University of Groningen Speeding up biochemistry …...probe thousands of DNA base pairs per binding event instead of going through repeated cycles of binding to a single, random site

University of Groningen

Speeding up biochemistry by molecular sledding along DNATurkin, Alexander Anatoliy

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Chapter 5

Towards biotechnological and pharmaceutical applications of pVIc

DNA-processing enzymes are vital for ensuring genomic stability and integrity.

Not only are these enzymes omnipresent in natural systems, they also found

numerous applications in molecular biology. In both natural and artificial

systems, these enzymes need to find and process their targets on DNA amidst a

large number of non-specific binding sites. Using three-dimensional search

mechanisms can be time-consuming and inefficient. Previously, we demonstrated

that association with DNA targets can be speeded up by functionalising the

binding partners with a DNA sliding peptide pVIc and thus enabling one-

dimensional sliding along DNA. Such a mechanism allows binding partners to

probe thousands of DNA base pairs per binding event instead of going through

repeated cycles of binding to a single, random site on DNA, dissociating and

rebinding somewhere else. Here, we apply this approach to DNA-processing

enzymes. Firstly, we attempted to enhance the processivity of DNA polymerase

Pfu by functionalising it with the molecular sled pVIc. Secondly, we set out to

improve topoisomerase inhibitors – widely used antibiotic and anticancer

chemotherapeutics. These initial experiments will serve as a basis for the future

development of applications of our concept of dimensionality reduction as a

means to accelerate molecular processes.

Chapter 5

132

5.1. Introduction

The concept of reduction of dimensionality to speed up search and target

recognition has been put forth more than half a century ago.1,2 Since then, a

number of DNA-interacting proteins, such as DNA-repair enzymes, transcription

factors, and DNA polymerases were shown to be able to one-dimensionally (1D)

diffuse along DNA.3-5 Such a process helps them find their targets more rapidly

compared to them diffusing in 3D. Several studies demonstrated that 1D diffusion

could be supported by either ‘sliding’, diffusion along the DNA while in

continuous electrostatic contact with the DNA, or by ‘hopping’, moving along the

DNA by frequent microscopic hops off and on the DNA. For reasons of simplicity

we will use here ‘sliding’ to describe any mechanism that supports the diffusive

movement of a protein along DNA in a 1D fashion.

Until recently, the use of this concept of dimensionality reduction was only

realised in natural systems. However, in a proof-of-principle reaction we showed

that association between arbitrary binding partners can be speeded up by allowing

the binding partners to slide along DNA. To our knowledge, our experiment

based on the association of biotin and streptavidin demonstrated the first artificial

system in which association was accelerated using the principle of sliding along

DNA. Our generic approach of enhancing reaction rates by reduction in

dimensionality can in principle be applied to numerous in vitro and in vivo

systems used in a variety of biotechnological and pharmaceutical processes, from

PCR to the development of antibiotics. In fact, we applied the concept of 1D

sliding along DNA to a conventional PCR, in which we functionalised the primers

to allow them to slide along DNA, thereby significantly reducing the overall

duration of the reaction. These developments are described in Chapter 3.

The key goal of this chapter is to explore the possibility of adapting our

approach to the speed up of reactions that are of biotechnological interest. The

small size of the DNA sled (the 11-amino acid peptide pVIc) makes it an ideal

candidate for a molecular building block that can be fused to a macromolecule


133

and enable that molecule to move efficiently along DNA. The general approach is

that the binding partners in any bimolecular reaction can be equipped with the

molecular sled and that DNA can be used as a “search catalyst” in solution to

reduce the dimensionality of search and speed up bimolecular association.

The first logical step in the development of such 1D biochemistry is

accelerating associations in the systems that naturally involve interactions with

DNA. After establishing a proof of concept with the biotin-streptavidin-pVIc

system and applying the concept to speed up PCR, we describe here attempts to

enhance the efficiency of the Pyrococcus furiosus (Pfu) polymerase, widely used in

PCR, by equipping it with the pVIc peptide that would facilitate search by the

polymerase for the primer-template site. In addition to the utilisation of pVIc as a

means of speeding up recognition processes in vitro, the molecular sled offers a

multitude of potential applications for in vivo processes. We describe here our

initial attempts to increase the efficiency of chemotherapeutics that interfere with

the bacterial/cellular DNA machinery. In particular, we performed conjugations

of pVIc with DNA gyrase inhibitors (antibiotics) and human topoisomerase

inhibitors (anticancer pharmaceuticals) and tested the effect of such a

functionalisation on their efficiency. These first steps with regards to using our

approach to enhance different in vitro and in vivo processes are described below.

5.2. Results and discussion

5.2.1. DNA Polymerase

DNA polymerases are enzymes that utilise nucleotide building blocks to

polymerise DNA in a template-directed fashion. In a cellular context, DNA

polymerases are responsible for the copying of DNA that needs to occur for

replication, i.e. the “reading” of the sequence of an existing DNA template strand

and the synthesis of a new DNA molecules with the complementary sequence.

The process of a template-directed copying of DNA is used for an important

molecular biology technique called Polymerase Chain Reaction (PCR) (see Text

box 3, Chapter 1). There, DNA polymerases are used to create an exponentially

Chapter 5

134

growing number copies of a DNA sequence starting the reaction with only one or

a few template molecules. PCR technology has become indispensable and is used

in various areas of research, forensics, and diagnostics.

Due to the importance of PCR technology, significant effort has been made

to improve its efficiency. One of the approaches is to improve the processivity of a

polymerase by increasing its affinity to DNA, which has been done by fusing a

DNA-binding domain to the enzyme (Fig. 5.1a and 5.1b). A good example of a

system where this strategy has been successfully realised is the Pfu polymerase

(from Pyrococcus furiosus). It was shown that its processivity and fidelity are

increased dramatically by fusing a DNA-binding domain (the Sso7d protein from

Sulfolobus solfataricus) to its carboxy terminus.6 Later, this chimera (Pfu-Sso7d)

became commercially available as “Phusion” polymerase, which is currently the

gold standard for high-performance PCR. Analogous to this approach, here we

attempted to fuse the pVIc molecular sled to the Pfu polymerase. The advantage

compared to the Pfu-Sso7d chimera would be that the pVIc chimera would not

only improve affinity to the DNA, but that it would still allow the protein to freely

diffuse along the DNA and search for the primer-template sites. As such, this

chimera would display improved thermodynamic affinity to the DNA, while

preventing it becoming kinetically trapped (Fig. 5.1c).

Figure 5.1 | Approaches to improve enzyme target search. (a) Original enzyme.

(b) Enzyme affinity to DNA is enhanced by fusing a DNA-binding protein to it.

(c) Enzyme affinity to DNA is enhanced by fusing a DNA-sliding protein or

peptide to it. Beyond an increase in affinity to DNA, this approach allows the

enzyme to scan a stretch of DNA in search for its target per binding event.

(a) (b) (c)EnzymeDNA-bindingprotein

DNA-slidingprotein


135

Since polymerases used in PCR need to function at elevated temperatures,

we first confirmed that pVIc sliding still occurs at those higher temperatures.

Conducting the biotin-streptavidin binding assay (Chapter 3) at 60oC (Fig. 5.2),

demonstrated that reaction speed-up was achieved in the presence of DNA,

suggesting that pVIc sliding is able to confer a kinetic advantage also at elevated

temperatures. The obtained reaction times τ (for definition see Chapter 3) were

13.15 s and 0.56 s without and with DNA respectively.

0 20 40 60 80 1000.0

0.2

0.4

0.6

0.8

1.0

Inte

nsity

Time (s)

60oC, DNA (-) 60oC, dsDNA (+)

Figure 5.2 | Biotin-streptavidin association at elevated temperatures. Reaction

speed-up was observed in the presence of DNA (2686 bp, 300 pM) at 60oC.

Encouraged by our observations, we designed a Pfu polymerase conjugate

with pVIc, whose performance we set out to compare with three polymerases:

commercially available wild type (WT) Pfu, commercially available “Phusion”

from NEB (Pfu-Sso7d) and an in-house expressed Pfu-Sso7d. Sso7d is a small

protein from Sulfolobus solfataricus that binds double-stranded DNA without

sequence specificity.7 The NEB “Phusion” protein has improved activity in PCR

because of the ability of the Sso7d domain to drive the polymerase-DNA

association equilibrium towards the DNA-bound state. The schematic of the

protein constructs we used are presented in Fig. 5.3.

Chapter 5

136

Figure 5.3 | Pfu polymerase constructs. (a) WT polymerase. (b) Pfu-Sso7d

polymerase, a chimera between Pfu polymerase and Sso7d DNA-binding protein.

(c) Pfu-pVIc polymerase, a chimera between Pfu polymerase and pVIc peptide.

The N-termini of the proteins contain a His-tag for purification purposes.

Pfu-Sso7d and Pfu-pVIc proteins were expressed by transforming E. coli

Rossetta cells with the corresponding plasmids and inducing protein expression

with IPTG. Subsequently, the His-tagged proteins were purified by nickel affinity

chromatography and heparin affinity chromatography where needed. The correct

protein molecular weight (MW) was confirmed after purification using sodium

dodecyl sulfate Polyacrylamide gel electrophoresis (SDS-PAGE) and Matrix-

assisted laser desorption/ionisation (MALDI) mass spectrometry (MS) (see

experimental section).

We tested the expressed and purified Pfu-pVIc and Pfu-Sso7d together with

commercial WT Pfu and “Phusion” (Pfu-Sso7d) using a real-time PCR assay

(qPCR). The experiments were performed using commercially available buffers

specifically designed for Phusion and Pfu polymerases (HF Phusion buffer, NEB

and Pfu buffer, Promega). Interestingly, commercial Pfu-Sso7d as well as the in-

house purified one exhibited high amplification rate in both buffers, while WT

Pfu was active only in the Pfu buffer and not in the HF Phusion buffer. Lastly, no

amplicon formation using Pfu-pVIc polymerase in any of the buffers was

detected, suggesting that fusion of a pVIc domain is detrimental to the function of

the polymerase.

PfuHis

PfuHis Sso7d

PfuHis pVIc

(a)

(b)

(c)


137

0 5 10 15 20 25 30 35 40 450

100

200

300

400

500

Inte

nsity

(a.u

.)

Cycle number

HF Phusion buffer Phusion Pfu-Sso7d Pfu-pVIc Pfu

(a)

0 5 10 15 20 25 30 35 40 45

0

200

400

600

800

1000

(b)

Inte

nsity

(a.u

.)

Cycle number

Pfu buffer Phusion Pfu-Sso7d Pfu-pVIc Pfu

Figure 5.4 | Polymerase performance in PCR. (a) qPCR using four polymerases

in HF Phusion buffer (NEB). (b) qPCR using four polymerases in Pfu buffer

(Promega).

The pVIc motif of the Pfu-pVIc polymerase contains a cysteine, which could

potentially make the protein construct prone to dimerisation. Although the

protein was stored with a high concentration of a reducing agent in solution (2

mM DTT), dilution of the protein into the qPCR reaction mixture could lead to

oxidation and dimer formation. To exclude this possibility we performed a series

of qPCR experiments with 10 mM DTT in the reaction buffer. However, no

improvement in performance of Pfu-pVIc was observed (data not shown).

It is not clear why addition of only 11 amino acids inactivates the

polymerase. Further work might include fusion of pVIc to the other terminus (N-

terminus) of the Pfu and/or using a different linker between Pfu and pVIc.

Alternatively, screening different PCR reaction buffer compositions might be

attempted since buffer composition is crucial for the function of polymerase.

5.2.2. Topoisomerase inhibitors

Topoisomerase inhibitors are potent pharmaceuticals and are a popular

choice in the treatment of infections (antibiotics, bacterial DNA gyrase inhibitors)

and cancer (antineoplastics, human DNA topoisomerase I and II inhibitors).8

Topoisomerase inhibitors act by interfering with the action of topoisomerases,

enzymes that introduce changes in DNA topology by breaking and rejoining of

Chapter 5

138

the phosphodiester backbone of DNA strands during the normal cell cycle. The

mode of action of topoisomerase inhibitors is the stabilisation of the cleavage

complexes in an open form resulting in the generation of chromosome breaks.

However, these pharmaceuticals possess severe side effects, including spontaneous

tendon rupture, arrhythmia, peripheral neuropathy for gyrase inhibitors, and

cardiomyopathy and typhlitis for human topoisomerase inhibitors. Therefore,

reducing the dosage of these pharmaceuticals without loss of their function is

highly desirable. For that reason, numerous studies have focused on the creation

of better topoisomerase inhibitors.9-11

In this study, we use the fact that topoisomerase inhibitors exert their

function on DNA and attempt to enhance their efficiency by functionalising them

with pVIc. Such modification would allow the drug molecule to 1D move along

DNA, reaching and inactivating topoisomerases faster as compared to 3D

diffusion.

Gyrase inhibitors: gemifloxacin

The most widely used class of gyrase inhibitors is that of the

fluoroquinolones, broad-spectrum antibiotics used against both gram-negative

and gram-positive bacteria. Our first choice of antibiotic from this class was

gemifloxacin (4th-generation gyrase inhibitor, Fig. 5.5a), which possesses an easily

modifiable primary amine that is well separated from the pharmacophore scaffold

(Fig. 5.5b), important in ensuring that modification does not interfere with drug

action. Substituents in all positions except 2, 3 and 4 (otherwise antibiotic loses its

activity) can vary. Position 6 almost always has a fluorine atom. Positions 1, 5, 7

and 8 are frequently modified in search of more potent compounds. Also,

inspection of the crystal structure of a gyrase-DNA complex with gyrase inhibitor

(Fig. 5.5c-e) reveals that position 7 on the pharmacophore is facing outward with

respect to DNA, suggesting that modifications at this position will not interfere

with binding to DNA.


139

Figure 5.5 | Fluoroquinolones and

their mode of action. (a)

Gemifloxacin. (b) Fluoroquinolone

pharmacophore. (c), (d), (e) Gyrase-

DNA complex with gyrase inhibitor.

Note the outward facing with respect

to the gyrase of the piperazine moiety

(ring shown in green with two nitrogen

atoms in blue) at position 7 of the

gyrase inhibitor (ciprofloxacin).

In a two-step reaction we attached pVIc to the amino group of gemifloxacin

using a PEG linker that would allow enough conformational freedom for the

antibiotic to bind to the gyrase on DNA. Also, as a negative control we used a

scrambled pVIc peptide (peptide S, SFRRCGLRQVK) that showed no reaction

N N

OH

OO

F

N

H2N

NO X N

R1

OH

OO

R6

R7

R5

R8

(a) (b)

(c) (d)

(e)

Chapter 5

140

enhancement as compared to pVIc in our previous PCR experiments. We used a

bifunctional 6-unit PEG linker with a maleimide (reactive to thiols) on one end

and a succinimide (reactive to amines) on the other. First, we attached the linker

to the primary amine of the gemifloxacin (see Experimental Section). Second, the

resulting compound was attached to the cysteine of the peptides. The synthesis

route is depicted in Fig. 5.6.

Figure 5.6 | Gemifloxacin-peptide conjugate synthesis. During step I, a 6-unit

PEG linker bearing a maleimide reactive group is conjugated to the primary

amine of the gemifloxacin. In step II, gemifloxacin-PEG6-maleimide is conjugated

to the cysteine of the peptide (pVIc or peptide S).

After the synthesis, the antimicrobial activity of the conjugates was tested

against E. coli ATCC 25922, which is a standard strain to evaluate the efficiency of

antibiotics.12 Two methods, the determination of the Minimal Inhibitory

Concentration (MIC) (Fig. 5.7) and the Kirby-Bauer Disk Test (Fig. 5.8), were

used (see Experimental Section).13-15

0.01 0.1 1 100.0

0.2

0.4

0.6

0.8 GEM GEM-pVIc GEM-S

OD 60

0

Concentration (µM)

Figure 5.7 | MIC test. Gemifloxacin

coupled to the S peptide and the

antibiotic conjugated to pVIc both

showed antibacterial activity only at

concentrations that are 1000 higher

than the MIC of the original

antibiotic. Error bars indicate ±SD;

n = 3.

N N

OH

OO

F

N

H2N

NO N N

OH

OO

F

N

HN

NO

O

ONH 6

O

N

O

O

pVIc or peptide S

GEM-pVIcGEM-Sm-PEG6-NHS

III


141

Figure 5.8 | Kirby-Bauer disk test. (a)

Gemifloxacin-inhibited growth of

E. coli, as indicated by the dark discs.

(b) Gemifloxacin conjugates with the

scrambled peptide S showed no

inhibitory activity against bacteria. (c)

Gemifloxacin conjugates with pVIc

showed no inhibitory activity against

bacteria.

However, no antibiotic activity of the gemifloxacin conjugates was detected.

To determine whether chemical modification itself inactivated gemifloxacin or

whether functionalisation with pVIc rendered the compound unable to be taken

up by the cells, we used an in vitro gyrase inhibition assay. In this assay, relaxed

DNA is used as a substrate for DNA gyrase, which is able to relax positive

supercoils and introduce negative supercoils. Addition of a gyrase inhibitor into

the reaction mixture stabilises the cleavage DNA-gyrase complexes with various

degree of supercoiling. We used E. coli gyrase to supercoil a relaxed DNA plasmid,

either in the presence or absence of gyrase inhibitors. Subsequently, the

differences in DNA topology (relaxed vs. supercoiled) were detected using DNA

(a)

(b)

C=1.2 μM

C=12 μMC=120 μM

no GEM

(b)

C=1.2 μM

C=12 μMC=120 μM

(c)

C=1.2 μM

C=12 μMC=120 μM

Chapter 5

142

agarose gel electrophoresis (Fig. 5.9a). Figure 5.9b shows that none of the

conjugates showed gyrase-inhibiting activity. Moreover, the gemifloxacin

conjugated with the PEG linker (compound obtained in step II, Fig. 5.6) was also

inactive.

Figure 5.9 | Gyrase inhibition assay. (a) General schematic of the assay. A

comparison between the reaction mixtures is obtained by separating the products

on an agarose DNA gel. DNA molecules with different topologies (relaxed vs.

supercoiled) will run at different speeds on the gel. (Image in the left panel,

copyright Joaquim Roca, Molecular Biology Institute of Barcelona) (b) Gyrase

inhibition assay results for gemifloxacin and its conjugates. The three right-hand

lanes corresponding to the three gemifloxacin conjugates were obtained within

the same experiment but were not placed sequentially after the first four lanes of

the DNA gel.

relaxed (R) plasmid

supercoiled (S) plasmid

+ + + relaxed (R) plasmid– + + gyrase– – + gyrase inhibitor

(a)

relaxed plasmid

supercoiled plasmid

(b)

3000 bp

2000 bp

1500 bp


143

Subsequently, we used a substantially smaller linker (linker 1, Fig. 5.10a) to

functionalise gemifloxacin, potentially providing insight on whether the size of

the linker is an important factor for antibiotic activity. It is important to note that

not only the size of the linker could contribute to the overall activity of the

antibiotic. The particular type of chemistry (succinimide ester to primary amine)

that was used converts the protonatable primary amine into an amide.

Figure 5.10 | Gemifloxacin conjugates. (a) AMAS (1) and CH3CO-NHS (2)

linkers. (b) Final conjugates with gemifloxacin. (c) Both gemifloxacin-AMAS and

gemifloxacin-COCH3 showed no gyrase inhibition activity in the inhibition assay.

NO

O

O

O

O

O

ON

O

O

O

1

2

(a)

N N

OH

OO

F

N

HN

NO

N N

OH

OO

F

N

HN

NO

N

O

O

O

O

GEM-AMAS

GEM-COCH3

(b)

relaxed plasmid

supercoiled plasmid

(c)

Chapter 5

144

This reaction results in electric charge removal, which could have an adverse

effect on binding to the gyrase on DNA. A related antibiotic ciprofloxacin, which

has a piperazine moiety at position 7 (Fig. 5.5b), was shown to fully retain its

activity upon amide bond formation (see next section “Ciprofloxacin”). Thus, to

test such a scenario we used the same type of chemical conjugation to introduce

the smallest possible (linker 2, Fig. 5.10a) modification to the primary amine of

the gemifloxacin. In this case, the size of the modification would no longer be an

issue since only two carbons will be attached to the primary amine. However, the

nitrogen will no longer be protonatable. As such, this type of modification will

help us determine whether the basic amine is required for the activity of

gemifloxacin. The in vitro gyrase inhibition assay showed that both conjugates

(Fig. 5.10b) had no activity against gyrase (Fig. 5.10c). This result strongly

suggests that the chosen combination of antibiotic and conjugation strategy is

unlikely to produce active compounds.

Gyrase inhibitors: ciprofloxacin

Since various modifications of gemifloxacin were unable to inhibit the

gyrase, we switched to a different antibiotic, namely ciprofloxacin (CIP, 2nd

generation gyrase inhibitor, Fig. 5.11a). Ciprofloxacin is probably the

fluoroquinolone that is most intensely studied and most frequently chemically

modified. The various types of reported conjugation chemistry are almost

exclusively targeted at the secondary amine, which is available at the piperazine of

Figure 5.11 | Ciprofloxacin. (a) Ciprofloxacin structure. (b) CIP-COCH3

conjugate structure. CIP-COCH3 showed comparable activity to the original

compound against gyrase as observed in the inhibition assay.

N

OH

OOF

NN

N

OH

OOF

NHN

(a) (b)

O

CIP CIP-COCH3

relaxed plasmid

supercoiled plasmid


145

ciprofloxacin.16-23 Therefore, the reactive position that we chose to modify was the

secondary amine of the piperazine moiety. To confirm that the basicity of this

amine is not critical for antibiotic function we converted the secondary amine to

the methylamide group (Fig. 5.11b). The resulting inhibitory activity against

gyrase was comparable to that of the original compound and consistent with what

is reported in literature.16

Next, we attempted conjugating several linkers to ciprofloxacin using the

strategy described above. Even though successful conjugation of linker number 1

and 3 was achieved (Fig. 5.12a), the resulting compounds possessed no gyrase

inhibitory activity (Fig. 5.12b). We were unable to find the appropriate reaction

conditions to achieve successful chemical conjugation between ciprofloxacin and

linker number 2.

Figure 5.12 | Ciprofloxacin conjugates. (a) Linkers m-PEG6-NHS (1), AMAS (2)

and 6MC-PFP (3). (b) CIP-PEG6 and CIP-6MC showed no gyrase inhibition

activity. No successful conjugation of AMAS to CIP was achieved.

Human topoisomerase II inhibitor: doxorubicin

Having tested several compounds from the class of gyrase inhibitors, we

continued our efforts within a different class of topoisomerase inhibitors, namely

human topoisomerase II inhibitors called anthracyclines. We selected

O

N

O

OO

F

F

F

F

F

NO

O

O

O

O

O

NHN

O ON

O

O

O

OO

6O1

2

3

(a)

relaxed plasmid

supercoiled plasmid

(b)

Chapter 5

146

doxorubicin, the most widely used representative of this class of pharmaceuticals.

Attachment of a peptide vector to doxorubicin as a means of increasing its

bioavailability and tumour-targeting is an established strategy.24-28 However,

within these approaches peptide are conjugated to the C3’-amine doxorubicin via

an amide bond, which is later cleaved in human body by proteolysis to release

doxorubicin. Such prodrug strategy is necessary to release doxorubicin with a

protonatable C3’-amine because it is crucial for binding to the DNA-

topoisomerase complex and therefore drug action.29

Here, we adopted the approach of C. Sun and colleagues who modified

doxorubicin by converting the primary amine to a secondary amine, ensuring that

the basicity of the amine is retained.29 Figure 5.13 shows the reaction scheme for

doxorubicin modification. The first two steps involve a synthesis of the

maleimide-functionalised linker bearing an aldehyde group, which is

subsequently conjugated to doxorubicin using a reductive amination reaction.

The authors conjugated the resulting maleimide-functionalised compound to

several amino acids and dipeptides and tested their activity using a human

topoisomerase II inhibition assay. Several of such doxorubicin modifications

showed activity comparable to the parent compound. Thus, this methodology is a

viable approach to functionalising doxorubicin.

Figure 5.13 | Doxorubicin modification scheme. In step I, an aldehyde-bearing

linker is synthesised from N-(3-hydroxypropyl)maleimide. In step II, the aldehyde

group is used for conjugation to doxorubicin in a reductive amination reaction. In

step III, DOX-mal is conjugated to the cysteine of the peptide (pVIc or S).

N

O

O

HO N

O

O

H

O

I

OO

O

OH

OH

O

HO

O

HO

O

HO

HN N

O

O

II III DOX-pVIcDOX-S


147

Following the published protocol, we attempted to first synthesise the

doxorubicin-maleimide conjugate. We successfully prepared the linker for

subsequent conjugation to the primary amine of doxorubicin by converting the

hydroxyl group of the N-(3-hydroxypropyl)maleimide to aldehyde (step I

Fig. 5.13, for details see Experimental Section). Conjugation of the resulting linker

to doxorubicin (step II Fig. 5.13, for details see Experimental Section) was

somewhat successful, although no solid conclusion could be drawn if the resulting

compound was indeed compound 3 in Fig. 5.13. Although Thin Layer

Chromatography (TLC) and silica chromatography revealed that conjugation did

occur, we were unable to identify the identity of the compounds by mass

spectrometry (MS) or Nuclear Magnetic Resonance (NMR) spectrometry

measurements (see Experimental Section). In view of time limitations, no further

trouble-shooting and characterisation was attempted.

5.3. Conclusions

Encouraged by our proof of concept biotin-streptavidin reaction, showing a

speed up in bimolecular association of more than an order of magnitude, we

aspired to extend the concept of 1D diffusion along DNA to biotechnologically

and pharmaceutically relevant systems. In Chapter 3, we showed that

conventional PCR can be speeded up significantly by functionalising the primers

with a pVIc peptide that allows them to 1D slide along DNA and find the

annealing sites faster as compared to search in 3D.

We embarked on a number of strategies to implement the concept of 1D

diffusion as a means to improve DNA-based processes. We prepared a

polymerase-pVIc chimera, which we expected to exhibit an improved efficiency as

compared to the parent polymerase. Unfortunately, the protein became

inactivated upon addition of a pVIc domain. Also, we conjugated the pVIc

molecular sled to antibiotics and antineoplastics that inactivate DNA-bound

topoisomerases within bacteria or malignant cells. We expected to achieve a

reduction of the required dose of these pharmaceuticals, thereby ultimately

Chapter 5

148

decreasing the possibility of side effects in clinical use. However, functionalisation

of the compounds described above resulted in their inactivation.

The general trend of a decrease in bioactivity of topoisomerases with

increasing lipophilicity of the substituents at position 7 of the pharmacophore is

generally accepted.16 While this might provide explanation why relatively

lipophilic AMAS and 6MC-PFP linkers inactivated the gyrase inhibitors, it does

not explain why hydrophilic PEG6 inactivated gemifloxacin and ciprofloxacin.

The size of the substituent at position 7 also plays a role. Cornier et al. conducted

Kirby Bauer disk testing against E. coli of ciprofloxacin conjugates that were

acylated at position 7.16 The authors varied the length of the substituent from

methyl to nonyl and discovered that the conjugates until hexyl were active while

heptyl-bearing ciprofloxacin exhibited no activity in E. coli. Further addition of

one carbon to the chain (octyl) completely restored the antibiotic activity of the

compound. Subsequent lengthening of the chain to nonyl inactivated the

compound again. Thus, the behaviour of the antibiotic conjugate can be

somewhat unpredictable even with systematic varying of the substituent length of

the same nature. In view of the time and/or technical limitations, no further

optimisation other than described in the corresponding sections was attempted.

The described approaches of chemical conjugation and molecular biology

are far from being exhaustive. Also, the tested pharmaceuticals are only a minor

part of the corresponding classes of compounds, therefore thorough compound

screenings using chemical libraries are required. Potential applications of the

molecular sled are numerous. The efficiency of restriction enzymes, ligases,

nucleases, RNAi could potentially be enhanced by conjugation of pVIc, which

renders molecules able to slide along DNA and find their targets faster than just

relying on 3D diffusion. However, as this Chapter clearly demonstrates, the

experimental strategies towards development of these applications are not

straightforward and will require a carefully balanced combination of rationally

designed synthesis approaches and high-throughput screening.


149

5.4. Experimental section

5.4.1. Polymerase

Materials:

Stock solutions and buffer solutions

General buffers

Potassium Phosphate buffer pH 7.5

(Incompatible with divalent cations)

1 L of 1 M potassium phosphate buffer

(pH 7.4):

802 mL of 1M K2HPO4 (MW 174.1)

198 mL of 1M KH2PO4 (MW 136.08)

Tris buffer, 1M Volume (L) Tris (g) HCl (ml)

pH 7.0 1 121.1 75-77

pH 7.5 1 121.1 60-62

pH 8.0 1 121.1 40-42

Antibiotics

Ampicillin stock 100 mg/ml in H2O, filtered

Working concentration 100 μg/ml

Chloramphenicol stock 34 mg/ml in Ethanol, filtered

Working concentration 34 μg/ml

Cell culture

IPTG (isopropyl-beta-D-

thiogalactopyranoside) (protein

expression induction)

Stock: 1 M in H2O

Working concentration: 0.5 mM

LB medium (freshly autoclaved): 5 g / 200mL

Protease inhibitors

EDTA

(metalloproteases)

Stock: 0.5 M

Working concentration: 1-10 mM

Leupeptin (MW 475.59)

(cysteine and serine proteases)

Stock: 10 mg/ml in H2O

Working concentration: 1-2- ug/ml

Pepstatin A (MW 685.89)

(aspartic proteases)

Stock 1 mg/ml in ethanol (eventually heat up

to 60oC)

Working concentration: 1 μg/ml

PMSF (MW 174.19)

(serine proteases)

Stock 200 mM in isopropanol (unstable in

H2O, Half-life = 1 hr. at pH 7.5) = 34.8 mg/ml.

Working concentration 0.1–1 mM

E64 (MW 357.41)

(cysteine peptidases)

Stock: 1 mM in H2O

Working concentration: 1-10 μM

Chapter 5

150

Other

NaCl (MW 58.44) 5 M

Imidazole (MW 68.07) 1M, pH 7.5

DTT (1,4-dithiothreitol) (MW

154.253)

1 M in H2O, filtered

BME (2-mercaptoethanol) 14 M

Plasmid preparation

The plasmid construct of Pfu-Sso7d was kindly provided by Morten

Nørholm.30 We used PfuX7 plasmid which corresponds to Pfu-Sso7d with a V93Q

mutation. This mutation improves the performance of the polymerase in uracil-

excision DNA engineering and has no additional effect on the overall

performance of the polymerase.

The plasmid was amplified in a competent E. coli Dh5α cells and further

confirmed by sequencing using the following primers: Pfu_2100_F and

T7_prom_F (see Table 5.1).

To substitute the Sso7d DNA sequence for pVIc

1. Sso7d was excised from the plasmid using BlpI (10 U) and KpnI-HF

(10 U) restriction enzymes at 37oC for 1.5 h.

2. pVIc insert was prepared by hybridising (10 min 99oC, gradient –

1oC/min, final temperature 21oC) F_KpnI_link_pVIc_BlpI and

R_KpnI_link_pVIc_BlpI (see table 1) in TE buffer (10 mM NaCl). The

inset is designed to have sticky ends for subsequent ligation to the

restricted plasmid, obtained in 1.

3. We used a 10-fold excess of the resulting pVIc insert to ligate it into the

cut plasmid (see 1) using a T4 ligase (NEB). The resulting plasmid was

used to transform competent E. coli Dh5α cells. To confirm successful

plasmid ligation we used colony PCR using Pfu_2100_F and R_in_pVIc

as primers (see Table 5.1) which resulted in the amplification of 312 bp

DNA. Moreover, sequencing of several colonies was performed.

4. The plasmid was then amplified using competent E. coli Dh5α cells.


151

The polymerases were expressed and purified as follows:

Purification Pfu-X7 from E. coli Rosetta/plysS/De3

Buffer Composition

Lysis buffer (filter and degas) 50 mM Phosphate buffer pH 7.5

300 mM NaCl

10 mM imidazole

10 % glycerol

His trap wash buffer (filter and degas) 50 mM Phosphate buffer pH 7.5

300 mM NaCl

20 mM imidazole

10 % glycerol

His trap elution buffer (filter and degas) 50 mM Phosphate buffer pH 7.5

300 mM NaCl

500 mM imidazole

10 % glycerol

Heparin wash buffer (filter and degas) 50 mM Phosphate buffer pH 7.5

50 mM NaCl

10 % glycerol

0.1 mM EDTA

Heparin elution buffer (filter and degas) 50 mM Phosphate buffer pH 7.5

1M NaCl

10 % glycerol

0.1 mM EDTA

Dialysis I buffer 50 mM Phosphate buffer pH 7.5

50 mM NaCl

10 % glycerol

0.5 mM EDTA

1 mM DTT

Storage buffer (Dialysis II buffer) 100 mM Tris pH 8

50 % glycerol

0.2 mM EDTA

2 mM DTT

0.1 % NP40

0.2% tween20

Chapter 5

152

Protocol

Add BME (10 mM) or DTT (1 mM, but not for Histrap) to all buffers freshly. If

degradation is observed: add PMSF to all buffers freshly (half life time in H2O: 30

min). Keep everything cold / on ice

1. Transform E. coli Rossetta cells with plasmid (10 ng)

This E. coli strain has another plasmid with chloramphenicol resistance + the

sequences for synthesis of tRNAs for rare codons in E. coli.

• Incubate 100 μl cells with plasmid on ice, 10 min

• Heat shock: 1 min at 42oC

• Incubate on ice, 10 min

• Add LB medium 600 μl, shake 30-45 min at 37oC

• Plate with antibiotics

2. Protein expression

• Pick a colony and inoculate 2 ml LB + ampicillin + chloramphenicol

• Grow overnight (o/n) at 37oC

• Inoculate 500 ml freshly autoclaved medium + antibiotics with 0.5-1 ml of

the o/n culture

• Grow to OD(600 nm) of about 0.6 (Take sample for SDS-PAGE)

• Induce protein expression with 0.5 mM IPTG

• Grow for about 3 h (OD600 up to 1-2)

• Harvest cells: centrifuge 10 min, 6000 g, JA9100 rotor

• Resuspend cell pellet in lysis buffer (20 ml) and snap-freeze. Store at

–80oC

3. Lysis:

• Thaw cells on ice

• Add protease inhibitors and BME (10 mM)

• Sonicate: put 50 ml falcon tube on ice during sonication,

• Use the thick stick, set cycle to 7 min, 10 s on/10 s off, 70% amplitude

• Clear lysate: centrifuge at 15000 rpm, Ja25.50 rotor at 4oC for 30 min.

4. Nickel affinity chromatography: HisTrap (GE healthcare, 1ml)

• Wash Akta system with H2O

• Wash tubings first with elution buffer, then with wash buffer

• Equilibrate column with 5 ml wash buffer

• Add imidazole to lysate to final 10 mM and load

• (Collect flowthrough and all wash steps)


153

• Wash with about 15 ml (until Abs280 is flat again)

• Wash with about 5 ml of 50 mM imidazole

• Elute: run gradient to 500 mM imidazole, 10-20x of column volume,

collect 1 ml fractions

• Pool peak fractions

• Clean column with H2O, store in 20 % Ethanol

If heparin chromatography is necessary: either dialyse into a low salt buffer

after HisTrap or use HisTrap buffers with less salt (lysis and wash buffer

150 mM, elution buffer max. 50 mM NaCl)

5. Optional: Dialysis o/n into low salt buffer

6. Heparin affinity chromatography: (GE healthcare, 1 ml)

• Wash Akta system with H2O

• Wash tubing first with elution buffer, then with wash buffer

• Equilibrate column with 5 ml wash buffer

• Load lysate

• Collect flowthrough

• Run gradient to 1 M NaCl, at least 20x column volume, collect 1 ml

fractions

• Clean column with H2O, store in 20 % Ethanol

7. Dialysis: into storage buffer, o/n at 4oC (prepare buffer of at least 300x volume

of sample). Store protein at –20oC.

Table 5.1. Sequencing primers.

Name DNA sequence

Pfu_2100_F 5’-AGT-TAA-AAT-AAA-GCC-AGG-AA-3’

T7_prom_F 5’-TAA-TAC-GAC-TCA-CTA-TAG-GG-3’

F_KpnI_link_pVIc_BlpI 5’-CGG-CGG-TGG-CGG-TGG-CGT-GCA-GAG-CCT-

GAA-ACG-CCG-CCG-CTG-CTT-TTA-GGC-3’

R_KpnI_link_pVIc_BlpI 5’-TCA-GCC-TAA-AAG-CAG-CGG-CGG-CGT-TTC-

AGG-CTC-TGC-ACG-CCA-CCG-CCA-CCG-CCG-

GTA-C-3’

sticky end, linker, pVIc, stop codon

Chapter 5

154

qPCR assay

Real time PCR (qPCR) experiment was performed using Bio-Rad iQ5 Real-

Time PCR System (Bio-Rad Laboratories, Richmond, USA). 25 μL reaction

mixtures contained forward and reverse primers at 0.2 μM, DNA template (2.5 ng

of pNZ-mEos3.2 plasmid), SYBR Green I (1×), dNTPs at 0.4 mM. Different

polymerase concentrations were screened to obtain the best amplification. The

SYBR Green I fluorescence was monitored at the end of each cycle. Commercially

available polymerases Phusion and Pfu were used at 1 U concentration. The

following polymerase buffers and corresponding qPCR protocols were used:

HR buffer + MgCl2 to bring its

total concentration to 2 mM

98oC (30 s)

45 cycles of [98oC (15 s), 72oC (45 s)]

72oC (10 min)

4oC (hold)

Pfu buffer 95oC (90 s)

45 cycles of [95oC (45 s), 60oC (30 s) 72oC (3 min)]

72oC (5 min)

4oC (hold)

5.4.2. Topoisomerase inhibitors

Gemifloxacin & Ciprofloxacin

Materials:

Antibiotics: Gemifloxacin (GEM) mesylate (BOC Sciences), Ciprofloxacin

(CIP) (Sigma-Aldrich); linkers: (succinimidyl-[(N-maleimidopropionamido)-

hexaethyleneglycol] ester) (m-PEG6-NHS), N-α-maleimidoacet-oxysuccinimide

ester (AMAS) (Piercenet), N-Succinimidyl Acetate (CH3CO-NHS) (Santa Cruz

Biotechnology), 6-Maleimidocaproic acid PFP ester (6MC-PFP) (Broadpharm);

other: Triethylamine (TEA), Dimethylformamide (DMF), Triofluoroacetic acid

(TFA).

All conjugates of the gyrase inhibitors were purified by HPLC

chromatography according to the following protocol:


155

Column RESOURCE RPC 1 mL

Buffer A 0.1 % TFA in H2O

Buffer B 0.1 % TFA in 80% CH3CN

Flow 1 mL/min

Detection a) GEM conjugates: Absorption at 220 nm, 272 nm, 343 nm

b) CIP conjugates: Absorption at 220 nm, 272 nm, 321 nm

1 Inject the sample into the injection loop. Load the sample on the

column with 10 mL of buffer A

2 Gradient 0% Æ 30% B // 5 min

3 Gradient 30% Æ 50% B // 60 min. Collect 1 mL fractions. Pool

the peak fraction together.

4 Gradient 50% Æ 100% B // 3 min

100% B // 7 min

Gradient 100% Æ 0% B // 3 min

0% B // 7 min

Wash the column using a steep gradient 0% Æ 100% B // 10 min

Equilibrate the column with 10 CV of buffer A.

GEM-PEG6-pVIc

10.7 mg of GEM, 73 μL of 250 mM mal-PEG6-NHS, 15 µL TEA were mixed

in 400 µL DMF. The reaction vial was placed on a nutator and was allowed to

proceed o/n at room temperature. The resulting GEM-PEG6 conjugate was

purified by HPLC chromatography using the protocol mentioned above. The

product was confirmed by electrospray ionisation (ESI) mass spectrometry, m/z

876.38 (MH+, required 876.37). Subsequently, the buffer was evaporated using

rotary evaporator. The resulting oily substance was redissolved in 770 µL DMF

and aliquoted in two vials. pVIc (5 mg in 100 µL DMF + 15 µL H2O) and S-

peptide (5 mg in 100 µL DMF + 15 µL H2O) were added to the vials. The reaction

vial was placed on a nutator and was allowed to proceed o/n at room temperature.

HPLC purification was carried out as described above. The product was

confirmed by MALDI mass spectrometry, m/z 2225.59 (MH+, required 2225.12).

Subsequently, the buffer was evaporated using rotary evaporator and the resulting

substance was redissolved in di H2O. The concentration was determined using

Chapter 5

156

optical absorption spectroscopy (272 nm and 343 nm, pure GEM was used as a

reference).

GEM-AMAS

10 mg of GEM, 121 μL of 250 mM AMAS, 15 µL TEA were mixed in 350 µL

DMF. The reaction vial was placed on a nutator and was allowed to proceed o/n at

room temperature. The resulting GEM-AMAS conjugate was purified by HPLC

chromatography using the protocol mentioned above. The product was

confirmed by ESI mass spectrometry, m/z 525.15 (MH–, required 525.16).




reference).

GEM-COCH3

2.7 mg of GEM, 28 μL of 250 mM CH3CO-NHS, 3 µL TEA were mixed in

75 µL DMF. The reaction vial was placed on a nutator and was allowed to proceed

o/n at room temperature. The resulting GEM-COCH3 conjugate was purified by

HPLC chromatography using the protocol mentioned above. The product was

confirmed by ESI mass spectrometry, m/z 432.17 (MH+, required 432.16).




reference).

CIP-COCH3

2.2 mg of CIP, 32 μL of 250 mM CH3CO-NHS, 3 µL TEA were mixed in

167 µL DMF. As opposed to the primary amine of the GEM, the secondary amine

of the CIP is less reactive. Therefore the reaction required harsher conditions. The

reaction was allowed to proceed in a shaker at 60oC for 5 h and was left o/n at

room temperature. The resulting CIP-COCH3 conjugate was purified by HPLC

chromatography using the protocol mentioned above. The product was




157


optical absorption spectroscopy (272 nm and 321 nm, pure CIP was used as a

reference).

CIP-PEG6

2 mg of CIP, 24 μL of 250 mM m-PEG6-NHS, 3 µL TEA were mixed in

271 µL DMF. The reaction was allowed to proceed in a shaker at 60oC for 5 h and

was left o/n at room temperature. The resulting CIP-COCH3 conjugate was

purified by HPLC chromatography using the protocol mentioned above. The

product was confirmed by ESI mass spectrometry, m/z 816.35 (MH–, required

816.35). Subsequently, the buffer was evaporated using rotary evaporator and the

resulting substance was redissolved in di H2O. The concentration was determined

using optical absorption spectroscopy (272 nm and 321 nm, pure CIP was used as

a reference).

CIP-AMAS

The same synthesis and purification protocol was used as for CIP-PEG6,

except m-PEG6-NHS was substituted by the AMAS linker. The HPLC

chromatogram showed two peaks, one of which corresponded to the starting

material (CIP). However, ESI mass spectrometry in both negative and positive

mode did not yield the desired peak.

CIP-6MC

2.4 mg of CIP, 32 μL of 250 mM 6MC-PFP, 3 µL TEA were mixed in 165 µL

DMF. The reaction was allowed to proceed in a shaker at 60oC for 5 h and was left

o/n at room temperature. The resulting CIP-6MC conjugate was purified by

HPLC chromatography using the protocol mentioned above. The product was




optical absorption spectroscopy (272 nm and 321 nm, pure CIP was used as a

reference).

Chapter 5

158

Doxorubicin

Materials:

Doxorubicin (DOX) (MedChem Express), N-(3-hydroxypropyl)maleimide

(mal-C3-OH) (Organix Inc), Dess–Martin periodinane (DMP) (Sigma-Aldrich),

ethylacetate (EtOAc), acetic acid (AcOH), tetrahydrofuran (THF).

The following protocol of doxorubicin modification was adopted from

C. Sun et al.29

Step Description

I 1. mal-C3-OH (200 mg, 1.29 mmol) was dissolved in 5 mL CH2Cl2.

2. DMP (15% wt in CH2Cl2, 4 mL, 1.93 mmol) was added in one

portion and the mixture was stirred for 2 h.

3. 2-Propanol (3 mL) was added and the solution was stirred for 30

min.

4. The resulting solution was filtered through a silica gel pad eluted

with EtOAc and the filtrate was concentrated.

5. The crude product was purified by silica gel chromatography eluting

with EtOAc-hexane (2/1) providing mal-COH (110 mg, 0.72 mmol,

55.7% yield), which was used immediately for step II. Clean NMR

spectrum can be obtained if measured immediately after synthesis,

although precipitate can be formed after 15 min.

II 1. To a stirred solution of DOX (100 mg, 0.172 mmol), mal-COH

(68.2 mg, 0.446 mmol) and glacial AcOH (20 μL, 195 mol%) in

CH3CN-H2O (2/1, 5 mL) was added a 1 M solution of NaCNBH3 in

THF (57 μL, 0.33 mol%). The mixture was stirred under nitrogen

atmosphere in the dark at room temperature for 1 h.

2. The solution was then concentrated in vacuo to give a residue,

which was diluted with an aqueous 5% NaHCO3 solution and

extracted with CH2Cl2.

3. Concentration of the organic solution and purification of the

resulting residue by silica gel chromatography eluting with CH2Cl2-

CH3OH (20/1) provided 26 mg of DOX-mal (21% yield).

III 1. DMSO, 30-60 min.

2. HPLC purification.


159

Successful mal-COH synthesis (step I) was confirmed by 1H NMR spectroscopy

(CDCl3, 400 MHz): δ 9.74 (t, 1H), 6.69 (s, 2H), 3.84 (t, 2H), 2.77 (dt, 2H). The

linker was immediately used for step II.

Following the published protocol, we attempted to synthesise the DOX-mal

conjugate (step II) for subsequent peptide attachment. Thin Layer

Chromatography (TLC) showed that new compounds were formed during step II.

The reaction mixture was loaded on a silica gel column and several fractions were

collected. However, neither ESI mass spectra (no desired MH+ 681.22 peak found)

nor NMR spectra of any of the fractions could be assigned to the desired DOX-

mal conjugate (data not shown).

In vivo antibiotics tests:

Kirby-Bauer test + minimum inhibitory concentration (MIC) test

Materials:

1. 6mm blank paper disc (BD, #231039)

2. 96 wells, 2 mL (Whatman, 7701-5205)

3. 96 wells, 200 μL, for absorption spectrum measurement

4. Müller-Hinton (MH) medium

5. Agar

Kirby-Bauer test

1. Work under laminar flow with all the precautions, MH medium has no

antibiotic nor specificity to prevent bacteria from growing. 22 g MH +

15 g Agar in 1 L of di H2O is autoclaved. It is important to use the same

volume of medium for all the plates. The pipette is filled up to 25 mL and

released until 2 mL (~23 mL per plate). Start from furthest left corner in

the fume hood (for right handed people) in order to prevent

experimenters hand from moving above the plate. It is handy to put the

plate on its lid. Let the plates dry for 45 min-1 h, label them and store at

4oC.

Chapter 5

160

2. Colony plates. E.coli ATCC25922 strain is kept at –80oC in 50% glycerol.

Dip a streaking loop into the cell stock and spread it over the agar surface.

The plate is kept in an incubator o/n at 37oC.

3. Put 20 mL of MH medium into a 100 mL autoclaved flask. Pick one thick

colony from a plate with a pipette tip, dip it into the medium and pipette

several times up and down. Put into a shaker at 250 RPM at 37oC until

OD600=0.132 is reached (~3h). If OD is higher, dilute with extra medium

and use in further experiments.

4. While the culture is growing prepare antimicrobial discs and let them dry

in a Petri dish under laminar flow fume hood. 30 μL of the antibiotic

solution is applied to the disc, different concentrations are scanned. A

good starting point is 0.5 mM for antibiotic sock solution and subsequent

1:10 dilutions. Use a control with di H2O. The discs dry in around 3 h (by

the time the culture is ready).

5. Pre-heat the necessary amount of empty MH plates in an incubator.

6. When the culture is ready/diluted, transfer the pre-heated plates under a

laminar flow fume hood and put 800 uL of culture on each of them. The

culture is spread by circularly lifting the edge of the Petri dish. A swab,

streaking loop or a spreader can be used as well. Whole surface must be

covered. Wait until the plates are dry.

7. Spot the antibiotic discs on the plates. 4 discs per plate is enough,

although a larger number may be used. A paper template with disc

arrangement chart is very handy, put it under the plate.

8. Put the plates in the incubator at 37oC o/n.

9. Ruler may be used for measurements but ImageJ is more accurate.

Calibration: lid 90 mm, bottom 85 mm.


161

MIC test

1. 96 well x 2 mL plate is used to screen different antibiotic concentrations.

1:2 decrease of concentration is used well to well, a duplicate of each

concentration is used. At this step 400uL of medium is put in all wells,

final volume in each well will be 500 μL.

2. A serial 1:2 dilution of antibiotic is prepared in a distant lane. For a

triplicate the first well is filled with 300 μL of antibiotic. All subsequent

wells are filled with 150 μL of H2O. 150 μL from well #1 is transferred to

well #2, mixed, 150 μL from well #2 is transferred to well #3 etc.

3. 50 μL of the antibiotic solution at a given concentration is added to

400 μL of MH medium in an appropriate well.

4. 50 μL of culture with OD600=0.132 is added into each well.

5. C+ is 400 µL of medium inoculated with 50 μL of water and 50 μL of

culture.

6. C– is 450 µL of medium inoculated with 50 μL of water.

7. The plate is put in the shaker at 350 RPM, 37oC, o/n.

8. 100 μL from each well is transferred to a similar 96 well plate (smaller) to

read the OD600.

In vitro gyrase inhibition assay

Inhibitory activity of the test compounds against DNA gyrase was tested

using an in vitro gyrase inhibition assay. In this assay an E. coli gyrase is used to

supercoil a relaxed DNA plasmid. Subsequently, the differences in DNA topology

(relaxed vs. supercoiled) are detected using DNA agarose gel electrophoresis.

DNA gyrase with 5X DNA Gyrase reaction buffer (M0306), gyrase substrate

(relaxed circular pUC19 plasmid, N0471) were purchased from NEB.

5X DNA Gyrase reaction buffer: 3 μl

DNA Gyrase (M0306): 0.2 μl (2 U)

DNA (relax, circular N0471): 0.25 μl (500 ng)

Gyrase inhibitor Final concentration 10 μM

H2O: to a total of 15 μl

Chapter 5

162

Incubate the mixture for 30 min at 37°C. In case a time-course measurement

is required, the reaction can be stopped by adding a 20-fold excess of EDTA with

respect to MgCl2 ions. Imaging of the results is performed using DNA agarose gel

(no ethidium bromide (EtBr)!) electrophoresis, 0.8%, 110 V, 1 h. It is important

to stain the gel with EtBr after running the electrophoresis since the dye influences

the superhelicity of the DNA.


163

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