∆𝐼+,(𝑇𝐴𝑀)

∆𝐼−,(𝐹𝑙𝑢𝑜𝑟)×

∆𝐼+,(𝐹𝑙𝑢𝑜𝑟)

∆𝐼−,(𝑇𝐴𝑀),

and the prove for this claim is included in chapter 6.

After the calculation, we obtain 𝛽 = 1.04, indicating that there is very slight forward binding

bias in the system.

0 20 40 60 80 100 120

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Inte

nsity (

a.u

.)

Time (min)

iFluorT (+)

TAMRA (-)

0 20 40 60 80 100 120

-3.0

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Be

ta

Time (min)

M12

(switch dye)

47

5.2.2 Binding of M9 under choline chloride-Mg2+ buffer

An similar experiment is carried out for the motor with 9 nucleotides leg, M9. The control

calibrated intensity plot for the normal dye and switch dye placement are shown in figure (20)

and (21) respectively. From these graphs, we can see that binding occurs for M9 as well.





The beta graphs are plotted for both normal dye and switch dye placement. Using the beta plot

we calculate the beta values using the last 100 minutes of the data and obtained 𝛽1 = 1.08

(normal dye) and 𝛽2 = 1.00 (switch dye), combining them using 𝛽 = √𝛽1×𝛽2, we obtain 𝛽 =

1.04 which is similar to the result obtained for M12.

0 20 40 60 80 100 120

0.0

0.2

0.4

0.6

0.8

1.0

1.2In

tensity (

a.u

)

Time (min)

iFluorT (-)

TAMRA (+)

0 20 40 60 80 100 120

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Inte

nsity (

a.u

.)

Time (min)

iFluorT (+)

TAMRA (-)

48

The raw data and the beta plots of M9 system in choline chloride-Mg2+ buffer included in

appendix (6).

In summary, triplex binding between the track and the motor leg is established in both M9 and

M12. From the calculated results, it appears that there is a slight binding bias in the system

with choline chloride-Mg2+ buffer. The forward binding bias calculated here is the intrinsic

property of the motor system, as mentioned in section 2.1.2, it will be a useful property for the

motor system to cushion the effect of any unwanted behaviour.

5.2.3 Operation of M12 under choline chloride-Mg2+ buffer

The operation experiment involves the addition of fuel into a motor-track incubated system.

The aim of this experiment is to test for any dissociation bias in the system after the fuel is

added.

The figure below shows the raw data obtained when the fuel is added into a motor-track

incubated system. During the first 10 minutes, the sample consists only the track-motor

complex and fuel is added in at the 10th minute. It can be seen clearly from the graph that the

intensity of the both dye rises upon fuel addition.

Figure 22: Raw operation data for M12 system under normal dye (choline chloride-Mg2+ buffer)

Upon calibrating with the control experiment, we obtained the results shown in figure (23).

0 20 40 60 80

0

5

10

15

20

25

Inte

nsity (

a.u

.)

Time (min)

iFluorT (-)

TAMRA(+)

49

Figure (23): Control calibrated operation data for M12 system under normal dye (choline chloride-

Mg2+ buffer)

Dissociation of the motor and track is observed from figure (23). This dissociation can be

tracked by the increase in the intensity signal of the dyes upon fuel addition. The initial shape

increase in the data upon fuel addition is likely to be an artefact of the shaking of the cuvette

before measurement. Judging from the intensity change, we can see that the positive end

dissociates more than the negative end. To have a numerical approximate, the dissociation bias

can be calculated from the operation data by the following formula:

𝛼 =∆𝐼+

∆𝐼−

An value that is more than 1 indicates that there this system has a front leg dissociation bias,

whereas an value less than 1 indicates a rear leg dissociation bias. Using the average of the

first 10 minutes of the track-motor complex signal as reference initial intensity, we obtain a

plot for the with respect to time, shown in figure (24) below.

0 20 40 60 80

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

Inte

nsity (

a.u

.)

Time (min)

iFluorT (-)

TAMRA (+)

50

0 20 40 60 80

-20

-18

-16

-14

-12

-10

-8

-6

-4

-2

0

2

4

6

8

10

12

14

16

18

20

Alp

ha

Time (min)

M12

(normal dye)

Figure (24): Alpha plot for M12 system with normal dye placement (choline chloride-Mg2+ buffer)

Differing from the binding experiment, the average is taken from the first 10 minutes upon fuel

addition. This is because in the autonomous system, the walking mechanism is expected to

proceed rather fast so we are only interested to find the binding bias for the initial timings. The

alpha value obtained from this experiment is 𝛼1 = 1.09, verifying that there is a forward leg

dissociation bias.

Similarly, the reverse dye experiment is carried out for the operation experiment. The control

calibrated result and the alpha plot is shown in figure (25) and figure (26) below respectively.

The raw data is included in appendix (6).

Figure (25): Control calibrated operation data for M12 system with switch dye placement (choline


0 20 40 60 80

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

Inte

nsity (

a.u

.)

Time (min)

iFluorT (+)

TAMRA (-)

51

Figure (26): Alpha plot for M12 with switch dye placement (choline chloride-Mg2+ buffer)

As seen from figure (25), there is a very large increase in the fluorescence signal at the positive

site as compared to the negative site after fuel addition. This large increase is supported by the

large alpha value of 𝛼2 = 6.12 though the calculation. This alpha value is also calculated from

the alpha values in first 10 minutes of fuel addition.

Comparing this result with the one obtained with normal dye placement, we can see that both

sets of experiments indicates that the system experiences forward leg dissociation bias. Even

though the two sets of experiments allow us to make the same conclusion, the large difference

in the alpha value indicates that dye effect is likely to be present in the experiment. To eliminate

this dye effect we use the following formula to get an resultant alpha value for this system:

𝛼 = √𝛼1×𝛼2 = √∆𝐼+,(𝑇𝐴𝑀)



∆𝐼−,(𝑇𝐴𝑀)

With the above formula we obtain an alpha value of 𝛼 = 2.58, indicating that there is a front

leg dissociation bias after accounting for the dye effect.

5.2.4 Operation of M9 under choline chloride-Mg2+ buffer

Similar to the M12, the entire set of experiment is conducted for the motor system with M9.

Figure (27) and (28) are the control calibrated operation graph of M9 with the normal dye and

switch dye placement on the track. From the two graphs, we can observe that front leg

dissociates more than the rear leg in both cases.

0 20 40 60 80

-20

-18

-16

-14

-12

-10

-8

-6

-4

-2

0

2

4

6

8

10

12

14

16

18

20

Alp

ha

Time (min)

M12

(switch dye)

52

Figure 27: Control calibrated operation data for M9 system with normal dye placement (choline


Figure 28: Control calibrated operation data for M9 system with switch dye placement (choline


The alpha graphs are plotted for both the normal dye and switch dye placement and the alpha

values are calculated using the same analysis as M12. Taking the average from 11th minute to

the 21th minute, we obtained 𝛼1 = 1.42 (normal dye) and 𝛼2 = 2.18 (switch dye), combining

them using 𝛼 = √𝛼1×𝛼2, we obtain 𝛼 = 1.76 which also allows us to conclude that forward

binding bias occurs upon fuel addition.

The raw operation data and alpha plot for this experiment are included in the appendix (6) for

reference.

0 20 40 60 80

0.00

0.05

0.10

0.15

0.20

0.25

Inte

nsity (

a.u

.)

Time (min)

iFluorT (-)

TAMRA (+)

0 20 40 60 80

0.0

0.1

0.2

0.3

0.4

0.5

0.6

Inte

nsity (

a.u

.)

Time (min)

iFluorT (+)

TAMRA (-)

53

5.2.5 Summary of the motor systems in choline chloride-Mg2+ buffer

The table below summarises the alpha and beta values obtained in this set of experiments with

choline chloride-Mg2+ buffer.

M12 system M9 system Conclusion

Binding bias () 1.04 1.04 Very weak forward binding bias for

both motor systems

Dissociation bias () 2.58 1.76 Relatively strong front leg

dissociation bias for both motor

system

Table (3): Summary on the motor systems in choline chloride-Mg2+ buffer

Comparing the values calculated from table (3), we see that M12 system has a higher front leg

dissociation bias as compared to the M9 motor. This observation can be account by the weaker

binding energy between the M9 legs and the track. Having a weaker binding, both legs can

dissociate more readily as compared to M12. As a result, the front leg dissociation bias is

limited by the relatively higher rear leg dissociation. A higher dissociation bias can better

ensure the directionality of the motion, thus the M12 system is a more preferred system for the

inchworm motor.

In addition, under this buffer, we can see that the motor behaves as expected under shearing

and unzipping force as described in section 3.1.2. The front leg that is designed to experience

unzipping force upon fuel intrusion dissociates more as compared to the rear leg which

experiences shearing force.

54

5.2.6 Binding of M12 under TAE-Mg2+ buffer

TAE-Mg2+ is another type of buffer which can support triplex formation at pH5. In this section,

we will analyse the binding results of the M12 systems in this TAE-Mg2+ buffer.

Figure (29) below shows the raw data obtained from the binding experiment of the M12 system

with normal dye placement. In this experiment, the track-only signal is recorded for the first

10 minutes of the run, the motor is added into the system at the 10th minute. Judging from the

drop in the intensity, we can conclude that, like the choline chloride-Mg2+ buffer, leg-track

binding is formed. However, in this buffer, the intensity emitted by Fluorescein dT is very

lower and it is significantly lower than the intensity emitted by TAMRA. This dye behaviour

arises because the fluorescein dT is sensitive to pH change and at pH below 7, protonation of

the dye molecule occurs and this causes a reduction in the intensity.

0 20 40 60 80 100 120

0

20

40

60

80

100

120

Inte

nsity (

a.u

.)

Time (min)

iFluorT (-)

TAMRA (+)

Figure (29): Raw binding data for M12 system with normal dye placement (TAE-Mg2+ buffer)

A control experiment is done and used to calibrate the raw data giving us a control calibrated

result in figure (30). As seen from figure (30), the shape of the two lines are far from symmetry

and it appears that the front leg binds more to the track as compared to the rear leg.

55


buffer)

The binding bias is plotted and shown in figure (31). The beta value is calculated by taking

average of the last 100 minutes of the beta plot. Upon calculating, we obtain a value of 𝛽1 =

3.65, indicating a strong forward binding bias.

0 20 40 60 80 100 120

-10

-8

-6

-4

-2

0

2

4

6

8

10

Beta

Time (min)

M12

(normal dye)

Figure (31): Beta plot for M12 with normal dye placement (TAE-Mg2+ buffer)

The switch dye experiment is carried out to compare the dye effects that might be present in

the system. The raw data obtained is shown in figure (32) and from the result, we can see a

consistent low intensity for fluorescein dT dye in this experiment. The control calibrated graph

obtained is shown in figure (33).

0 20 40 60 80 100 120

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Inte

nsity (

a.u

.)

Time (min)

iFluorT (-)

TAMRA (+)

56

Figure (32): Raw binding data for M12 system with switched dye placement (TAE-Mg2+ buffer)


buffer)

From the control calibrated plot, we can conclude that binding occurs for the two leg. The

binding bias for every point is plotted in figure (34) and we obtain a beta value of 𝛽2 = 0.87

by taking average of the last 100 minutes in the beta plot. This result indicates a backward

binding bias which is contradicts to the result obtained in normal dye placement.

0 20 40 60 80 100 120

0

20

40

60

80

100

120

Inte

nsity (

a.u

.)

Time (min)

iFluorT (+)

TAMRA (-)

0 20 40 60 80 100 120

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Inte

nsity (

a.u

.)

Time (min)

iFluorT (+)

TAMRA (-)

57

Figure (34): Beta plot for M12 with switched dye placement (TAE-Mg2+ buffer)

The two sets of experiments give contradicting beta values as summarised in table (4). The

normal dye placement experiment gives a beta value which represents forward binding bias but

its counterpart gives a value that represents backward binding bias. The contradicting beta

value indicates a significantly large dye effect in the system.

𝛽1 (normal dye placement) 3.65

𝛽2 (switched dye placement) 0.87

𝛽 1.78

Table (4): Summary of the beta values for M12 in TAE-Mg2+

This dye effect is likely contributed by those present in the choline chloride buffer and the

effect due to low fluorescein dT intensity. Assuming that the low intensity can still reflect the

behaviour of system accurately, we can eliminate the dye effect by using 𝛽 = √𝛽1×𝛽2. The

value obtained from this calculation is 𝛽 = 1.78. After the attempt to remove the dye effect,

we can conclude that there the system has an intrinsic forward binding bias.

0 20 40 60 80 100 120

-10

-8

-6

-4

-2

0

2

4

6

8

10

Be

ta

Time (min)

M12

(switch dye)

58

5.2.7 Binding of M9 under TAE-Mg2+ buffer

The normal dye and switch dye experiment is repeated for M9 system and the control calibrated

results are shown in figure (35) and figure (36).

0 20 40 60 80 100 120

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

Inte

nsity (

a.u

.)

Time (min)

iFluorT (-)

TAMRA (+)


buffer)


buffer)

Like the M12 system, fluorescein dT emits a low intensity due to the low pH in these two sets

of experiments. The beta values are calculated for the last 100 minutes of the plots and the

values are summarised in table (5) below. The raw binding graphs and the beta plots are

included in appendix (7) for reference.

0 20 40 60 80 100 120

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Inte

nsity (

a.u

.)

Time (min)

iFluorT (+)

TAMRA (-)

59

𝛽1 (normal dye placement) 2.59

𝛽2 (switched dye placement) 1.04

𝛽 1.64

Table (5): Summary of the beta values for M9 in TAE-Mg2+

Even though there is no contradiction in the conclusion between the 𝛽1 and 𝛽2 , the large

difference also indicates the presence of dye effect on the system. However, upon removing

the dye effect, we can conclude from the value that the binding bias tends towards the forward

direction.

In summary, there is a rather strong forward binding bias for both M9 and M12 in this buffer.

This strong forward binding bias will allow the recovery of directionality when an undesired

behaviour occurs in the motor. However, the low fluorescein dT intensity creates a concern on

the reliability of the results. True enough, due to the low intensity in one dye, we lose the ability

to derive conclusive results from individual set of data because the experimental signal gives

an opposite trend when the dye position is switched. However, upon combining the normal dye

and switched dye experiment, we can still extract conclusive results on the bias.

5.2.8 Operation of M12 under TAE-Mg2+ buffer

During the operation experiment, we will first measure the intensity of the motor-track

incubated system for 10 minutes. The fuel is added into the system at the 10th minute and the

sample is monitored for another 80 minutes.

The figure (37) and (38) shows the control calibrated operation data obtained from the

experiment under normal dye placement and switched dye placement. The raw data of these

graphs are included in appendix (7) for reference.

60

Figure (37): Control calibrated operation data for M12 system under normal dye (TAE-Mg2+ buffer)

0 20 40 60 80 100

0.0

0.2

0.4

0.6

0.8

1.0

Inte

nsity (

a.u

.)

Time (min)

iFluorT (+)

TAMRA (-)

Figure (38): Control calibrated operation data for M12 system under switched dye (TAE-Mg2+ buffer)

From figure (37), we can observe a very strong rear leg dissociation bias in the system when

the fuel is added. However, the results show otherwise in the switched dye experiment. This

contradiction again indicates a strong dye effect that occur in the system and the individual

plots will not be reliable for us to obtain a conclusive result.

To remove such dye effect, we need to calculate the alpha value for each plot to get the resultant

alpha calculated by 𝛼 = √𝛼1×𝛼2. The results are summarised in table (5) below. The alpha

plot for both experiments are included in the appendix (7) for reference.

0 20 40 60 80 100

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Inte

nsity (

a.u

.)

Intensity (a.u)

iFluorT (-)

TAMRA (+)

61

𝛼1 (normal dye placement) 0.04

𝛼2 (switched dye placement) 4.05

𝛼 0.40

Table (6): Summary of the alpha values for M12 in TAE-Mg2+

The resultant calculated by 𝛼 = √𝛼1×𝛼2 gives us a value that is less than 1, which means

that rear leg dissociation bias is present in the system. This result is opposite to the result

obtained in choline chloride buffer and is also contradicting to the motor design.

5.2.9 Operation of M9

The experiment is repeated for the M9 system and the control calibrated data for the normal

dye placement and switched dye placement are shown in figure (39) and (40) below.

Figure (39): Control calibrated operation data for M9 system under normal dye (TAE-Mg2+)

0 20 40 60 80 100 120

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Inte

nsity (

a.u

.)

Time (min)

iFluorT (+)

TAMRA (-)

Figure (40): Control calibrated operation data for M9 system under switched dye (TAE-Mg2+)

0 20 40 60 80 100

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Inte

nsity (

a.u

.)

Time (min)

iFluorT (-)

TAMRA (+)

62

Again, we see the contradicting results upon fuel addition. Figure (39) shows a strong rear leg

dissociation bias while figure (40) shows a strong front leg dissociation bias. To extract

conclusive result from the experimental data, the alpha graphs are plotted and the alpha values

are calculated. Table (6) below summarises the alpha values obtained from the taking average

of the first 10 minutes of the operation (the average is calculated from the 11th to 21st minute).

𝛼1 (normal dye placement) 0.04

𝛼2 (switched dye placement) 20.6

𝛼 0.91

Table (7): Summary of the alpha values for M9 in TAE-Mg2+

As seen from the table, even though 𝛼1 and 𝛼2 are contradicting values, upon eliminating the

dye effect, the result indicates a rear leg dissociation bias, a similar conclusion as M12 system

in the same buffer.

63

5.2.10 Summary of the motor systems in TAE-Mg2+ buffer and comparison with choline

chloride-Mg2+ buffer

The table below summarises the alpha and beta values obtained in this set of experiments

with TAE-Mg2+ buffer.

M12 system M9 system Conclusion

Binding bias () 1.78 1.64 Rather strong forward binding bias

for both motor systems

Dissociation bias () 0.40 0.91 Relatively strong rear leg

dissociation bias for both motor

system

Table (8): Summary on the motor systems in TAE-Mg2+ buffer

Even though the results obtained under TAE-Mg2+ buffer contradicts to what we expect for

an inchworm motor, the bias trend fits into the requirement of a hand-on-hand motor, which

will also drive the motor on the track. Comparing the values calculated from table (8), we see

that M12 has a higher forward binding bias as well as rear leg dissociation bias as compared to

the M9 system. The M12 system will be a better candidate for a motor that moves in the hand-

on-hand manner.

From the entire set fluorescence experiment, we can see that buffers have a great influence in

the behaviour of the system. In the experiment involving choline-chloride buffer, we concluded

that both the motors have weak forward binding bias but a relatively strong forward

dissociation bias. On the contrary, the experimental results under TAE-Mg2+ buffer show that

both motor has relatively strong forward bind bias and rear leg dissociation bias. This result

show that the motor can be a versatile motor where its motion can be easily switched over just

by changing the buffer.

From the two experiment, we can also see the dependence of the motor leg’s length on the

behaviour of the motor system. In the inchworm system, although both motor systems have the

same forward binding bias, M12 has a greater front leg dissociation bias which is more

preferred. Similarly, M12 system under TAE-Mg2+ performs better as a hand-on-hand motor

as explained above. One possible reason to account for this varied dissociation bias might be

due to the higher stability in the bonding between the 12-nucleotide leg and the track. The 9-

nucleotide leg might experience more random dissociation on both legs, decreasing ratio of the

ratio between forward leg dissociation and rear leg dissociation.

64

From all the binding experiments, we can see that leg-track binding occurs in the experiment.

Absorbance test is also carried out to test the presence of such binding and the results support

the formation of these bonds. The results of the absorbance test are in the following section.

5.3 Absorbance

In this project, the absorbance experiment is carried out to verify the formation of leg-binding.

Absorbance at 260nm is carried out for M12 in TAE-Mg2+ buffer and choline chloride- Mg2+

buffer and M9 in choline chloride-Mg2+ buffer. The results below are normalised with the

control experiment.

The first 10 minutes of the experiment involves the absorbance measure for the buffer. The

track is added at the 10th minute and the signal is measured for another 10 minutes. Following,

the motor (either M12 or M9) is added in and the absorbance is measured for 40 minutes. The

raw absorbance measurements are normalised with the control experiment. In the control

experiment, everything is held the same as the operation except that the buffer for the motor is

added instead.

Figure (41): Absorbance at 260nm for M12 in TAE- Mg2+ buffer

0 20 40 60

0.112

0.114

0.116

0.118

0.120

0.122

0.124

0.126

0.128

0.130

0.132

Absorb

ance

Time

M12 (260nm)

65

0 20 40 60

1.06

1.08

1.10

1.12

1.14

1.16

1.18

1.20

1.22

1.24

1.26A

bso

rban

ce

Time (min)

M12 (260nm)

Figure (42): Absorbance at 260nm for M12 in choline chloride- Mg2+ buffer

0 20 40 60

0.94

0.96

0.98

1.00

1.02

1.04

1.06

1.08

1.10

1.12

Ab

so

rban

ce

Time (min)

M9 (260nm)

Figure (43): Absorbance at 260nm for M9 in choline chloride- Mg2+ buffer

Consistent for all the three sets of data, after the addition of motor at the 20th minute, there a

gradual drop in the absorbance is observed. As discussed in section 2.4, the formation of bond

between DNA will reduce the absorbance at 260nm and this observation is seen all the three

experiments conducted. With this, we can conclude that indeed motor-track binding occurs in

the binding experiment, supporting the fluorescence binding data.

66

Chapter 6: Discussion In this chapter, we will discuss some other findings and analysis from the experimental results

we obtained from the experiment. Also, we will discuss some potential areas of improvement

and future works.

6.1 Further discussion on experimental results

6.1.1 Comment on dyes

Through the experiment, we found out that the dyes which are introduced into the system as

the probe are the major limiting factor to the project. The pH sensitivity of fluorescein dT has

induced large dye related effect to the system, especially in the TAE-Mg2+ buffer, masking the

real signal of the system. As such, it is no longer reliable to depend on the result a single

experiment for conclusion in this project. A switched dye analyse must be done so that we can

account for the dye effects that are present.

In section 5.2, we claimed that the dye effect can be eliminated from the biases just by using

the following two formula, 𝛽 = √𝛽1×𝛽2 or 𝛼 = √𝛼1×𝛼2 where the ratio 𝛼𝑥 or 𝛽𝑥 just the

ratio of intensity change. Here, we attempt to prove the claim. As seen from the formula below,

in the presence of dye effect, each ∆𝐼 term will be influenced by both the real dissociation

signal (∆𝐼𝑇𝐴𝑀 or ∆𝐼𝐹𝑙𝑢𝑜𝑟 ) or and the dye effect signal (∆𝐼+ or ∆𝐼−). Notice that by using the

formula introduced, we can effectively cancel off the signal due to dye effect giving us a real

signal of the system.

√∆𝐼+,(𝑇𝐴𝑀)



∆𝐼−,(𝑇𝐴𝑀)= √

∆𝐼+×∆𝐼𝑇𝐴𝑀

∆𝐼−×∆𝐼𝐹𝑙𝑢𝑜𝑟×

∆𝐼+×∆𝐼𝐹𝑙𝑢𝑜𝑟

∆𝐼−×∆𝐼𝑇𝐴𝑀= √

∆𝐼+

∆𝐼−×

∆𝐼+

∆𝐼−

As mentioned in section 2.6, studies have shown that strong bonds can be formed between

some dye and quencher pairs and the strength of these bonds is pair dependent. These effects

are undesirable in the system as this additional attractive force will mask the actual binding

trend of the leg and motor.

Analysing the experimental results obtained from the choline chloride-Mg2+ buffer, we can

make the inference that the attractive force between the TAMRA and the Iowa Black FQ is

slightly higher than fluorescein dT with this quencher. As observed in the operation experiment,

the dissociation of the front leg consistently lesser for both M9 and M12 system when TAMRA

is placed at the positive end. The intensity jump is much higher when the fluorescein dT is

67

placed there. In addition, we can also get a consistent conclusion from the binding experiment.

It is observed that the binding curve takes a symmetrical shape under the switched dye

placement, where fluorescein dT is placed at the positive end. However, the binding curve takes

a slightly different shape under the normal dye placement where the TAMRA and fluorescein

dT is attached at the positive and negative end respectively. Specifically, in the case for binding,

the front leg always binds faster than then rear leg. A faster binding rate for the site with

TAMRA dye indicates that the attractive force between TAMRA and the quencher is stronger

than the fluorescein dT.

It is also worthy to note that the protonation of the fluorescein dT under acidic pH will enhance

the attractive force between the negatively charged DNA and the positively charge dye.

Nonetheless, from our analysis, the effect of the attractive forces are not that significant to

hinder the reliability of the results.

6.1.2 Stability of DNA triplex under shearing and unzipping force

The motivation of the triplex binding site arises from a study which has reported that there is

varied stability of LNA-DNA triplex structure under shearing and unzipping forces. Such

varied stability for pure DNA triplex has yet to be reported.

The motor system is deliberately designed such that the addition of fuel strands into the motor

system will induce a unzipping and shearing force in the front and rear leg respectively. From

the results obtained from the experiment, we can see that such asymmetry indeed exist in DNA

triplex but it is likely to be buffer dependent. In choline chloride-Mg2+ buffer, the triplex is less

stable under the force in unzipping direction, thus giving us front leg dissociation bias.

6.2 Areas for improvements

In this experiment, the alpha and beta values, which represent the dissociation and binding bias

respectively, are calculated from only one fluorescence experiment. These values are calculated

to give us an idea on the behaviour of the motor system. However due to time limitation, we

did not manage to perform more set of experiments to minimise random errors. It will be good

to repeat these experiments several times for average taking for future publications, especially

for the TAE-Mg2+ buffer as the normal dye and switch dye experiment shows contradicting

results. For the experiments done under choline chloride, the reverse dye experiment also

serves the purpose to verify the behaviour of the motor system. Since a same trend is observed,

we can deduce that the trend for binding bias we conclude is very likely to be accurate. But in

68

order to get a reliable alpha and beta value, we will still have to repeat the experiments several

time.

In addition, acid of higher concentration can be used to minimise the additional volume added

into the buffer. In this experiment, an acid concentration of 0.5N is used and we require a larger

volume of acid to achive the desired pH. This will alter the true concentration of the salt in the

buffer.

Also, due to the limitation of time, we did not manage to test the upper limit of pH where triplex

can still be formed. In future work, it will be good to test this upper limit because pH not only

limits the intensity of the dye, it may also limit the activity of the enzyme. The cutsmart buffer

has a pH value of 7.9 and since this buffer is always used optimise the activity of the enzyme,

Nt.BbvCI, we can infer that the active pH of the enzyme should be maximised at neutral or

slight alkaline pH. Since acidic pH may cause denaturation of the enzyme causing inactivity,

important to explore a working pH which can support both the triplex formation and enzyme

activity.

6.3 Future works

In this experiment, we carried out experiment with the two site track. We managed to find out

the intrinsic binding bias of the motor as well as the binding bias upon fuel addition. In order

to for us to see the entire behaviour of the motor system, we need to extent the experiment and

carry out some experiments on a three-site and five-site track. For the inchworm motor to work,

we require two dissociation bias. The first involve the front leg dissociation bias upon fuel

addition and the second involves the rear leg dissociation upon enzyme activity. We have yet

to explore the latter and three site track will be useful for this. One possible way to determine

this bias is to first add motor-fuel incubated sample into the three-site track sample, and

subsequently the enzyme. By tracking this signal, we can determine the effect of enzyme on

the system.

Other than the three site track, we can also obtain a real time signal for the behaviour of the

hairpin engine by attaching FRET pair to the two ends of the hairpin. When the hairpin is at

the close state, quenching will occur and the intensity of the acceptor dye will increase; the

opening of the hairpin will increase the intensity of the donor dye. By tracking the intensity it

provides us with another way to analyse the behaviour of the motor system.

69

Chapter 7: Conclusion

The project sets up with the aim to design a new autonomous motor which can advance on an

overhang free track in an inchworm manner. The overhang-free track is advantageous as it can

minimise possible interactions with other DNA molecules present in the system and instead of

the conventional duplex motor track binding, we explore the option of triplex in this experiment.

Through experimentation, we found out that two buffer can support such bindings between the

motor legs and track. The two buffers are choline chloride-Mg2+ at pH6.1 and TAE-Mg2+ at

pH5. These two buffers are subsequently used for the testing of the motor’s behaviour.

Other than testing for triplex formation, we tested the motion and behaviour of the motor under

two-site track. Using the experimental results, we calculated the binding bias and dissociation

bias for the two motor systems (M12 and M9) under the two buffers. The results showed that

the motor’s motion is dependent on the leg length and from comparing the results, we

concluded that M12 system is a better candidate as it shows a higher binding and dissociation

bias in both buffers.

An interesting finding from this experiment is that the behaviour of the motor system is buffer

dependent. The motor demonstrates inchworm behaviour under choline chloride-Mg2+ buffer

and it experiences strong front leg dissociation bias upon fuel addition. However, it

demonstrates hand-on-hand behaviour under TAE-Mg2+ buffer as results reveal that the motor

system has a strong forward binding bias and a strong rear leg dissociation bias. Coupled with

the forward binding bias, the motor system might be versatile one which can adopt different

motion depending on the buffer used.

In addition, we also verified that the new engine designed for this motor works well and will

be able to support the autonomous motion of this motor through the electrophoresis experiment.

In conclusion, experimental results support that the motor designed in this project is a potential

system that can make advancement on an overhang-free track. Further experimentation on three

site track is still required for us to understand this motor system more extensively, nonetheless,

the FYP project goals have been met.

70

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of Sciences of the United States of America, 87(23), 9436–9440.

17) Escudé, C., Mohammadi, S., Sun J., Nguyen, C., Bisagni, E., Liquier, L, Taillandier,

E., Garestier, T., & Hélène, C. (1996). Ligand-induced formation of Hoogsteen-paired

parallel DNA. Chemistry & Biology¸ 3(1), 57-65. doi:10.1016/S1074-5521(96)90085-

X

18) Marras, S. A. E., Kramer, F. R., & Tyagi, S. (2002). Efficiency of fluorescence

resonance energy transfer and contact-mediated quenching in oligonucleotide probes.

Nucleic Acids Research, 30(21), e122.

19) Chen, Y., Lee, S., Mao, C. (2004). A DNA nanomachine based in a duplex-triplex

transition. Angew. Chem. Int. Ed. 43(40). 5335-5338. doi: 10.1002/anie.200460789

72

Appendix (1)

The table below summarises the amount of TE buffer added into each tube to create a 100m

stock. The amounts are indicated in the specification sheets from IDT.

Single stranded DNA Representation Amount of TE

buffer (L)

2-site track Track strand 1 T1 44

Track strand 2 T2 55

Track strand 2

(switch dye)

T2 (sd) 100





Fuel Fuel 86

73

Appendix (2)

The composition of individual samples used in experiment to test for duplex structure in

electrophoresis. Each sample here is placed into one well of agarose gel. In general, 1l of

loading dye is added to each sample and the DNA oligonucleotide that is within each sample

has a mass of 200ng.

Track (T/T1/T2) of 5M (l) TE buffer (l) Dye (l)

T sample 2.05 2.95 1

T1 sample 4.18 0.82 1

T2 sample 4.03 0.97 1

Motor 12nt leg of 5M (l) TE buffer (l) Dye (l)

M12 sample 1.39 3.61 1

MS1 (12nt) sample 2.10 2.90 1

MS2 (12nt) sample 4.06 0.94 1

Motor 9nt leg of 5M (l) TE buffer (l) Dye (l)

M9 sample 1.48 3.52 1

MS1 (9nt) sample 2.21 2.79 1

MS2 (9nt) sample 4.47 0.53 1

Fuel of 5M (l) TE buffer (l) Dye (l)

Fuel sample 3.32 1.68 1

Ladder (l) TE buffer (l) Dye (l)

Ladder sample 4 1 1

74

Appendix (3)

The following is the composition of each lane for the gel that test for the fuel-enzyme cycle. This

experiment is carried out to test out if the hairpin engine works. The fuel and motor complex samples

are incubated for one hour and the samples with enzyme are incubated for 1.5 hours before the

enzyme is denatured by incubating the sample in 80C for 20 minutes.

Lane 1

(MS1 only)

Composition Volume (l)

MS1 (12nt) 2.10

Cutsmart buffer 0.55

DI water 2.85

Dye 1

Lane 2

(MS1 + enzyme)


MS1 (12nt) 2.10

Enzyme 0.55


DI water 2.30

Dye 1

Lane 3

(Fuel only)


Fuel 3.32


DI water 1.60

Dye 1

Lane 4

(Fuel + enzyme)


Fuel 3.32

Enzyme 0.55


DI water 1.05

Dye 1

Lane 5

(MS1 and fuel only)


MS1 (12nt) 1.08

Fuel 1.62


DI water 2.25

Dye 1

Lane 6

(MS1 and fuel + enzyme)


MS1 (12nt) 1.08

Fuel 1.62

Enzyme 0.55


DI water 1.70

Dye 1

Ladder Lane


Ladder 4.00

75


DI water 0.95

Dye 1

Appendix (4)

The figure below shows the electrophoresis result to test for the fuel-enzyme cycle in the

single hairpin engine under TE buffer.

Instead of using cutsmart buffer, in lane 1 to 6, TE buffer is used instead. Lane 7 is the only

lane that consist of cutsmart buffer. As we can see from lane 6, there is no enzyme activity as

the bands that appeared in lane 6 corresponds to those that appeared in lane 5, a lane without

enzyme.

The difference between lane 6 and 7 is just the buffer variation, TE buffer is added in lane 6

while cutsmart buffer is added into lane 7. The results show that the enzyme activity does

depend on the buffer used.

76

Appendix (5)

The follow table includes the composition of the buffers that are used in this experiment. A

successful buffer in this project is defined to be the buffer where triplex is effectively formed.

In this experiment, several operation buffers are explored. The table below shows the various

buffers used in this experiment. The pH is lowered by adding HCl, note that the concentration

below is the raw buffer before pH adjustment

Tris-acetate-EDTA-Mg2+ Concentration (mM)

1 Tris buffer (pH8) 40

2 Acetic acid 20

3 EDTA 2

4 Mg(CH3COO)2 (Magnesium

Acetate)

12.5

At pH5, the motor and track shows binding. This buffer at pH5 is then used as operation buffer.

No other pH is tested for this buffer under this system.

Choline chloride-Mg2+ Concentration (mM)


2 Na2EDTA 1

3 choline chloride 4000


Acetate)

50

pH 9.5, 7.0 and 6.1 are tested for this buffer. Significant binding is observed under pH6.1,

this buffer is also used as operation buffer in the experiment. No significant binding is

observed for pH 9.5 and 7.0.

Choline chloride-Mg2+ (diluted) Concentration (mM)


2 Na2EDTA 1

3 choline chloride 2000


Acetate)

50

pH 9.5 and 7.0 are tested for this buffer with 50nM magnesium acetate, no significant binding

is observed for both pH values. A similar experiment can be conducted at pH6.1 to test the

effect of choline chloride concentration on the system.

TE-NaCl Concentration (mM)

1 NaCl 200

2 TE buffer -

pH 3.5, 5.5, 6.2 and 6.5 are tested, no significant motor-track binding is observed under this

buffer for all the pH values.

77

TE-MgCl2 Concentration (mM)

1 MgCl2 10/50

2 TE buffer -

Two different concentration of MgCl2 are tested for this buffer at pH8. No significant binding

is observed for this buffer.

The cutsmart buffer that ensures the activity of the enzyme is provided by the from New

England BioLabs Inc. The composition is summarised in the table below. This buffer has a

pH of 7.9.

Cutsmart buffer Concentration (mM)

1 Tris-acetate 20

2 Magnesium acetate 10

3 Potassium acetate 50

4 BSA 100 g/ml

78

Appendix (6)

Figure (1): Raw binding data for M12 system with switch dye placement (choline chloride-

Mg2+ buffer)


buffer)

0 20 40 60 80 100 120

0

20

40

60

80

100

120

140

160In

tensity (

a.u

.)

Time (min)

iFluorT (+)

TAMRA (-)

0 20 40 60 80 100 120

0

20

40

60

80

100

120

140

Inte

nsity (

a.u

.)

Time (min)

iFluorT (-)

TAMRA (+)

79

Figure (3): Beta plot for M9 with normal dye placement (choline chloride-Mg2+ buffer)

0 20 40 60 80 100 120

0

20

40

60

80

100

120

140

160

Inte

nsity (

a.u

.)

Time (min)

iFluorT (+)

TAMRA (-)

Figure (4): Raw binding data for M9 system with switch dye placement (choline chloride-Mg2+

buffer)

0 20 40 60 80 100 120

-3.0

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Be

ta

Time (min)

M9

(normal dye)

80

Figure (5): Beta plot for M9 with switch dye placement (choline chloride-Mg2+ buffer)

Figure (6): Raw operation data for M12 system with switch dye placement (choline chloride-Mg2+

buffer)

0 20 40 60 80 100 120

-3.0

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Beta

Time (min)

M9

(switch dye)

0 20 40 60 80 100

0

2

4

6

8

10

12

14

16

18

20

22

24

Inte

nsity (

a.u

.)

Time (min)

iFluorT (+)

TAMRA (-)

81

0 20 40 60 80

0

5

10

15

20

Inte

nsity (

a.u

.)

Time (min)

iFluorT (-)

TAMRA (+)

Figure (7): Raw operation data for M9 system with normal dye placement (choline chloride-Mg2+

buffer)

Figure (8): Alpha plot for M9 with normal dye placement (choline chloride-Mg2+ buffer)

0 20 40 60 80 100

-10

-8

-6

-4

-2

0

2

4

6

8

10

Alp

ha

Time (min)

M9

(normal dye)

82

0 20 40 60 80 100 120

0

5

10

15

20

25

30

35

40

45

Inte

nsity (

a.u

.)

Time (min)

iFluorT (+)

TAMRA (-)

Figure (9): Raw operation data for M9 system with switch dye placement (choline chloride-Mg2+

buffer)

Figure (10): Alpha plot for M9 with switch dye placement (choline chloride-Mg2+ buffer)

0 20 40 60 80 100 120

-10

-8

-6

-4

-2

0

2

4

6

8

10

Alp

ha

Time (min)

M9

(switch dye)

83

Appendix (7)

0 20 40 60 80 100 120

0

20

40

60

80

100In

tensity (

a.u

.)

Time (min)

iFluorT (-)

TAMRA (+)

Figure (1): Raw binding data for M9 system with normal dye placement (TAE-Mg2+ buffer)

Figure (2): Beta plot for M9 with normal dye placement (TAE-Mg2+ buffer)

0 20 40 60 80 100 120

-10

-8

-6

-4

-2

0

2

4

6

8

10

Beta

Time (min)

M9

(normal dye)

84

0 20 40 60 80 100 120

0

10

20

30

40

50

60

70

80

Inte

nsity (

a.u

.)

Time (min)

iFluorT (+)

TAMRA (-)

Figure (3): Raw binding data for M9 system with switched dye placement (TAE-Mg2+ buffer)

0 20 40 60 80 100

-10

-8

-6

-4

-2

0

2

4

6

8

10

Be

ta

Time (min)

Beta

Figure (4): Beta plot for M9 with switched dye placement (TAE-Mg2+ buffer)

85

0 20 40 60 80 100

0

2

4

6

8

10

12

14

16

18

20

Inte

nsity (

a.u

)

Time (min)

iFluorT (-)

TAMRA (+)

Figure (5): Raw operation data for M12 with normal dye placement (TAE-Mg2+ buffer)

Figure (6): Alpha plot for M12 with normal dye placement (TAE-Mg2+ buffer)

0 20 40 60 80

-1.0

-0.8

-0.6

-0.4

-0.2

0.0

0.2

0.4

0.6

0.8

1.0

Alp

ha

Time (min)

M12

(normal dye)

86

Figure (7): Raw operation data for M12 system with switch dye placement (TAE-Mg2+ buffer)

0 20 40 60 80

-10

0

10

20

Alp

ha

Time (min)

Alpha

Figure (8): Alpha plot for M12 with switch dye placement (TAE-Mg2+ buffer)

0 20 40 60 80 100 120

0

2

4

6

8

10

12

14

Inte

nsity (

a.u

.)

Time (min)

iFluorT (+)

TAMRA (-)

87

0 20 40 60 80 100 120

0

2

4

6

8

10

12

14

16

Inte

nsity (

a.u

.)

Time (min)

iFluorT (-)

TAMRA (+)

Figure (9): Raw operation data for M9 with normal dye placement (TAE-Mg2+ buffer)

0 20 40 60 80

-2.0

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

Alp

ha

Time (min)

M9

(normal dye)

Figure (10): Alpha plot for M9 with normal dye placement (TAE-Mg2+ buffer)

88

0 20 40 60 80 100 120

0

5

10

15

20

Inte

nsity (

a.u

.)

Time (min)

iFluorT (+)

TAMRA (-)

Figure (11): Raw operation data for M9 with switched dye placement (TAE-Mg2+ buffer)

0 20 40 60 80

-100

-80

-60

-40

-20

0

20

40

60

80

100

Alp

ha

Time (min)

Alpha

Figure (12): Alpha plot for M9 with switched dye placement (TAE-Mg2+ buffer)

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