Selected Topics in Aptamer Research - University of …mysite.science.uottawa.ca/mberezov/team/bsc-theses/2014_bsc_thesis...Selected Topics in Aptamer ... forming stable three-dimensional

UNIVERSITY OF OTTAWA, FACULTY OF SCIENCE

April 15, 2014

Authored by: Suzanne Elizabeth Kosteniuk, 5282464

Research Supervisor: Dr. Maxim V. Berezovski

Selected Topics in Aptamer Research

Research report submitted in partial fulfillment of the requirements for the course BPS4006

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Abstract

Aptamer research is a young, promising, and highly versatile field. Colloquially referred to as “artificial

antibodies” due to their practical similarities, aptamers are single-stranded oligonucleotides capable of

forming stable three-dimensional structures and binding to targets with high affinity and specificity.

However, aptamers have a number of advantages over antibodies. Aptamers are relatively inexpensive, easy

to synthesize and modify, stable under high temperatures, do not permanently denature, and are versatile

and easy to select. The process through which aptamers for a given target are selected in known as

Systematic Evolution of Ligands by Exponential enrichment (SELEX). SELEX involves repeating rounds of

partitioning a single stranded oligonucleotide pool according its binding to a target, amplifying the group

with the desired binding characteristics, and isolating the amplified single stranded oligonucleotides. The

following report documents three separate projects, each dealing with DNA aptamer research but with

distinctly different focuses and applications.

In the first project, a Polymerase Chain Reaction (PCR) protocol capable of selectively and efficiently

amplifying single stranded DNA oligonucleotides was developed and optimized. The successful

development of this protocol meant that two of the three key steps of SELEX – oligonucleotide

amplification and single stranded oligonucleotide isolation – would be combined into one quick and

efficient step. This project was successful, and the method that was developed was shown to be superior to

Berezovski research group’s previously employed protocol in terms of its efficiency, limit of detection, and

resistance to side-product formation.

The second project dealt with oncolytic viruses, a process known as AptaVIP, and chemical modification of

aptamers. Oncolytic viruses are diverse group of viruses capable of selectively killing cancer cells.

However, their use in therapeutic applications is challenging because when used in vivo they are subject to

degradation mediated by neutralizing antibodies (nAbs). In the past, Berezovski research group has selected

aptamers capable of binding to the oncolytic virus Vesicular Stomatitis Virus ∆51 (VSV∆51) and shielding it

from nAbs-mediated destruction, a concept known as Aptamer-facilitated Virus Protection (AptaVIP).

However, aptamers are subject to in vivo degradation by nucleases, thus aptamer modifications are required

to make AptaVIP feasible from a clinical perspective. In this project, we sought to extend the in vivo half-life

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of these AptaVIP-performing aptamers through conjugation to a 20 kDa polyethylene glycol (PEG moiety).

A method for producing and isolating PEG-modified aptamers was successfully developed and is described

herein. However, the project as a whole was unsuccessful, as it was shown that the aptamers Berezovski

research group previously developed to protect VSV∆51 from nAbs isolated from one antibody-producing

animal are not capable of protecting the virus from nAbs prepared from a different antibody-producing

animal.

In the third and final project, SELEX was employed to select oligonucleotide pools capable of binding to

Vero cells, a common continuous mammalian cell line. Selection of Vero cell-binding aptamers has many

potential applications, including cancer biomarker discovery and enabling novel chemotherapeutic drug

delivery research. Though this project is not yet completed, the oligonucleotide pools that have been

produced in five rounds of SELEX show superior affinity to Vero cells compared to random DNA

oligonucleotide libraries, which is a highly promising result.

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Acknowledgements

First and foremost, I would like to thank my supervisor, Dr. Maxim Berezovski, for giving me the

opportunity to do my honours project in his lab. Dr. Berezovski’s knowledge, generosity, and infectious

enthusiasm for his work has made this an amazing experience for me.

I would also like to thank all of the members of Berezovski lab for making the past year so excellent. I feel

extremely fortunate to have spent the year with such kind, passionate, and wickedly smart people, who are

as comfortable discussing the thermodynamic principles behind oligonucleotide folding as they are debating

whether or not goats are capable of yelling like humans. In particular, I am exceedingly grateful to Darija

Muharemagic, whose mentorship and insight has been absolutely invaluable to me.

I am deeply indebted to my mother, Elizabeth Manning, and my boyfriend, Travis Stewart, for their

support and love throughout this process. They put up with me when my experiments weren’t working and

I was a grumpy jerk, and for that they have my sincerest gratitude.

Finally, I feel I ought to thank Vince Gilligan, George R. R. Martin, and the people of Youtube for

providing me with much needed entertainment for the past year. Without their creativity and talent, I

probably would have finished this project months earlier.

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Table of Contents

ABSTRACT ................................................................................................................................................. I

ACKNOWLEDGEMENTS .............................................................................................................................. III

TABLE OF CONTENTS ................................................................................................................................. IV

INTRODUCTION ........................................................................................................................................ 1

DESIGN OF LINEAR-AFTER-THE-EXPONENTIAL PCR FOR SELEX .................................................................... 3

BACKGROUND ......................................................................................................................... 3

RESULTS AND DISCUSSION ........................................................................................................... 5

CONCLUSIONS ......................................................................................................................... 9

MATERIALS AND METHODS ........................................................................................................ 10

IMPROVING IN VIVO STABILITY OF ONCOLYTIC VIRUS-PROTECTING APTAMERS THROUGH PEGYLATION ........20

BACKGROUND ....................................................................................................................... 20

RESULTS AND DISCUSSION ......................................................................................................... 22

CONCLUSIONS ....................................................................................................................... 26


VERO CELL SELEX ....................................................................................................................................42

BACKGROUND ....................................................................................................................... 42

RESULTS AND DISCUSSION ......................................................................................................... 44

CONCLUSIONS ....................................................................................................................... 45


REFERENCES .............................................................................................................................................50

APPENDIX ...............................................................................................................................................52

FIGURES ............................................................................................................................... 52

PROTOCOL – LATE-PCR FOR SINGLE STRANDED OLIGONUCLEOTIDE POOL AMPLIFICATION ......................... 72

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Selected Topics in Aptamer Research

Introduction

In 1952, James Watson and Francis Crick elucidated the structure of genomic DNA and revolutionized the

fields of chemistry and biology1. Though that discovery occurred scarcely 60 years ago, the technologies

that have been developed in its wake have been astounding. While deoxyribonucleic acid (DNA) and other

nucleic acid molecules in nature serve to transmit genetic information of living organisms, the advent of

technologies to manipulate nucleic acids has led to the use of these molecules in novel, man-made

applications. Aptamers are synthetic single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA)

molecules that form stable three-dimensional structures capable of binding to specific protein, small

molecule, virus, or cell targets, much in the way proteins and small molecules do. The first articles on

aptamer research were published in 1990, shortly after the invention of polymerase chain reaction (PCR) 2,

3, 4.

Aptamer research is still in its infancy, but it is a rapidly growing field with enormous potential. Aptamers

are often compared to antibodies, and can be applied to research and medicine nearly everywhere

antibodies can. The list of current and potential applications for aptamers is substantial. Most notably,

aptamers have applications as therapeutic molecules, like the drug Macugen® for treatment of macular

degeneration, as well as in biosensing, biomarker discovery, biological assays, enhanced drug delivery, and

diagnostic medicine.

Aptamers are discovered via a process known as Systematic Evolution of Ligands by Exponential

Enrichment (SELEX) 2, 3, 4.

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Scheme 1: Basic scheme of SELEX

SELEX begins with a pool of random oligonucleotides flanked with constant sequences at both the 5’ and 3’

ends. The random pool, commonly referred to as a library, is incubated with the desired target. Targets are

extremely varied, and are typically small molecules, proteins, viruses, or cells. The oligonucleotides that do

not bind to the target are washed away, and the binding oligonucleotides are isolated. PCR is used to

amplify the oligonucleotides with desirable binding properties, then the dsDNA PCR product is converted

to ssDNA. That ssDNA makes up the oligonucleotide pool for the subsequent round of selection. The cycle

is repeated approximately eight to twelve times, until a pool of approximately one to one hundred aptamer

sequences are obtained. Sequencing is performed on the newly discovered aptamers, and they can then be

used in applications ranging from pharmaceuticals, biosensing, or biomarker discovery 2, 3, 4.

My honours project consisted of three separate projects, each dealing with a different aspect of aptamer

research. First, I designed and optimized a PCR protocol capable of amplifying ssDNA oligonucleotides

selectively in one step, thereby simplifying the SELEX process. Second, I chemically modified a known and

potentially therapeutic aptamer in an attempt to increase its in vivo stability. Finally, I performed SELEX in

order to discover aptamer pools capable of binding to Vero cells, a well-known immortal mammalian cell

line.

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Design of Linear-After-The-Exponential Polymerase Chain Reaction for SELEX

Background

One of the most challenging steps of SELEX is the isolation of ssDNA from the dsDNA produced during

PCR. Traditionally this step is done using expensive and time-consuming techniques such as exonuclease

degradation or separation via biotinylated primers 5. In the past, Berezovski research group used a two-step

process for amplification and isolation of ssDNA – first traditional symmetric PCR was employed to amplify

dsDNA, then the symmetric PCR product was subjected to asymmetric PCR was used to selectively

amplify ssDNA. While this method was less expensive and time-consuming than most, purification steps

were required and side products were frequently formed.

The goal of this research was to design a novel PCR protocol capable of amplifying ssDNA oligonucleotides

in a single PCR step for use in SELEX. To do this, a technique known as Linear-After-The-Exponential PCR

(LATE-PCR) was employed. LATE-PCR is a special type of asymmetric PCR that allows for efficient

ssDNA production 6, 7, 8. In LATE-PCR, the limiting primer's melting temperature is set higher than the

excess primer's melting temperature. The discrepancy between the limiting primer’s Tm and the excess

primer’s Tm (TmL-Tm

X) allows the limiting primer to bind completely to the DNA template during the

annealing phase. Early in LATE-PCR the amplification curve resembles symmetric PCR showing

exponential growth, and when the limiting primer is depleted the curve becomes linear, resembling

asymmetric PCR 6, 7, 8. This relationship is demonstrated in figure 1, a graph of asymmetric quantitative PCR

(qPCR) data sets in which TmL-Tm

X was variable. In figure 1, the blue curve which represents a TmL-Tm

X of

+5°C is representative of LATE-PCR. It clearly shows a period of exponential growth, followed by a

plateauing linear phase. As well, it shows superior amplification to its counterparts with lower TmL-Tm

X

values, particularly early on in the PCR. Conversely, the yellow, green, and orange curves, which

respectively represent TmL-Tm

X values of +3°C, 0°C, and -3°C do not display as strong an inflection as the

blue curve, and cannot be characterized as LATE-PCR.

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FIGURE 1: QUANTITATIVE PCR DATA MEASURING AMPLIFICATION AS A FUNCTION OF NUMBER OF PCR CYCLES. EACH

CURVE REPRESENTS A SPECIFIC PRIMER SET, EACH WITH A DIFFERENT TML-TM

X. FROM PIERCE ET AL. 2005 6.

It was theorized that the use of LATE-PCR for ssDNA amplification during SELEX could reduce the time

and money required to perform SELEX, contribute to a SELEX protocol that could be automated i.e.

performed by a robotic station. Optimization of LATE-PCR consisted of manipulating variables such as the

ratio of forward primer to reverse primer concentration, total number of PCR cycles, annealing

temperature used during PCR, and type of DNA polymerase using during PCR. The resulting optimized

protocol for a LATE-PCR was not only more efficient and cost-effective than the previous method

employed by Berezovski research group, it tended to produce less undesirable side products and was capable

of amplifying smaller initial concentrations of DNA more effectively than the previously employed method.

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Results & Discussion

Prior to experimentation with LATE-PCR, the ssDNA amplification and isolation method previously

employed by Berezovski research group was assessed. In this method, dubbed “symmetric-then-asymmetric

PCR,” oligonucleotides are first amplified by symmetric PCR to form dsDNA, then the symmetric PCR

product is subjected to asymmetric PCR to selectively amplify ssDNA. We sought to determine the lower

limit of starting DNA concentration it could apparently amplify (limit of detection, LOD), the extent of

amplification, and the extent of side-product formation. The results of this experiment would provide a

basis to judge the effectiveness of future experiments with LATE-PCR.

The results are displayed in figure 2 in the appendix. They show that the LOD of the symmetric-then-

asymmetric method is a starting DNA concentration of 100 pM. PCR reaction mixtures with starting DNA

library concentrations below showed no apparent amplification. At concentrations greater than 100 pM the

symmetric-then-asymmetric method amplified DNA library very effectively, with well over 50% of the

forward primer becoming incorporated into the amplicons. Interestingly, while side product formation was

minimal when the starting DNA concentration was 100 pM, at higher DNA concentrations (1 nM) the

symmetric-then-asymmetric method produced a significant amount of side product. The exact reason for

this is not known, but a possible explanation is that a higher template and amplicon concentration

encourages the formation of complexes – two or more oligonucleotide molecules bound to one another –

which have different electrophoretic mobility than individual oligonucleotides.

Prior to beginning LATE-PCR experiments, a review of literature discussing LATE-PCR was conducted to

determine reasonable experimental conditions to use for the first round of experiments 6, 7, 8. The

theoretical basis behind LATE-PCR is that if the melting temperature of the limiting primer is higher than

that of the excess primer, then during the annealing step of PCR nearly all molecules of the limiting primer

will bind to their template strand, resulting in efficient amplification until the limiting primer has been

exhausted. The general consensus throughout the literature reviewed was that difference in Tm between the

limiting and excess primer (TmL-Tm

X) should be approximately 5-10°C, and that the ideal annealing

temperature used in PCR varies. Using Applied Biosystems® Tm Calculator and knowledge of the typically

used PCR primer sequences for the 40N DNA library, ideal excess (forward) primer and limiting (reverse)

primer sequences for LATE-PCR were designed. The reverse primer typically used for PCR of the 40N

DNA library, a 20 nucleotide sequence with a Tm of 54.03°C, was determined to be appropriate for LATE-

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PCR. The forward primer we decided to use for LATE-PCR is a truncated version of the normal 20

nucleotide primer; it is a 17 nucleotide sequence with a Tm of 48.29°C. The TmL-Tm

X of this primer pair is

5.74°C, which fits the criteria for LATE-PCR.

In addition, the literature reviewed suggested that ratios of excess to limiting primer concentrations can

range from 10 to 100, with 20 to 40 being typical.

The first LATE-PCR experiment consisted of an assay of PCR reactions with varying excess primer to

limiting primer ratios (ratios of 1/20 to 1/80 were analysed) and different annealing temperatures (48-

56°C). The PCR products were analysed with agarose gel electrophoresis, and the gel image is annotated in

figure 3 in the appendix.

The results of this experiment were promising and not unexpected. When the ratio of limiting to excess

primer is small, product formation is low. However, while increasing the ratio of limiting to excess primer

increased product amplification, it also seems to increase the amount of side product produced. Thus a

fairly moderate limiting to excess primer ratio, between 1/40 and 1/20, is likely optimal. As well,

decreasing annealing temperature seemed to slightly increase the yield of both desirable product and side

product. This effect was relatively minor, though annealing temperature required optimization as well.

Another experimental variable emphasized by LATE-PCR literature was the number of PCR cycles used.

The number of cycles used in LATE-PCR is greater than that used in traditional symmetric or asymmetric

PCR, though it is subject to a great deal of variability. The articles reviewed studied the results of LATE-

PCR that ranged from 35 to 85 PCR cycles. Thus our second LATE-PCR experiment consisted of an assay

of PCR reactions with varying numbers of PCR cycles (55-80) and different annealing temperatures (48-

56°C). Due to the concern of side product amplification at very high numbers of PCR cycles, a duplicate

experiment was performed in which none of the PCR mixtures contained any template DNA. This large

negative control experiment serves to determine a “safe” upper limit of PCR cycles in which only template

DNA is amplified. The PCR products were analyzed with agarose gel electrophoresis, and the gel images

are annotated in figures 4 and 5 in the appendix.

The experiments reveal that although higher numbers of cycles yield greater amplification, at 60-65 cycles

unwanted side products begin to be noticeably amplified. Thus 55 was tentatively set as the optimal number

of cycles for LATE-PCR of 40N DNA library.

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The most interesting result from this project came about when the tentatively optimized LATE-PCR

procedure was performed using the truncated 17 nucleotide forward primer, designed especially for LATE-

PCR, and with the normal 20 nucleotide forward primer side-by-side. The only differences in these two

reaction mixtures was the length of the forward primer used and the annealing temperate – the mixture

containing the truncated primer had its annealing time set lower to suit the shorter primer’s decreased Tm.

The mixtures were analysed using agarose gel electrophoresis and the gel image is annotated in the appendix

as figure 6.

It was expected that the PCR mixture containing the specially designed 17 nucleotide LATE-PCR primer

would amplify the template DNA more effectively. However, no noticeable difference existed between the

two PCR mixtures.

The reason that the 20 nucleotide forward primer worked equally as well as the 17 nucleotide forward

primer is still not fully understood. It is possible that at 55 cycles, both of the PCR mixtures’ amplification

reached a linear plateau, and the difference in the extent of amplification is not distinguishable when

quantifying DNA concentration with a method as insensitive as agarose gel electrophoresis. Figure 1 seems

to support this idea. In figure 1, the LATE-qPCR shows superior amplification to the other reactions early

on in PCR, during the LATE-PCR’s exponential phase. However, once the limiting primer of the LATE-

PCR had been exhausted and the slope had become linear, the other reactions’ level of amplification caught

up with that of the LATE-PCR. This idea could be tested with quantitative PCR (qPCR), unfortunately we

did not have the equipment necessary to perform a qPCR experiment.

Following optimization of these essential parameters, a direct comparison of the old symmetric-then-

asymmetric method versus the new LATE-PCR method at varying starting DNA template concentrations

was made. This experiment yielded extremely exciting results. The new LATE-PCR procedure was

superior to the symmetric-then-asymmetric in terms of its amplification yield and LOD, and did not suffer

from an increase in side product formation.

Finally, to ensure that the results of this experiment would be practical and have benefit to PCR, we tested

the newly developed LATE-PCR protocol using a master mix kit from a different company. The reasoning

behind this is that different varieties of DNA polymerase have different strengths. One issues that has been

encountered by members of Berezovski research group is the selection of aptamers and aptamer pools with

high levels of the nucleotides guanine and cytosine, commonly referred to as “high GC”. Although high GC

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oligonucleotides tend to form secondary structures more successfully than oligonucleotides with even

nucleotide distribution, making them likely to be excellent aptamers, their tendency to fold and their

relatively high melting temperature make them difficult to amplify with PCR 9. Some DNA polymerase

varieties are superior at amplifying high GC DNA compared to competitors, and I wanted researchers using

this LATE-PCR protocol to have a back-up option, in case the GoTaq© Polymerase cannot successfully

amplify their aptamer pools due to high GC content. We chose to use the KAPA2G Robust Hotstart PCR

Kit from Kapa Biosystems due to recommendations from other lab members and its relatively low cost. The

results from experimentation with KAPA2G are annotated in figure 8 in the appendix. The KAPA2G DNA

polymerase was shown to be a feasible alternative to GoTaq©’s polymerase, with comparable amplification

and no noticeable side product formation.

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Conclusions

Overall this project was successful. The newly designed LATE-PCR protocol was shown to have superior

efficiency, cost-effectiveness, limit of detection, and level of side product formation compared to the old

symmetric-then-asymmetric PCR method. Following experimentation, formal protocols for LATE-PCR

were written. They can be found in the appendix.

The LATE-PCR protocol was used in Berezovski research group for SELEX experiments for a short time,

though currently we are isolating ssDNA in SELEX experiments by performing exonuclease digestion

following symmetric PCR. The exonuclease method appears to be more robust when it comes to difficult-

to-amplify pools, and is fairly rapid and inexpensive.

In the future, we hope to incorporate LATE-PCR into automated SELEX that will be performed in

Berezovski lab. In automated SELEX, aptamer selection is performed extremely rapidly by a robotic

station. In this circumstance, LATE-PCR will be absolutely ideal for oligonucleotide amplification and

ssDNA isolation because it significantly reduced the number of steps involved in SELEX and streamlines the

process.

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Materials and Methods

Primers and 40N DNA library were obtained from Integrated DNA Technologies (Coralville, USA). The

40N DNA library consists of a mixture of DNA oligonucleotides, each composed of a 40 nucleotide random

sequence flanked by constant 20 nucleotide sequences at each end that serve as primer regions. Both the full

forward primer (5’-CTC CTC TGA CTG TAA CCA CG-3’) and truncated forward primer (5’- CTC CTC

TGA CTG TAA CC-3’) were labelled with FAM on their 5’ ends, whereas the reverse primer (5’-GGC-

TTC-TGG-ACT-ACC-TAT-GC-3’) was unlabelled.

Primer annealing temperatures were calculated with Applied Biosystems® Tm Calculator

(www6.appliedbiosystems.com/support/techtools/calc) from Life Technologies (Carlsbad, USA).

Several varieties of DNA polymerases and master mixes were used throughout this project. The most

frequently used was the GoTaq© Hot Start PCR Kit from Promega (Madison, USA). The master mixes

prepared from this kit contained nuclease-free ddH2O, 1X GoTaq© Flexibuffer, 2.5 mM MgCl2, 250 μM

dNTPs, 0.025 UμL-1 GoTaq© Hot Start DNA Polymerase, and varying amounts of forward and reverse

primer. In addition to the Promega master mixes and DNA polymerase, the KAPA2G Robust Hotstart PCR

Kit from Kapa Biosystems (Wilmingtom, USA) was used. Manufacturers’ instructions were followed when

preparing the KAPA2G master mixes. All PCR reactions were carried out using the vapo.protect

Mastercycler proS from Eppendorf (Hamburg, Germany).

Gel electrophoresis experiments were performed in 0.5X TAE buffer (20 mM Tris, 10 mM acetic acid, and

0.5 mM EDTA), which had been prepared in-lab. UltraPureTM Agarose was obtained from Invitrogen

(Carlsbad, USA). Gels were visualized using the Fluorochem® Q from Alpha Innotech (Santa Clara, USA).

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Assessment of Current Method – Symmetric-then-Asymmetric PCR

Two master mixes were prepared – one for symmetric PCR, one for asymmetric PCR. Their components

are as follows:

Symmetric master mix:

1X Colourless GoTaq® Flexi Buffer (Promega), from 5X concentration stock

2.5 mM MgCl2 (Promega)

300 nM reverse DNA primer (sequence: 5'-GGC TTC TGG ACT ACC TAT GC-3')

300 nM 5' FAM-labelled forward primer (sequence: 5'-CTC CTC TGA CTG TAA CCA CG-3')

200 uM dNTPs

0.025 UuL-1 Gotaq® Hot Start DNA Polymerase (Promega)

Asymmetric master mix:

1X Green GoTaq® Flexi Buffer (Promega), from 5X concentration stock



1 µM 5' FAM-labelled forward primer (sequence: 5'-CTC CTC TGA CTG TAA CCA CG-3')

200 uM dNTPs


Eight 45 µL aliquots of the symmetric master mix were placed into 0.2 mL PCR tubes. 5 µL of DNA

library solution was placed into each of the eight master mix aliquots. The DNA concentration for each

DNA library solution used varied (1 aM, 10 aM, 1 fM, 100 fM, 1 pM, 100 pM, 1 nM, and 0 (negative

control) were used).

The following PCR program was used for both symmetric and asymmetric amplification:

Initial denaturation: 2 minutes at 94º C

Followed by 15 cycles of the following steps:

Denaturation: 30 seconds at 94º C

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Annealing: 15 seconds at 56º C

Extension: 15 seconds at 72º C

Hold at 4º C

Eight 45 µL aliquots of the asymmetric master mix were placed into 0.2 mL PCR tubes. 5 µL of each

symmetric PCR product was placed into one of the eight asymmetric master mix aliquots. Asymmetric

amplification was completed using the same PCR program as was used for symmetric amplification.

A 3% agarose gel in 0.5X TAE buffer was prepared, and 10 µL of each symmetric-then-asymmetric PCR

products was loaded into the gel. The gel was then run at 150 V for 30 minutes. The gel image is annotated

in figure 2 in the appendix.

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Initial Optimization of Primer Ratios

An assay was performed in which the limiting (reverse) primer concentration and the annealing

temperature were simultaneously varied. Three master mixes were prepared, described below:



12.5 nM, 25 nM, or 50 nM reverse DNA primer (sequence: 5'-GGC TTC TGG ACT ACC TAT

GC-3')


400 uM dNTPs


Five 12.5 µL aliquots of each master mix were placed into 0.2 mL PCR tubes. 2.5 µL 100 nM DNA library

was placed into four out of five aliquots of each master mix, and to the remaining DNA library-free master

mix aliquots 5 µL ddH2O was added.

Using the gradient function of the vapo.protect Mastercycler proS, the PCR program was set such that the

annealing temperature varied. The PCR program is described below:




Annealing: 20 seconds at 48, 50, 52, 54 or 56º C


Hold at 4º C

Note that for the negative controls the annealing temperature was set at 52°C.

A total of 18 samples were prepared, each with a different combination of primer ratio and annealing

temperature.

A 3% agarose gel in 0.5X TAE buffer was prepared, and 10 µL of each PCR products was loaded into the

gel. The gel was then run at 150 V for 45 minutes. The gel image is annotated in figure 3 in the appendix

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Initial Optimization of Number of PCR Cycles

An assay was performed in which the number of PCR cycles and the annealing temperature were

simultaneously varied. One master mix was prepared, described below:




1 µM 5' FAM-labelled forward primer (sequence: 5'-CTC CTC TGA CTG TAA CC-3')

400 uM dNTPs


A total of 48 samples were prepared. 48 12.5 µL aliquots of master mix were prepared. 2.5 µL 100 nM

DNA library was added to each of 24 master mix aliquots. 2.5 µL ddH2O was added to the remaining

aliquots.




Followed by 55 to 80 cycles of the following steps:


Annealing: 20 seconds at 48, 50, 52, or 54 º C


Hold at 4º C

Two 3% agarose gels in 0.5X TAE buffer were prepared, and 10 µL of each PCR products was loaded into

a gel. The gels were then run at 150 V for 55 minutes. The gel images are annotated in figures 4 and 5 in

the appendix.

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Selection of a Primer Pair

Two master mixes were prepared. The only difference between them was the type of forward primer each

contained. One contained the regular 20 nucleotide primer, the other contained the truncated 17

nucleotide forward primer designed for LATE-PCR. The master mix components are listed below.




1 µM 5' FAM-labelled forward primer (sequence: either 5'-CTC CTC TGA CTG TAA CC-3' or 5'-CTC

CTC TGA CTG TAA CCA CG-3')

400 uM dNTPs


Two 12.5 µL aliquots of each master mix were prepared. 2.5 µL 100 nM DNA to one aliquot of each

master mix variety. 2.5 µL ddH2O was added to each of the remaining two master mix aliquots.






Annealing: 20 seconds at 52 or 56 º C (note: the samples containing the 20 nucleotide

primer had their annealing temperature set to 56° C, the samples with the 17 nucleotide

primer had an annealing temperature of 52° C.)


Hold at 4º C

A 3% agarose gels in 0.5X TAE buffer was prepared, and 10 µL of each PCR products was loaded into the

gel. The gel was then run at 150 V for 25 minutes. The gel image is annotated in figure 6 in the appendix.

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Direct Comparison of LATE-PCR and Symmetric-then-Asymmetric PCR

In this experiment, both LATE-PCR and symmetric-then asymmetric PCR were performed, and their

results were assessed side-by-side using agarose gel electrophoresis.

For the LATE-PCR, the first step was to make a master mix, whose components are listed below:





400 uM dNTPs


Seven 25 µL aliquots of this master mix were prepared. Each aliquot was combined with 5 µL of a DNA

library solution. The DNA concentration for each DNA library solution used varied (10 fM, 100 fM, 1 pM,

10 pM, 100 pM, 1 nM, and 0 (negative control) were used).

The seven PCR mixtures were amplified according to the following PCR program:




Annealing: 20 seconds at 56 º C


Hold at 4º C

The LATE-PCR mixtures were then set aside briefly while the symmetric-then-asymmetric PCR mixtures

were prepared.

For the symmetric-then asymmetric-PCR, two master mixes were prepared – one for symmetric PCR, one

for asymmetric PCR. Their components are as follows:

Symmetric master mix:

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1X Colourless GoTaq® Flexi Buffer (Promega), from 5X concentration stock



300 nM 5' FAM-labelled forward primer (sequence: 5'-CTC CTC TGA CTG TAA CCA CG-3')

200 uM dNTPs


Asymmetric master mix:





200 uM dNTPs


Seven 25 µL aliquots of the symmetric master mix were placed into 0.2 mL PCR tubes. 5 µL of DNA

library solution was placed into each of the seven master mix aliquots. The DNA concentration for each

DNA library solution used varied (10 fM, 100 fM, 1 pM, 10 pM, 100 pM, 1 nM, and 0 (negative control)

were used).

The following PCR program was used for both symmetric and asymmetric amplification:






Hold at 4º C

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Eight 45 µL aliquots of the asymmetric master mix were placed into 0.2 mL PCR tubes. 5 µL of each

symmetric PCR product was placed into one of the eight asymmetric master mix aliquots. Asymmetric

amplification was completed using the same PCR program as was used for symmetric amplification.

A 3% agarose gel in 0.5X TAE buffer was prepared, and 10 µL of each of the 14 samples was loaded into

the gel. The gel was then run at 150 V for 50 minutes. The gel image is annotated in figure 7 in the

appendix.

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Verification of LATE-PCR Feasibility with a Different Variety of DNA Polymerase

A master mix was prepared with the following components:

1X KAPA2G Buffer A, from 5X concentration stock


1 uM 5' FAM-labelled forward primer (sequence: 5'-CTC CTC TGA CTG TAA CCA CG-3')

400 uM dNTPs

0.025 UuL-1 KAPA2G Robust Hot Start DNA Polymerase

Two 25 µL aliquots of the master mix was prepared. To one aliquot, 5 µL 100 nM DNA library was added.

To the other, 5 µL ddH2O was added.

The two PCR mixtures were subjected to the following PCR program:

Initial denaturation: 30 seconds at 95º C





Hold at 4º C

A 3% agarose gel in 0.5X TAE buffer was prepared, and 10 µL of each of the two samples was loaded into

the gel. The gel was then run at 150 V for 30 minutes. The gel image is annotated in figure 8 in the

appendix.

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Improving In Vivo Stability of Oncolytic Virus-Protecting Aptamers through PEGylation

Background

The first effective cancer-treating drugs were discovered in the early 20th century, and millions of lives have

been saved by chemotherapy since that time. Early chemotherapies such as nitrogen mustard and

methotrexate, derived from the warfare agent mustard gas, were notoriously toxic, brutal drugs to be

treated with 10. While modern chemotherapies are not as toxic as many of their predecessors, they still carry

side effects lists that include extreme nausea, immune suppression, and secondary cancers. The extreme

side effects of chemotherapies derive from the fact that they are designed to kill cancers cells, but do so with

imperfect selectivity, thus they cause a high level of damage to healthy cells as well.

Modern research into new cancer treatments is largely focused on improving selectivity of drugs to cancer

cells, and one novel and extremely promising approach is the use of oncolytic viruses (OVs). OVs are a

diverse group of wild-type and genetically engineered viruses that are capable of selectively destroying

cancer cells in a variety of ways 11, 12. One such virus is enhanced vesicular stomatitis virus (VSVΔ51).

VSVΔ51 belongs to the Rhabdoviridae family of viruses, and contains a mutation causing deletion of a single

amino acid in its viral matrix protein which confers its ability to selectively kill cancerous cells. While

VSVΔ51 is capable of infecting mammals, because it is extremely sensitive to interferons, a class of proteins

produced in response to pathogens, infections are typically mild. However, because cancer cells often have

defective interferon pathways, VSVΔ51 is able to cause lethal infections in a wide range of cancer cells 13, 14.

The primary challenge of working with OVs as cancer therapeutics in poor in vivo half-life due to

destruction by neutralizing antibodies (nAbs) of the immune system. To overcome this problem, Berezovski

research group has investigated Aptamer-Facilitated VIrus Protection (AptaVIP), which involves the use of

aptamers capable of binding to VSVΔ51’s outer surface and shielding virus particles from neutralization by

nAbs. Several aptamer sequences capable of shielding VSVΔ51 from nAbs have been elucidated by

Berezovski research group14. Past cell-based viral infectivity assays performed by Berezovski research group

have shown that these aptamer sequences are capable of shielding VSVΔ51 from nAbs in vitro, enabling

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VSVΔ51 to infect and kill interferon-deficient cells 14. However, aptamers are subject to degradation by

nucleases when used in vivo. This degradation is rapid, and reduces the half-life of aptamers to only a few

hours, making unmodified aptamers unfeasible for in vivo applications 16, 17. Fortunately, many methods of

modifying aptamers exist to confer in vivo stability to aptamers, allowing them to be used therapeutically 16,

17.

This project involved the covalent attachment of a polyethylene glycol (PEG) moiety to the VSVΔ51-

binding aptamer ZMYK-23. The covalent attachment of a PEG group to another molecule is commonly

referred to as PEGylation. PEGylation is a common modification in pharmaceutical science, being used to

improve delivery and stability of small molecule, peptide, protein, and oligonucleotide drugs 17, 18.

Numerous PEGylated drugs are currently on the market, notable among them are Pegasys® (Hoffman-La

Roche, Inc., USA), an interferon for hepatitis treatment, Neulasta® (Amgen, USA), a protein used to

stimulate neutrophil growth in patients undergoing chemotherapy, and Macugen® (OSI Pharmaceuticals,

USA), an aptamer for treatment of macular degeneration 17, 18, 19.

This project involved several phases with the ultimate goal of producing PEGylated aptamers capably of

effective VSVΔ51 AptaVIP in vivo. First, a protocol for PEGylating aptamers efficiently was designed and

optimized. Following that, the PEGylated aptamers needed to be isolated and purified. Then cell-based

viral infectivity assays were performed to determine whether or not PEGylated aptamers could successfully

perform AptaVIP in vitro. In addition, flow cytometry was employed to study the aptamers’ binding

characteristics. Finally, had in vitro experiments been successful, we would have performed in vivo

experiments to show if VSVΔ51 AptaVIP with PEGylated aptamers has therapeutic potential.

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The first task required for this project was to design an organic reaction capable of efficiently linking the

VSVΔ51-binding aptamer ZMYK-23 to PEG. Factors including the type of linkage, reactant concentrations,

solvent systems, pH, and reaction time and temperature had to be optimized. Several publications7, 8, 9, 10

were reviewed in order to determine appropriate starting conditions. Based on articles describing similar

PEG conjugation reactions and PEGylation in pharmacology, the reactants selected were 20 kDa mPEG

modified with succinimidyl carbonate (mPEG-SC 20K) and 3’ amino modified aptamer. The conjugation

mechanism shown in scheme 2.

In the early part of this task, results were modest. Some factors, such as buffer, reaction mixture pH, and

acetonitrile concentration seem to have a small effect on the extent of aptamer PEGylation (see figures 10,

11, 12, and 14 in the appendix). At the same time, many factors that we predicted could impact

PEGylation, such as reaction time, temperature, and agitation seemingly had no effect on PEG-aptamer

yield (see figures 9 and 13 in the appendix). Eventually a plateau seemed to have been reached, as

PEGylation yield never rose above ~25%, despite experimentation with several different variables. While

these results were frustrating while attempting to design a reaction with a high PEGylation yield, they were

quite informative regarding the nature of the reaction. The lack of impact of reaction temperature,

agitation, and time indicated that this reaction is kinetically very favourable, going to completion rapidly

and without the need for added internal energy to reach activation barriers. In addition, one might expect

that reagent degradation might contribute to the seemingly identical PEGylation yields of both 4 hour and

20 hour incubated reaction mixtures. Indeed, according to the manufacturers, the mPEG-SC used in this

experiments has a hydrolysis half-life of 20.4 minutes at pH 8, 25°C.

The breakthrough came when an assay of reaction mixtures with varying reagent concentrations was

assessed. Our 25% yield plateau was shown to be only a temporary phase, as increasing the concentration of

amino-modified ZMYK-23 to 4 to 10 µM pushed the PEGylation yield up to ~75% (see figure 15B and

15C). Interestingly, the correlation between reagent concentration and yield in this reaction is not

consistent, as figure 16 reveals that at increasing aptamer concentration above ~30 µM causes a drastic

reduction in PEGylation yield. The reason for this effect is not known, but some speculations can be made.

Le Chatalier’s Principle cannot explain the increased PEGylation yield at high reagent concentrations, as

both the aptamers and PEG concentrations were increased. It is possible that the PEGylation reaction is

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completing with mPEG-SC degradation, and by increasing reagent concentration, the number of collisions

between the aptamer’s amino group and the succinimidyl carbonate group on the PEG molecule is

increased and PEGylation yield in turn increases. The lack of reactivity at high reagent concentrations is

likely due to the effect of PEG on solution viscosity and reagent mobility, and possibly also due to inter-

aptamer complexation, leading to steric hindrance preventing the reaction between the aptamer’s amino

group and the PEG’s succinimidyl carbonate group.

Once a reasonable PEGylation yield could be achieved, the next step was to isolate and purify the

PEGylated aptamer. Many effective methods exist for the purification of unmodified nucleic acids.

However, the bulky PEG moiety on PEG-ZMYK-23 alters its physical and chemical properties, making

many conventional nucleic acid purification methods non-ideal, and making the task of PEG-ZMYK-23

isolation quite challenging. PEG-modified pharmaceuticals are typically isolated using preparative High

Performance Liquid Chromatography (prep HPLC) 17. However, prep HPLC facilities are expensive and

difficult to find. I hoped to devise a way of isolating an adequate amount of PEGylated aptamer for in vitro

experiments, and possibly look into prep HPLC if in vitro experiments were successful and in vivo

experiments were a logical next step.

The purification technique I found was electroelution, a fairly uncommon technique in which a slice of gel

from gel electrophoresis containing the desired biomolecule is subjected to an electric current, causing the

biomolecule in question to migrate out of the gel 21. Unlike most DNA purification techniques, which rely

on the solubility characteristics of DNA, electroelution relies only on charge to isolate DNA. This makes it

an ideal method for isolating nucleic acids that have been modified with bulky moieties such as 20 kDa PEG.

Figure 17 in the appendix shows the result of electroelution of PEGylated ZMYK-23. 50 µL 200 nM

PEGylated ZMYK-23 was successfully produced. While this was a good result, the total amount of

PEGylated aptamer collected was modest, and would not be sufficient for in vivo experimentation.

Therefore, we kept prep HPLC in mind as a possibility in the future.

Once we had developed a method for producing and isolating PEGylated aptamer, we moved on to cell-

based experimentation. The first step was to titre polyclonal anti-VSV∆51 nAbs, to determine an

appropriate dilution to use in cell-based viral infectivity assays. Unfortunately, the stock of anti-VSV∆51

nAbs previously used by Berezovski research group in VSV∆51 AptaVIP experiments, which had been

produced by bunny #2, has denatured and was no longer biologically active. We received a new stock of

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anti-VSV∆51 nAbs, produced by bunny #3, from Dr. John Bell and did all subsequent experimentation

with that stock. Results of the titring experiment are displayed in figure 18 in the appendix. They show that

a dilution of 1/5000 is the smallest concentration of nAbs capable of eliminating all infectivity from

VSV∆51 under the experimental conditions employed, therefore 1/5000 the ideal dilution of nAbs to use

in future experimentation.

Next, cell-based viral infectivity assays were performed to verify that unmodified aptamers were capable of

shielding VSV∆51 from nAbs. The results of this experiment can be found in figure 19 in the appendix.

This experiment was repeated many times under many different experimental conditions, but at no point

did the VSV-binding aptamers previously selected by Berezovski research group display any capacity to

shield VSV∆51 from nAbs. This result was disappointing and quite surprising. The aptamers in question had

previously been shown to perform VSV∆51 AptaVIP effectively in cell-based viral infectivity assays 14,

therefore it was assumed that they would continue to work throughout this project.

To attempt to explain this unexpected outcome, we considered the variables present in the experiment,

and what had changed between my cell-based viral infectivity assays and equivalent ones previously

performed in Berezovski lab. The most significant variable seemed to be the nAbs. The aptamers used in the

experiment had been selected using anti-VSV∆51 nAbs isolated from bunny #2 in Dr. John Bell’s lab.

However, I had performed cell-based viral infectivities using nAbs from a different animal, bunny #3. We

hypothesized that nAbs from the different animals may bind to different epitopes on the virus. If this was

the case, it is possible that while the aptamers being used could shield VSV∆51 from nAbs produced by

bunny #2, nAbs from other animals would not be hindered by those aptamers.

To test this idea, we performed a flow cytometric displacement assay. In this assay, VSV∆51 particles were

first incubated with a fluorescently labelled polyclonal mixture of VSV∆51-binding aptamers, and the

extent of binding was measured with flow cytometry. Then nAbs from bunny #3 were added to the

VSV∆51 and aptamer-containing sample, and flow cytometry was used to determine to what extent the

nAbs could displace the aptamers. A high level of displacement would indicate that the aptamers and the

nAbs from bunny #3 bound to the same epitopes on VSV∆51. However, if the VSV particles were still

bound to a significant amount of fluorescent aptamer after being incubated with nAbs, it is likely that the

nAbs bind to different epitopes than the aptamers. The flow cytometric histogram is displayed in figure 20

in the appendix.

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The extent of aptamer binding to the virus decreases after exposure to nAbs from bunny #3, but there is

still a significant amount of fluorescent aptamer still attached to VSV∆51 after incubation with nAbs.

Though this result is not incontrovertible, it is suggestive that the polyclonal aptamer mixture employed

binds to several different epitopes on the surface of VSV∆51 than do the anti-VSV∆51 nAbs isolated from

bunny #3. Some factors that might have contributed to the decreased level of fluorescence detected on the

VSV∆51 after incubation with nAbs include loss of aptamer from the mixture from the extra wash

following nAbs incubation, as well as some overlap in epitopes between the polyclonal aptamer mixture and

the polyclonal nAbs.

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Conclusions

We were unable to demonstrate the feasibility of using PEGylated VSV∆51-binding aptamers in AptaVIP,

thus we failed to achieve the goal of this research project. We did, however, glean a good deal of interesting

information and hopefully contribute to future research prospectives. The development of a method for

producing and isolating PEGylated aptamers will likely be valuable to future research projects. PEGylation

is a fairly common process in aptamer research, therefore it is not unreasonable to expect that in the future

a researcher with connections to Berezovski research group may be able to use our PEGylation

experimental results to successfully inform their own PEGylation experimental design.

The cell-based viral infectivity assay and flow cytometric displacement assay also yielded fascinating results

worth investigating further in the future. They demonstrated that the VSV∆51-binding and protecting

aptamers are not necessarily able to protect against nAbs they had not been selected to protect against. This

results encourages us to explore the binding that occurs between nAbs, VSV∆51-binding aptamers, and

epitopes on the surface of VSV∆51. The flow cytometric results suggest that there are differences between

the sites that known VSV∆51-binding aptamers bind and the epitopes that polyclonal nAbs from bunny #2

bind, however we have not determined if there are any overlapping sites. In the future, flow cytometric

displacement assays that analyze the displacement of individual monoclonal VSV∆51-binding antibodies by

anti-VSV∆51 nAbs produced by a variety of different animals could potentially yield extremely fascinating

and valuable information. Such experiments could determine which aptamers are the most effective at

protecting VSV∆51 from nAbs from a wide range of animals, which could eventually lead to development

of highly effective, versatile aptamer pools for VSV∆51 AptaVIP that could potentially be used clinically. It

also opens up the possibility of research directly into the epitopes that anti-VSV∆51 nAbs bind to. Such

research is within the field of proteomics and is quite ambitious, but discovery of epitopes important to

VSV∆51 neutralization makes possible novel research into designing smarter, more targeted methods for

extending VSV∆51’s in vivo half-life and increasing VSV∆51’s therapeutic potential.

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Materials & Methods

Polyethylene glycol (mPEG-succinimidyl carbonate 20 kDa) was obtained for Laysan Bio Inc. (Arab, USA).

All aptamers were obtained from Integrated DNA Technologies (Coralville, USA). Aptamer sequences are

displayed in table 1 below.

Table 1: List of all VSV-binding aptamer sequences used in this research. All sequences were discovered by members of

Berezovski research group.

Aptamer name Aptamer sequence (5’ – 3’)

ZMYK-21 CTC CTC TGA CTG TAA CCA CGC GGG AAC CAA ATC ACG TCC TAG

ATT GTG ATG AAC CTC GGC GCA TAG GTA GTC CAG AAG CC

ZMYK-22 CTC CTC TGA CTG TAA CCA CGG CGA CAA CAC GGA CGG TTG AGA

CTT TAA TTC TGC TCA CGG GCA TAG GTA GTC CAG AAG CC

ZMYK-23 CTC CTC TGA CTG TAA CCA CGG GGA CCT ATC AGG CGA TGT GAA

AAC TCT TAT ACC ACT GGG CAT AGG TAG TCC AGA AGC C

ZMYK-29 CTC CTC TCT GTA ACC ACG CAC ATC CTA CGT TTG CCA CGC GCT

ACT CCG CCA TCT ACC CGC ATA GGT AGT CCA GAA G

MS-50 CTC CTC TGA CTG TAA CCA CGC CAT CAC CCT ATT ATC TCA TTA

TCT CGT TTT CCC TAT GCG GCA TAG GTA GTC CAG AAG CC

SS-31 CTC CTC TGA CTG TAA CCA CGT GAC CCG AGA TTC TAG TGA TTG

CTT GTT CGG TAT GTT CGG CAT AGG TAG TCC AGA AGC C

SS-37 CTC CTC TGA CTG TAA CCA CGG CAT AGC GGG GGA GAT GGG GGA

TGA CTT GGG TGT GAT GGG GCA TAG GTA GTC CAG AAG CC

SS-39 CTC TCC TCT GAC TGT AAC CAC GGC ACT TCA CTT CTC CTC TGA

CTG TAA CCA CGC GCA TAG GTA GTC CAG AAG CA

Note that aptamers that were ordered with the intention of being conjugated with PEG were modified with

an amino group on their 3’ terminal.

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Gel electrophoresis experiments were performed in 0.5X TAE buffer (20 mM Tris, 10 mM acetic acid, and

0.5 mM EDTA), which had been prepared in-lab. UltraPureTM Agarose was obtained from Invitrogen

(Carlsbad, USA). Gels were visualized using the Fluorochem® Q from Alpha Innotech (Santa Clara, USA).

Several products were purchased for attempted PEGylated aptamer purification. VWR® Centrifugal Filters

(modified PES, 30K, 500 µL) were obtained from VWR International (Radnor, USA). ZebaTM Spin

Desalting Columns (40K MWCO, 2 mL) were obtained from Thermo Scientific (Waltham, USA). D-TubeTM

Dialyser Midi MWCO 6-8 kDa tubes were used for electroelution and were obtained from EMD Millipore

(Billerica, USA).

Original samples of Vero cells, VSV∆51-YFP, and polyclonal anti-VSV neutralizing antibodies from bunny

#2 were obtained from Dr. John C. Bell’s laboratory at the Ottawa Hospital Research Institute (Ottawa,

Canada). Samples of VSV∆51-YFP (conc. 1×1010 pfu/mL) were prepared by myself and Darija

Muharemagic by infecting Vero cells with VSV∆51-YFP and purifying the virus through sucrose cushion

purification, as described in Diallo et al., 2012 22.

The medium used for all cell cultures was Dulbecco’s Modified Eagle Medium (DMEM) + 10% FBS

(HyClone, Thermo Scientific, Waltham, USA), and the cultures were grown in HeracellTM 150i CO2

incubator (Thermo Scientific, Waltham, USA). Vero cell cultures were grown and maintained according to

Ammerman et al., 2008 23 with the exception that the cultures were grown in 150 mm dishes from BD

Biosciences (Franklin Lakes, USA) rather than 75 cm2 cell cultures flasks.

Flow cytometric analysis was performed using the Beckman Coulter Cytomics FC500 and flow cytometric

data was analysed using Kaluza® Flow Analysis Software from Beckman Coulter (Mississauga, Canada). Yeast

ribonucleic acid was obtained from Calbiochem® (San Diego, USA). Dulbecco’s phosphate buffered saline

(DPBS) with low Mg2+ and low Ca2+ (HyClone, Thermo Scientific, Waltham, USA).

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Optimization of Conjugation of PEG to ZMYK-23

First Trial – Determining the Effect of Reaction Time on Aptamer PEGylation

The reaction mixture was a solution of 2 μM ZMYK-23 (5’ FAM, 3’ amino) and 200 μM mPEG-SC 20K in

DPBS. One reaction mixture was prepared and incubated at 30°C and 400 rpm for 4 hours, and a second

reaction mixture was prepared and incubated at 30°C and 400 rpm for 20 hours. The PEGylation results

were assessed through agarose gel electrophoresis. The gel was 1.5% agarose in 0.5X TAE buffer and was

run at 150 V for 30 minutes. The gel image is annotated in figure 9 in the appendix.

Scheme 2: Mechanism of conjugation between primary amine and succinimidyl carbonate

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Determining the Effect of Reaction Mixture Buffer and pH on Aptamer PEGylation – Acetonitrile and

Sodium Carbonate


a solvent system of one half acetonitrile (commonly written as MeCN), one half sodium carbonate buffer

(100 mM Na2CO3, pH 11). It was incubated at ambient temperature for one hour, then characterized using

agarose gel electrophoresis. The gel was 1.5% agarose in 0.5X TAE buffer and was run at 150 V for 25

minutes. The gel image is annotated in figure 10 in the appendix.


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Determining the Effect of Reaction Mixture Buffer and pH on Aptamer PEGylation – Acetonitrile and

Borate Buffered Saline


a solvent system of one half acetonitrile, one half borate buffered saline (BBS) solution (10 mM Na2B4O7,

150 mM NaCl, pH 9). It was incubated at ambient temperature for one hour, then characterized using

agarose gel electrophoresis. The gel was 1.5% agarose in 0.5X TAE buffer and was run at 200 V for 25

minutes. The gel image is annotated in figure 11 in the appendix.


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Determining the Effect of Acetonitrile Concentration on Aptamer PEGylation

An assay of PEGylation reaction mixtures with varying concentrations of acetonitrile was assembled. The

reaction mixtures were solutions of 1 μM ZMYK-23 (5’ FAM, 3’ amino) and 100 μM mPEG-SC 20K in a

solvent systems of acetonitrile and BBS (10 mM Na2B4O7, 150 mM NaCl, pH 9). Three different

acetonitrile to BBS ratios were tested – 1:3, 1:1, and 3:1. The reaction mixtures were incubated at ambient

temperature for one hour, then characterized using agarose gel electrophoresis. The gel was 1.5% agarose

in 0.5X TAE buffer and was run at 200 V for 25 minutes. The gel image is annotated in figure 12 in the

appendix.


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Determining the Effect of Heat and Agitation on Aptamer PEGylation



150 mM NaCl, pH 9). It was incubated 50°C, 300 rpm in an Eppendorf Thermomixer for one hour, then

characterized using agarose gel electrophoresis. The gel was 1.5% agarose in 0.5X TAE buffer and was run

at 200 V for 20 minutes. The gel image is annotated in figure 13 in the appendix.


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Determining the Effect of Different Organic Solvents of Aptamer PEGylation

Two reaction mixtures were prepared. The first was a solution of 1 μM ZMYK-23 (5’ FAM, 3’ amino) and

100 μM mPEG-SC 20K in a solvent system of one half DMSO, one half borate buffered saline (BBS)

solution (10 mM Na2B4O7, 150 mM NaCl, pH 9). The second was a solution of 1 μM ZMYK-23 (5’ FAM,

3’ amino) and 100 μM mPEG-SC 20K in a solvent system of one half acetone, one half borate buffered

saline (BBS) solution (10 mM Na2B4O7, 150 mM NaCl, pH 9). They were incubated at ambient

temperature for one hour, then characterized using agarose gel electrophoresis. The gel was 1.5% agarose

in 0.5X TAE buffer and was run at 200 V for 30 minutes. The gel image is annotated in figure 14 in the

appendix.


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Determining the Effect of Reagent Concentration on Aptamer PEGylation

Three reaction mixtures were prepared. The first was a solution of 2 μM ZMYK-23 (5’ FAM, 3’ amino)

and 200 μM mPEG-SC 20K. The second was a solution of 4 μM ZMYK-23 (5’ FAM, 3’ amino) and 400

μM mPEG-SC 20K. The third was a solution of 10 μM ZMYK-23 (5’ FAM, 3’ amino) and 1 mM mPEG-

SC 20K. The solvent system for all three mixtures was one half acetonitrile, one half borate buffered saline

(BBS) solution (10 mM Na2B4O7, 150 mM NaCl, pH 9). They were each incubated for one hour at ambient

temperature, then characterized using agarose gel electrophoresis. The gels were 1.5% agarose in 0.5X

TAE buffer and were each run at 200 V for 20 minutes. The gel images are annotated in figure 15 in the

appendix.


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Determining the Maximum Reagent Concentration for Effective Aptamer PEGylation

Six reaction mixtures, all containing a solvent system of one half acetonitrile, one half borate buffered saline

(BBS) solution (10 mM Na2B4O7, 150 mM NaCl, pH 9) were prepared. Their reagent concentations are as

follows:

1. 13.3 μM ZMYK-23 (5’ FAM, 3’ amino) and 12 mM mPEG-SC 20K.

2. 21.5 μM ZMYK-23 (5’ FAM, 3’ amino) and 19 μM mPEG-SC 20K.





They were each incubated for one hour at ambient temperature, then diluted to a final aptamer

concentration of 200 nM and characterized using agarose gel electrophoresis. The gel was 1.5% agarose in

0.5X TAE buffer and was run at 200 V for 20 minutes. The gel image is annotated in figure 16 in the

appendix.


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Isolation of PEGylated ZMYK-23 Through Electroelution

The reaction mixture was a solution of 40 μM ZMYK-23 (5’ FAM, 3’ amino) and 2 mM mPEG-SC 20K in


150 mM NaCl, pH 9). It was incubated at 40°C for 90 minutes.

50 µL of the reaction mixture was loaded onto a 1.5% agarose gel in 0.5X TAE buffer and run at 150V for

45 minutes. The slice of gel containing the PEGylated aptamer was excised and placed into a D-TubeTM

Dialyser Midi MWCO 6-8 kDa tube along with 700 µL of 0.5X TAE buffer. Electroelution was performed

in a n electrophoresis developing tank at 150V for 25 minutes, then the polarity was reversed and it was run

at 125V for another 2 minutes.

The solution collected from electroelution was concentrated using a VWR® Centrifugal Filters (modified

PES, 30K, 500 µL). The solution was placed in a VWR® filter, which was then centrifuged for 10 minutes

at 3800 rcf. 50 µL DPBS was placed on the top of the column, allowed to incubate at ambient temperature

for 5 minutes, then collected.

The concentrated, purified PEG-ZMYK-23 solution was characterized using agarose gel electrophoresis. 2

µL of the solution was diluted to 10 µL with ddH2O and was loaded onto a 1.5% agarose gel in 0.5X TAE

buffer. 10 µL 200 nM FAM-labelled DNA library was loaded into another well as a control. The gel was

run at 200 V for 20 minutes. The gel image is annotated in figure 17 in the appendix.

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Viral Infectivity Assays

Titring Neutralizing Antibodies

Two 12-well plates containing ~95% confluent Vero cells in DMEM+10% FBS were prepared.

A series of solutions containing VSV∆51-YFP and polyclonal anti-VSV neutralizing antibodies (nAbs) in

DMEM were prepared. All solutions contained 400 pfu/mL VSV∆51-YFP, as well as various dilutions of

nAbs. The nAbs dilutions were as follows: 1/10000, 1/7500, 1/5000, 1/2500, 1/1250, 1/625, and

1/312.5, and 0 (no nAbs). The VSV∆51-YFP/nAbs solutions were each incubated at 37°C for one hour.

The medium was removed from the Vero cell-containing 12-well plates. 250 µL of each VSV∆51-

YFP/nAbs solution was added to each of 3 wells. The plates were then incubated at 37°C, 5% CO2 for one

hour.

An agarose overlay solution was prepared as follows: 15 mL of 1% agarose in ddH2O was heated until

homogenous. 15 mL 2X DMEM+10% FBS was added to the agarose solution. 1 mL of the solution was

gently placed on top of each well of the two 12-well plates. The plates were then incubated at 37°C, 5%

CO2 for 24 hours. Images of the plates were taken using the Fluorochem® Q from Alpha Innotech, and are

annotated in figure 18 in the appendix.

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Viral Infectivity Assay – Verifying Aptamer Pool’s Ability to Shield VSV from nAbs in vitro

Three 12-well plates containing ~95% confluent Vero cells in DMEM+10% FBS were prepared.

A 100 µM polyclonal aptamer pool containing equimolar amounts of the following VSV-binding aptamers

ZMYK-20, ZMYK-23, ZMYK-29, MS-50, SS-31, and SS-39 was prepared.

A series of solutions containing VSV∆51-YFP and aptamers were prepared. First, 30 µL 1.04e4 pfu/mL

VSV∆51-YFP was placed into each of 10 tubes (312 pfu per tube). Then, varying amounts of VSV-binding

aptamer was added to each tube as follows:

1. No aptamer

2. 8 µL 100 µM polyclonal aptamer pool (0.8 nmol total aptamer)




6. No aptamer

7. 8 µL 100 µM unlabelled ZMYK-23 (0.8 nmol total aptamer)




The tubes were incubated at 37°C for one hour. Following incubation, 30 µL of a 1/80 nAbs dilution in

DMEM was added to each of the 10 tubes, and they were again incubated at 37°C, 5% CO2 for one hour.

DMEM was added to the tubes such that each tube contained a total of 800 µL of liquid, then the tubes

were again incubated at 37°C, 5% CO2 for one hour.

The medium was removed from the Vero cell-containing 12-well plates. 250 µL of each VSV∆51-

YFP/aptamer/nAbs solution was added to each of 3 wells. The plates were then incubated at 37°C, 5%

CO2 for one hour.

An agarose overlay solution was prepared as follows: 15 mL of 1% agarose in ddH2O was heated until

homogenous. 15 mL 2X DMEM+10% FBS was added to the agarose solution. 1 mL of the solution was

gently placed on top of each well of the two 12-well plates. The plates were then incubated at 37°C, 5%

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CO2 for 24 hours. Images of the plates were taken using the Fluorochem® Q from Alpha Innotech, and are

annotated in figure 19 in the appendix.

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Flow Cytometric Competitive Binding Assay

A solution containing equimolar amounts of the 5’ FAM-labelled VSV-binding aptamers MS-50, SS-31, SS-

37, and SS-39, with a total aptamer concentration of 400 nM was prepared in DPBS.

Four tubes, each containing 2e9 pfu VSV∆51-YFP and 0.1 mg/mL yeast RNA in DPBS were prepared and

incubated at ambient temperature for 30 minutes. The tubes were labelled A-, A+, B-, and B+. The tubes

were centrifuged at 17200 rcf for 20 minutes, then the pellets were isolated.

50 µL of the 400 nM polyclonal VSV-binding aptamer solution was added to tubes A+ and B+. 50 µL

DPBS was added to A- and B-. The four tubes were again incubated at ambient temperature for 30 minutes,

centrifuged at 17200 rcf for 20 minutes, and the pellets were isolated.

The pellets of tubes B+ and B- were resuspended in 50 µL 2 mg/mL nAbs in DPBS and incubated at 37°C

for one hour. Tubes A+ and A- were resuspended in 300 µL DPBS and analysed with flow cytometry while

tubes B+ and B- were incubating with nAbs.

Following incubation of tubes B+ and B-, they were centrifuged at 17200 rcf for 20 minutes, then their

pellets were isolated and resuspended in 300 µL DPBS, and they were analysed with flow cytometry.

Histograms of the flow cytometric data compiled were analyzed using Kaluza® Flow Analysis Software from

Beckman Coulter and are displayed as figure 20 in the appendix.

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Vero Cell SELEX

Background

Aptamer research is still in its youth, and a wealth of new potential aptamer targets have yet to be explored.

In this project aptamer pools were selected for Vero cells, a novel target, through cell-SELEX.

Vero cells are one of the most widely used cell lines in research. Originally derived from the kidney

epithelial cells of an African green monkey (Cercopithecus aethiops), Vero cells are an anchorage-dependent,

continuous cell line. They have been invaluable in virology, parasitology, and oncology research since the

1960s 23. In Berezovski research group, Vero cells are an essential component of in vitro research into

oncolytic viruses.

The discovery of Vero cell-binding aptamers will serve as a proof of concept and will have many potential

benefits. One application of aptamers with high binding affinity to Vero cells is use in Aptamer-facilitated

BIomarker Discovery (AptaBID). In AptaBID, aptamers with high affinity and specificity to a particular cell

line are selected and used to discover cell surface biomarkers unique to the cell line 24. Cell surface

biomarkers are proteins that are expressed on the surfaces of some cells, and can be indicative of a cell’s

type, susceptibility to disease, or disease state. In recent years their use has grown in popularity, as they

have proven themselves to be invaluable tools in diagnostic medicine, with applications such as cancer

diagnosis and prognosis determination 25. AptaBID is accomplished by first selecting aptamers with the

desired binding characteristics, then isolating the aptamer-bound biomarker by lysing cells, and finally

analyzing the biomarker with mass spectroscopy to determine its structure 24.

Another application for Vero cell-binding aptamers is as a key component of a proof-of-concept experiment

dealing with enhanced VSV∆51 delivery currently planned in Berezovski research group. In the proposed

project, VSV∆51-binding aptamers and Vero cell-binding aptamers will be linked together via a bridging

oligonucleotide, which contains complementary sequences to both the virus-binding aptamer and the cell-

binding aptamer. Theoretically, by connecting the two aptamers, VSV∆51 will be able to reach its potential

host cells more rapidly and selectively, and will therefore be in circulation for a shorter amount of time, be

less likely to be neutralized, and be more potent. The development of Vero-cell binding aptamers is an

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essential component to this proof-of-concept project, and if the proof-of-concept is successful, similar

methods could one day be used clinically with human cancer cell-binding aptamers.

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Although this project was the shortest and the simplest of the three that make up my honours project, its

results were extremely promising. Unfortunately, due to time constraints, a full 8-12 rounds of SELEX plus

sequencing was not completed as hoped. Instead, five rounds of SELEX were performed and the resulting

oligonucleotide pools’ binding affinity to Vero cells was assessed through flow cytometry.

The procedure for the five rounds of selection that were performed were based on a protocol developed by

Shahrokh Ghobabloo, a fellow member of Berezovski research group. The rounds were successful,

producing adequate amounts of 100 nt oligonucleotide for analysis and subsequent analysis. Following the

five rounds of selection, oligonucleotide pools and a sample of Harvard DNA library were diluted to 25 nM

for use in flow cytometric analysis.

Results from the flow cytometric binding assay can be found in figure 21 in the appendix. The flow

cytometric histogram clearly shows that the oligonucleotide pools obtained through SELEX have increased

binding affinity to Vero cells compared to DNA library. All five pools show good binding affinity to Vero

cells, interestingly with the pools from rounds 1 and 2 having the weakest binding of the 5 rounds, and

round 3’s pool having the strongest. It is expected that each subsequent pool would have superior binding

than its predecessor, but that was not the case in this experiment. Though this results is unexpected, it is

not terribly concerning because the difference in binding between round 3’s and round 5’s pools is very

modest, and a number a variables can influence flow cytometric results on a small scale. This unexpected

result could be the result of slight oligonucleotide concentration differences, or differences in the way each

flow cytometry sample was handled (extent of washing, incubation time, heat, etc.).

Although the difference in Vero cell affinity between the DNA library and the SELEX-produced

oligonucleotide pools is modest, it is a promising and exciting results. A greater shift would have been

unlikely for two reasons – only five rounds of SELEX were performed, and the concentration of the

oligonucleotide pools used was quite low. Unfortunately, time constraints made it unfeasible to amplify,

isolate, and purify the five oligonucleotide pools obtained for a second time to obtain a greater mass of

DNA. Therefore we worked at a low oligonucleotide concentration during the experiment with flow

cytometry.

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Conclusions

This experiment is only partially complete, but it holds a great deal of promise for the future. The next

steps for this project will be to repeat the flow cytometric binding assay, this time using oligonucleotide

pools with a DNA concentration greater than 25 nM. This will provide greater and more valid insight into

the binding characteristics of each pools. Following that, SELEX will be completed. Five more rounds of

Vero cell SELEX should ideally be performed. In addition, three rounds of negative selection, which

involved selecting for the oligonucleotides that do not bind to a different cell line, should ideally be

performed to ensure that the aptamers discovered are specific to Vero cells. The resulting aptamer pools

should eventually be sequenced, synthesized, and again have their Vero-cell binding capacity assessed

through flow cytometry.

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Materials & Methods

Primers and Harvard DNA library were obtained from Integrated DNA Technologies (Coralville, USA). The

Harvard DNA library consists of a mixture of DNA oligonucleotides, each composed of a 60 nucleotide

semi-random sequence flanked by constant 20 nucleotide sequences at each end that serve as primer

regions. The forward primer (5’-CTC CTC TGA CTG TAA CCA CG-3’) was labelled with Cy5 on its 5’

end, whereas the reverse primer (5’-GGC-TTC-TGG-ACT-ACC-TAT-GC-3’) was modified with a reversed

5’ phosphate group.

Original samples of Vero cells were obtained from Dr. John C. Bell’s laboratory at the Ottawa Hospital

Research Institute (Ottawa, Canada). The medium used for all cell cultures was Dulbecco’s Modified Eagle

Medium (DMEM) + 10% FBS (HyClone, Thermo Scientific, Waltham, USA), and the cultures were grown

in HeracellTM 150i CO2 incubator (Thermo Scientific, Waltham, USA). Vero cell cultures were grown and

maintained according to Ammerman et al., 2008 22 with the exception that the cultures were grown in 150

mm dishes from BD Biosciences (Franklin Lakes, USA) rather than 75 cm2 cell cultures flasks.

KAPA2G Robust Hotstart PCR Kit from Kapa Biosystems (Wilmingtom, USA) was used to amplify

aptamer pools. Gel electrophoresis experiments were performed in 1X modified TAE buffer (40 mM Tris -

Acetate 100 µM EDTA, pH 8.2), which had been prepared in-lab. The vapo.protect Mastercycler proS

from Eppendorf (Hamburg, Germany) was used as the thermocycler in all PCRs. UltraPureTM Agarose was

obtained from Invitrogen (Carlsbad, USA). GelRedTM for visualizing bands on agarose gels was obtained

from Biotium (Hayward, USA). Gels were visualized using the Fluorochem® Q from Alpha Innotech (Santa

Clara, USA).

Amicon Ultra Filters (3 kDa, 2 mL) for cell lysate concentration and Ultrafree-DA Centrifugal Filter Units

were obtained from EMD-Millipore (Billerica, USA). Lambda Exonuclease and buffer were obtained from

New England Biolabs (Ipswitch, USA).

Flow cytometric analysis was performed using the Beckman Coulter Cytomics FC500 and flow cytometric

data was analysed using Kaluza® Flow Analysis Software from Beckman Coulter (Mississauga, Canada).

Dulbecco’s phosphate buffered saline (DPBS) with low Mg2+ and low Ca2+ was obtained from HyClone

(Thermo Scientific, Waltham, USA).

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Procedure for Vero cell SELEX

1. Prepare cells: Vero cells are cultured in 150 mm plates in DMEM + 10% FBS and grown to 85-100%

confluence prior to selection.

2. In low Ca2+, low Mg2+ DPBS, prepare Harvard DNA library (2000 μL, 2 μM) for the first round of

selection, or DNA pool (1000 μL, target concentration 500 ng/mL) for subsequent rounds by mixing

and heating at 95°C for 10 minutes, then cooling on ice for 5 minutes. This ensures the formation of

folded ssDNA.

3. Remove the medium from the plate containing Vero cells and gently apply the DNA library or pool.

Shake at 200 rpm, 37°C for 5 minutes.

4. Remove the liquid from the plate and washed three times with low Ca2+, low Mg2+ DPBS.

5. Apply 1500 μL 10 mM EDTA in low Ca2+, low Mg2+ DPBS to the plate. Incubate at 37°C, 5% CO2

for 10 minutes.

6. Pipette the liquid up and down on the plate to thoroughly detach Vero cells from the surface of the

plate (this takes about 10 minutes). Collect the liquid suspension.

7. Heat the suspension at 95°C for 10 minutes, then centrifuge at 13,100 rcf for 5 minutes and collect

the supernatant.

8. Using an Amicon Ultra Filter (3k, 2 mL), concentrate the lysate to 50 μL.

9. Prepare PCR mixture with forward Cy5-labelled and reversed 5’-phosphate group primers as follows:

1X KAPA GC buffer with 1.5 mM MgCl2

500 nM each primer

500 μM dNTPs

0.02 UμL-1 KAPA2G Robust HotStart DNA Polymerase

1/50 dilution of concentrated cell lysate (from step 8)

10. Run the following PCR program:

2 minutes at 95°C

35 cycles of:

5 seconds at 95°C

20 seconds at 56°C

10 seconds at 72°C

Hold at 4°C

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11. Load 50 μL of the PCR solution into each well of a 3% agarose + 1X GelRedTM gel in 1X modified

TAE buffer. Run the gel at 150 V for 45 minutes.

12. Visualize bands on the gel under UV light using the Fluorochem® Q and excise the gel slices

containing the 100 nucleotide dsDNA fragments. Purify using Ultrafree-DA Centrifugal Filter Units.

13. Confirm the product by running 10 μL of the purified dsDNA on a 3% agarose gel in 1X modified

TAE at 150 V for 25 minutes

14. Digest the product with Lambda exonuclease according to the protocol provided by New England

Biolabs.

The preceding protocol was repeated five time, resulting in five aptamer pools for Vero cells. The binding

affinity of these pools to Vero cells was assessed using flow cytometry.

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Procedure for Flow Cytometric Binding Assay

A 150 mm plate containing 95% confluent Vero cells was trypsinized and resuspended in 10 mL DMEM +

10% FBS. 500,000 cells were plates into each of 7 1.5 mL tubes, which were then centrifuged at 500 rcf for

5 minutes. The supernatant was removed from each tubes, then the tubes were labelled A-G, and the

following was added to each:

A. 200 µL low Ca2+, low Mg2+ DPBS

B. 200 µL 25 nM Cy5-labelled Harvard DNA library in low Ca2+, low Mg2+ DPBS

C. 200 µL 25 nM Cy5-labelled round 1 Vero cell SELEX aptamer pool in low Ca2+, low Mg2+ DPBS

D. 200 µL 25 nM Cy5-labelled round 2 Vero cell SELEX aptamer pool in low Ca2+, low Mg2+ DPBS

E. 200 µL 25 nM Cy5-labelled round 3 Vero cell SELEX aptamer pool in low Ca2+, low Mg2+ DPBS

F. 200 µL 25 nM Cy5-labelled round 4 Vero cell SELEX aptamer pool in low Ca2+, low Mg2+ DPBS

G. 200 µL 25 nM Cy5-labelled round 5 Vero cell SELEX aptamer pool in low Ca2+, low Mg2+ DPBS

The tubes were incubated at 37°C for 5 minutes, then centrifuged at 500 rcf for 5 minutes. The

supernatants were removed, all pellets were resuspended in 200 µL low Ca2+, low Mg2+ DPBS, and

centrifuged at 500 rcf for 5 minutes. Once again, the supernatants were removed. Then each pellet was

resuspended in 500 µL low Ca2+, low Mg2+ DPBS and each suspension was analyzed using flow cytometry.

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References

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4. Jayasena, Sumedha D. "Aptamers: an emerging class of molecules that rival antibodies in

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13. Stojdl, David F., et al. "Exploiting tumor-specific defects in the interferon pathway with a

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Appendix

Figures

FIGURE 2: AGAROSE GEL IMAGE OF LIMIT OF DETECTION ASSAY FOR SYMMETRIC-THEN-ASYMMETRIC PCR. 10 µL OF

EACH SYMMETRIC-THEN-ASYMMETRIC PCR MIXTURE WAS LOADED INTO A WELL. IN ADDITION, 5 µL MASSRULER LOW

RANGE DNA LADDER, READY-TO-USE FROM THERMO SCIENTIFIC WAS LOADED INTO THE CENTRE WELL. THE GEL WAS

3% AGAROSE IN 0.5X TAE BUFFER, AND WAS RUN AT 150 V FOR 45 MINUTES. THE GEL WAS VISUALIZED USING THE

FLUOROCHEM® Q FROM ALPHA INNOTECH ON THE CY2 FLUORESCENCE SETTING. FULL PROCEDURAL INFORMATION

CAN BE FOUND ON PAGES 11-12.

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FIGURE 3: AGAROSE GEL IMAGE OF FORWARD TO REVERSE PRIMER RATIO PLUS ANNEALING TEMPERATURE ASSAY, THE

FIRST EXPERIMENT OF LATE-PCR PROTOCOL OPTIMIZATION. FOLLOWING PCR, 10 µL OF EACH SAMPLE WAS LOADED

INTO A WELL, GROUPED ACCORDING TO THEIR PRIMER RATIOS. NOTE THAT A NEGATIVE CONTROL (I.E. NO TEMPLATE

DNA) FOR EACH PRIMER RATIO WAS PERFORMED, AND IS HERE DENOTED BY A NEGATIVE SYMBOL (-). IN ADDITION, 5

µL MASSRULER LOW RANGE DNA LADDER, READY-TO-USE FROM THERMO SCIENTIFIC WAS LOADED INTO A WELL.

THE GEL WAS 3% AGAROSE IN 0.5X TAE BUFFER, AND WAS RUN AT 150 V FOR 45 MINUTES. THE GEL WAS VISUALIZED

USING THE FLUOROCHEM® Q FROM ALPHA INNOTECH ON THE CY3 FLUORESCENCE SETTING. FULL PROCEDURAL

INFORMATION CAN BE FOUND ON PAGE 13.

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FIGURE 4: AGAROSE GEL IMAGE OF LATE-PCR CYCLE NUMBER PLUS ANNEALING TEMPERATURE ASSAY, THE SECOND

EXPERIMENT OF LATE-PCR PROTOCOL OPTIMIZATION. EACH PCR SAMPLE INITIALLY CONTAINED 17 NM TEMPLATE

DNA. FOLLOWING PCR, 10 µL OF EACH SAMPLE WAS LOADED INTO A WELL, GROUPED ACCORDING TO THE NUMBER

OF PCR CYCLES THEY HAD BEEN SUBJECT TO. THE GEL WAS 3% AGAROSE IN 0.5X TAE BUFFER, AND WAS RUN AT 150

V FOR 55 MINUTES. THE GEL WAS VISUALIZED USING THE FLUOROCHEM® Q FROM ALPHA INNOTECH ON THE CY2

FLUORESCENCE SETTING. FULL PROCEDURAL INFORMATION CAN BE FOUND ON PAGE 14.

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FIGURE 5: AGAROSE GEL IMAGE OF LATE-PCR CYCLE NUMBER PLUS ANNEALING TEMPERATURE NEGATIVE CONTROLS.

NONE OF THE PCR SAMPLES CONTAINED ANY TEMPLATE DNA. FOLLOWING PCR, 10 µL OF EACH SAMPLE WAS

LOADED INTO A WELL, GROUPED ACCORDING TO THE NUMBER OF PCR CYCLES THEY HAD BEEN SUBJECT TO. THE GEL

WAS 3% AGAROSE IN 0.5X TAE BUFFER, AND WAS RUN AT 150 V FOR 55 MINUTES. THE GEL WAS VISUALIZED USING

THE FLUOROCHEM® Q FROM ALPHA INNOTECH ON THE CY2 FLUORESCENCE SETTING. FULL PROCEDURAL

INFORMATION CAN BE FOUND ON PAGE 14.

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FIGURE 6: AGAROSE GEL IMAGE COMPARING PCR PRODUCTS AND NEGATIVE CONTROLS OF LATE-PCRS PERFORMED

USING A TRUNCATED (17 NT) FORWARD PRIMER AND A FULL-LENGTH (20 NT) FORWARD PRIMER. FOLLOWING PCR,

10 µL OF EACH PCR PRODUCT WAS LOADED INTO A WELL. IN ADDITION, 10 µL 200 NM N-40 DNA LIBRARY WAS

LOADED INTO THE CENTRE WELL. THE GEL WAS 3% AGAROSE IN 0.5X TAE BUFFER, AND WAS RUN AT 150 V FOR 25

MINUTES. THE GEL WAS VISUALIZED USING THE FLUOROCHEM® Q FROM ALPHA INNOTECH ON THE CY2


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FIGURE 7: AGAROSE GEL IMAGE OF LIMIT OF DETECTION ASSAY FOR LATE-PCR AND SYMMETRIC-THEN-ASYMMETRIC

PCR. FOLLOWING PCR, 10 µL OF EACH PCR PRODUCT WAS LOADED INTO A WELL. IN ADDITION, 10 µL 200 NM N-40 DNA LIBRARY WAS LOADED INTO THE CENTRE WELL. THE GEL WAS 3% AGAROSE IN 0.5X TAE BUFFER, AND WAS

RUN AT 150 V FOR 50 MINUTES. THE GEL WAS VISUALIZED USING THE FLUOROCHEM® Q FROM ALPHA INNOTECH ON

THE CY2 FLUORESCENCE SETTING. FULL PROCEDURAL INFORMATION CAN BE FOUND ON PAGES 16-18.

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FIGURE 8: AGAROSE GEL IMAGE OF LATE-PCR PRODUCTS MADE USING KAPA2G ROBUST HOTSTART DNA

POLYMERASE. BOTH A NEGATIVE CONTROL PCR MIXTURE (WITH NO TEMPLATE DNA) AND AN EXPERIMENTAL

MIXTURE (WITH 170 PM N-40 DNA LIBRARY TEMPLATE) WERE PREPARED AND ANALYZED. FOLLOWING PCR, 10 µL OF

EACH PCR PRODUCT WAS LOADED INTO A WELL. IN ADDITION, 10 µL 200 NM N-40 DNA LIBRARY WAS LOADED INTO

THE CENTRE WELL. THE GEL WAS 3% AGAROSE IN 0.5X TAE BUFFER, AND WAS RUN AT 150 V FOR 30 MINUTES. THE

GEL WAS VISUALIZED USING THE FLUOROCHEM® Q FROM ALPHA INNOTECH ON THE CY2 FLUORESCENCE SETTING.

FULL PROCEDURAL INFORMATION CAN BE FOUND ON PAGE 19.

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FIGURE 9: AGAROSE GEL IMAGE ASSESSING THE EFFECT OF REACTION TIME ON PEGYLATION YIELD OF A REACTION

MIXTURE CONTAINING THE 79 NUCLEOTIDE SSDNA APTAMER ZMYK-23 (5’ FAM, 3’ AMINO) AND 20 KDA MPEG-SC.

5 µL OF THE REACTION MIXTURE (TOTAL APTAMER CONCENTRATION 2 µM) WAS LOADED INTO THE GEL, ALONG WITH

25 µL 200 NM N40 DNA LIBRARY (80 NUCLEOTIDE SSDNA). THE GEL WAS 1.5% AGAROSE IN 0.5X TAE BUFFER, AND

WAS RUN AT 150 V FOR 30 MINUTES. THE GEL WAS VISUALIZED USING THE FLUOROCHEM® Q FROM ALPHA INNOTECH

ON THE CY2 FLUORESCENCE SETTING. FULL PROCEDURAL INFORMATION CAN BE FOUND ON PAGE 28.

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FIGURE 10: AGAROSE GEL IMAGE ASSESSING THE EFFECTIVENESS OF A ½ SODIUM CARBONATE (PH 11), ½

ACETONITRILE REACTION MIXTURE BUFFER SOLUTION ON SUPPORTING PEGYLATION OF A REACTION MIXTURE

CONTAINING THE 79 NUCLEOTIDE SSDNA APTAMER ZMYK-23 (5’ FAM, 3’ AMINO) AND 20 KDA MPEG-SC. 5 µL OF

THE REACTION MIXTURE (TOTAL APTAMER CONCENTRATION 1 µM) WAS LOADED INTO THE GEL, ALONG WITH 25 µL

200 NM N-40 DNA LIBRARY (80 NUCLEOTIDE SSDNA). THE GEL WAS 1.5% AGAROSE IN 0.5X TAE BUFFER, AND WAS


THE CY2 FLUORESCENCE SETTING. FULL PROCEDURAL INFORMATION CAN BE FOUND ON PAGE 29.

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FIGURE 11: AGAROSE GEL IMAGE ASSESSING THE EFFECTIVENESS OF A ½ BORATE BUFFERED SALINE (PH 9), ½

ACETONITRILE REACTION MIXTURE BUFFER SOLUTION ON SUPPORTING PEGYLATION OF A REACTION MIXTURE

CONTAINING THE 79 NUCLEOTIDE SSDNA APTAMER ZMYK-23 (5’ FAM, 3’ AMINO) AND 20 KDA MPEG-SC. 5 µL OF

THE REACTION MIXTURE (TOTAL APTAMER CONCENTRATION 1 µM) WAS LOADED INTO THE GEL, ALONG WITH 25 µL

200 NM N-40 DNA LIBRARY (80 NUCLEOTIDE SSDNA). THE GEL WAS 1.5% AGAROSE IN 0.5X TAE BUFFER, AND WAS


THE CY2 FLUORESCENCE SETTING. FULL PROCEDURAL INFORMATION CAN BE FOUND ON PAGE 30.

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FIGURE 12: AGAROSE GEL IMAGE ASSESSING THE EFFECT OF VARYING THE CONCENTRATION OF ACETONITRILE ON

APTAMER PEGYLATION IN A REACTION MIXTURE CONTAINING THE 79 NUCLEOTIDE SSDNA APTAMER ZMYK-23 (5’

FAM, 3’ AMINO) AND 20 KDA MPEG-SC IN AN ACETONITRILE AND BORATE BUFFERED SALINE (PH 9) BUFFER

SOLUTION. 5 µL OF EACH REACTION MIXTURE (TOTAL APTAMER CONCENTRATION 1 µM) WAS LOADED INTO THE GEL,

ALONG WITH 25 µL 200 NM N-40 DNA LIBRARY (80 NUCLEOTIDE SSDNA). THE GEL WAS 1.5% AGAROSE IN 0.5X

TAE BUFFER, AND WAS RUN AT 200 V FOR 25 MINUTES. THE GEL WAS VISUALIZED USING THE FLUOROCHEM® Q FROM

ALPHA INNOTECH ON THE CY2 FLUORESCENCE SETTING. FULL PROCEDURAL INFORMATION CAN BE FOUND ON PAGE

31.

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FIGURE 13: AGAROSE GEL IMAGE ASSESSING THE EFFECT OF HEAT AND AGITATION ON APTAMER PEGYLATION IN A

REACTION MIXTURE CONTAINING THE 79 NUCLEOTIDE SSDNA APTAMER ZMYK-23 (5’ FAM, 3’ AMINO) AND 20 KDA

MPEG-SC IN A ½ ACETONITRILE AND ½ BORATE BUFFERED SALINE (PH 9) BUFFER SOLUTION. THE REACTION MIXTURE

WAS INCUBATED AT 50°C, 300 RPM FOR ONE HOUR. FOLLOWING INCUBATION, 5 µL OF THE REACTION MIXTURE

(TOTAL APTAMER CONCENTRATION 1 µM) WAS LOADED INTO THE GEL, ALONG WITH 25 µL 200 NM N-40 DNA

LIBRARY (80 NUCLEOTIDE SSDNA). THE GEL WAS 1.5% AGAROSE IN 0.5X TAE BUFFER, AND WAS RUN AT 200 V FOR

20 MINUTES. THE GEL WAS VISUALIZED USING THE FLUOROCHEM® Q FROM ALPHA INNOTECH ON THE CY2


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FIGURE 14: AGAROSE GEL IMAGE ASSESSING THE EFFECT OF VARYING THE ORGANIC SOLVENT USED IN THE REACTION

BUFFER SOLUTION ON APTAMER PEGYLATION. THE REACTION MIXTURES CONTAINED THE 79 NUCLEOTIDE SSDNA

APTAMER ZMYK-23 (5’ FAM, 3’ AMINO) AND 20 KDA MPEG-SC IN A ½ BORATE BUFFERED SALINE (PH 9), ½

ORGANIC SOLVENT BUFFER SOLUTION. 5 µL OF EACH REACTION MIXTURE (TOTAL APTAMER CONCENTRATION 1 µM)

WAS LOADED INTO THE GEL, ALONG WITH 25 µL 200 NM N-40 DNA LIBRARY (80 NUCLEOTIDE SSDNA). THE GEL WAS

1.5% AGAROSE IN 0.5X TAE BUFFER, AND WAS RUN AT 200 V FOR 30 MINUTES. THE GEL WAS VISUALIZED USING THE


CAN BE FOUND ON PAGE 33.

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FIGURE 15: AGAROSE GEL IMAGES ASSESSING THE EFFECT OF REAGENT CONCENTRATION ON APTAMER PEGYLATION IN

REACTION MIXTURES CONTAINING THE 79 NUCLEOTIDE SSDNA APTAMER ZMYK-23 (5’ FAM, 3’ AMINO) AND 20 KDA

MPEG-SC IN ½ ACETONITRILE AND ½ BORATE BUFFERED SALINE (PH 9) BUFFER SOLUTIONS. EACH REACTION MIXTURE

CONTAINED A 100 X MOLAR EXCESS OF MPEG-SC OVER ZMYK-23. EACH REACTION MIXTURE WAS DILUTED TO AN

APTAMER CONCENTRATION OF 200 NM IN DDH2O, THEN 25 µL OF EACH DILUTE REACTION MIXTURE WAS LOADED

INTO THE GEL, ALONG WITH 25 µL 200 NM N-40 DNA LIBRARY (80 NUCLEOTIDE SSDNA). THE GEL WAS 1.5%

AGAROSE IN 0.5X TAE BUFFER, AND WAS RUN AT 200 V FOR 20 MINUTES. THE GEL WAS VISUALIZED USING THE



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FIGURE 16: AGAROSE GEL IMAGE ASSESSING THE EFFECT OF REAGENT CONCENTRATION ON APTAMER PEGYLATION IN A

REACTION MIXTURE CONTAINING THE 79 NUCLEOTIDE SSDNA APTAMER ZMYK-23 (5’ FAM, 3’ AMINO) AND 20 KDA

MPEG-SC IN ½ ACETONITRILE AND ½ BORATE BUFFERED SALINE (PH 9) BUFFER SOLUTIONS. EACH REACTION MIXTURE

CONTAINED A 900 X MOLAR EXCESS OF MPEG-SC OVER ZMYK-23. EACH REACTION MIXTURE WAS DILUTED TO AN

APTAMER CONCENTRATION OF 200 NM IN DDH2O, THEN 10 µL OF EACH DILUTE REACTION MIXTURE WAS LOADED

INTO THE GEL, ALONG WITH 10 µL 200 NM N-40 DNA LIBRARY (80 NUCLEOTIDE SSDNA). THE GEL WAS 1.5%

AGAROSE IN 0.5X TAE BUFFER, AND WAS RUN AT 200 V FOR 20 MINUTES. THE GEL WAS VISUALIZED USING THE



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FIGURE 17: AGAROSE GEL IMAGE OF PEGYLATED ZMYK-23 (5’ FAM) PURIFIED BY ELECTROELUTION. 10 µL OF THE

PURIFIED APTAMER WAS LOADED INTO THE GEL, ALONG WITH 10 µL 200 NM N-40 DNA LIBRARY (80 NUCLEOTIDE

SSDNA). THE GEL WAS 1.5% AGAROSE IN 0.5X TAE BUFFER, AND WAS RUN AT 200 V FOR 20 MINUTES. THE GEL WAS

VISUALIZED USING THE FLUOROCHEM® Q FROM ALPHA INNOTECH ON THE CY2 FLUORESCENCE SETTING. FULL

PROCEDURAL INFORMATION CAN BE FOUND ON PAGE 36.

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← No nAbs ← 1/2500

← 1/10000 ← 1/1250

← 1/7500 ← 1/625

← 1/5000 ← 1/312

FIGURE 18: IMAGES OF 12-WELL PLATES USED IN ANTI-VSV∆51 NABS T ITERING EXPERIMENT. EACH WELL CONTAINS

95% VERO CELLS, ALONG WITH 100 PFU VSV∆51 THAT HAD PREVIOUSLY BEEN INCUBATED WITH A PARTICULAR

DILUTIONS OF ANTI-VSV∆51 NABS. THE PLATES WERE VISUALIZED USING THE FLUOROCHEM® Q FROM ALPHA

INNOTECH ON THE CY2 FLUORESCENCE SETTING. FULL PROCEDURAL INFORMATION CAN BE FOUND ON PAGE 37.

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A A B C D E F G H I J K

FIGURE 19: IMAGES OF 12-WELL PLATES USED IN A VIRAL INFECTIVITY ASSESSING THE ABILITY OF VSV∆51-BINDING

APTAMERS TO PROTECT VSV∆51 FROM NABS. EACH WELL CONTAINS 95% VERO CELLS, ALONG WITH 100 PFU

VSV∆51 THAT HAD PREVIOUSLY BEEN INCUBATED WITH A PARTICULAR MIXTURE AND CONCENTRATION OF VSV∆51-BINDING APTAMERS, FOLLOWED BY ANTI-VSV∆51 NABS. THE CONTENTS OF CONTENTS OF EACH TRIPLICATE GROUP

OF WELLS ARE LISTED BELOW.

A: NEGATIVE CONTROL (NO VSV∆51-YFP, NABS, OR APTAMERS) B: POSITIVE CONTROL (100 PFU VSV∆51-YFP PER WELL) C: VSV∆51-YFP (100 PFU PER WELL), NABS D: VSV∆51-YFP (100 PFU PER WELL), NABS, POLYCLONAL VSV-BINDING APTAMER MIXTURE (0.25 NMOL PER WELL) E: VSV∆51-YFP (100 PFU PER WELL), NABS, POLYCLONAL VSV-BINDING APTAMER MIXTURE (0.625 NMOL PER WELL) F: VSV∆51-YFP (100 PFU PER WELL), NABS, POLYCLONAL VSV-BINDING APTAMER MIXTURE (0.9375 NMOL PER WELL) G: VSV∆51-YFP (100 PFU PER WELL), NABS, POLYCLONAL VSV-BINDING APTAMER MIXTURE (0.1875 NMOL PER WELL) H: VSV∆51-YFP (100 PFU PER WELL), NABS, MONOCLONAL ZMYK-23 (0.25 NMOL PER WELL) I: VSV∆51-YFP (100 PFU PER WELL), NABS, MONOCLONAL ZMYK-23 (0.625 NMOL PER WELL) J: VSV∆51-YFP (100 PFU PER WELL), NABS, MONOCLONAL ZMYK-23 (0.9375 NMOL PER WELL) K: VSV∆51-YFP (100 PFU PER WELL), NABS, MONOCLONAL ZMYK-23 (1.875 NMOL PER WELL)

THE PLATES WERE VISUALIZED USING THE FLUOROCHEM® Q FROM ALPHA INNOTECH ON THE CY2 FLUORESCENCE

SETTING. FULL PROCEDURAL INFORMATION CAN BE FOUND ON PAGE 38-39.

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Fluorescence Intensity

FIGURE 20: HISTOGRAM OF FLOW CYTOMETRIC DISPLACEMENT ASSAY DATA MEASURING THE ABILITY OF ANTI-VSV∆51

NABS PRODUCED BY BUNNY #2 TO DISPLACE A POLYCLONAL MIXTURE OF VSV∆51-BINDING APTAMERS FROM THE

SURFACE OF VSV∆51. FLOW CYTOMETRY WAS PERFORMED WITH THE BECKMAN COULTER CYTOMICS FC500 AND

FLOW CYTOMETRIC DATA WAS ANALYSED USING KALUZA® FLOW ANALYSIS SOFTWARE FROM BECKMAN COULTER.

FULL PROCEDURAL DATA CAN BE FOUND OF PAGE 40.

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Fluorescence Intensity

FIGURE 21: HISTOGRAM OF FLOW CYTOMETRIC BINDING ASSAY MEASURING THE BINDING OF CY5-LABELLED

OLIGONUCLEOTIDE POOLS SELECTED THROUGH VERO CELL SELEX AND OF HARVARD DNA LIBRARY TO BIND TO VERO

CELLS. EACH SAMPLE CONTAINED A TOTAL OF 500,000 VERO CELLS AND 5 NMOL OLIGONUCLEOTIDE. FLOW

CYTOMETRY WAS PERFORMED WITH THE BECKMAN COULTER CYTOMICS FC500 AND FLOW CYTOMETRIC DATA WAS

ANALYSED USING KALUZA® FLOW ANALYSIS SOFTWARE FROM BECKMAN COULTER. FULL PROCEDURAL DATA CAN BE

FOUND OF PAGE 48.

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Protocol – LATE-PCR for Single Stranded Oligonucleotide Pool

Amplification

Apparatus and Materials

PCR reaction mixture containers

0.2 mL PCR tubes

Thermocycler

Eppendorf Mastercycler® pro S

LATE-PCR using Taq Polymerase

Reagents and Chemicals

Distilled, deionized water (ddH2O)

Gotaq® Hot Start DNA Polymerase (Promega)

5X Green GoTaq® Flexi Buffer

Reverse DNA Primer: (5’-GGC TTC TGG ACT ACC TAT GC-3’)

5' FAM labelled Forward DNA Primer (5’-CTC CTC TGA CTG TAA CCA CG-3’)

dNTP mix (10 mM stock)

Protocol

Prepare a master mix containing the following components at the following concentrations:



25 nM reverse DNA primer (5'-GGC TTC TGG ACT ACC TAT GC-3')

1 uM 5' FAM-labelled forward primer (5'-CTC CTC TGA CTG TAA CCA CG-3')

400 uM dNTPs


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Combine this master mix with the oligonucleotide pool with a ratio of 1 volume oligonucleotide pool per 5

volumes master mix.

Use the following thermocycler program to amplify the oligonucleotide pools:






Hold at 4º C

LATE-PCR using KAPA DNA Polymerase

For more difficult amplifications, including those involving high GC content

Reagents and Chemicals

Distilled, deionized water (ddH2O)

KAPA2G Robust Hot Start DNA Polymerase (KAPA Biosystems)

5X KAPA2G Buffer A (KAPA Biosystems)

Reverse DNA Primer (5’-GGC TTC TGG ACT ACC TAT GC-3’)

5' FAM labelled Forward DNA Primer (5’-CTC CTC TGA CTG TAA CCA CG-3’)

dNTP mix (10 mM stock)

Protocol

Prepare a master mix containing the following components at the following concentrations:

1X KAPA2G Buffer A, from 5X concentration stock

25 nM reverse DNA primer (5'-GGC TTC TGG ACT ACC TAT GC-3')

1 uM 5' FAM-labelled forward primer (5'-CTC CTC TGA CTG TAA CCA CG-3')

400 uM dNTPs

0.025 UuL-1 KAPA2G Robust Hot Start DNA Polymerase

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Combine this master mix with the oligonucleotide pool with a ratio of 1 volume oligonucleotide pool per 5

volumes master mix.

Use the following thermocycler program to amplify the oligonucleotide pools:






Hold at 4º C

Documents

Selected Topics in Aptamer Research - University of …mysite.science.uottawa.ca/mberezov/team/bsc-theses/2014_bsc_thesis...Selected Topics in Aptamer ... forming stable three-dimensional