aptamer selection against ccr5 final manuscript 30 november 2011

Aptamer Selection Against CCR5 for the Prevention of HIV-1 Pathogenesis

Juan Herrejon

30 November 2011

Fall 2011

Pool: N34/N50 RNA Target: C-C Chemokine Receptor 5 (CCR5)

Herrejon Aptamer Selection Against CCR5 Page 0

Abstract

HIV, the human immunodeficiency virus, progressively weakens the body’s internal defense system by infecting cells

vital to the human immune system such as helper T-Cells, macrophages, and dendritic cells (Reviewed in Cunningham et al,

2010). HIV progresses to AIDS, the acquired immunodeficiency syndrome, a state of extremely low immune system

effectiveness that makes the body susceptible to infections that can result in death. A retrovirus, HIV binds to specific receptor

and coreceptor proteins on helper T-cells, macrophages, and dendritic cells in order to inject the cells with its virulent RNA.

C-C Chemokine Receptor type 5, CCR5, is the main coreceptor protein used by M-tropic strains of the HIV type 1

(HIV-1) virus. Humans who exhibit a mutation in the CCR5 gene coding region produce a nonfunctional, truncated protein

that is not transported to the surface of the cell membrane (Blanpain et al, 2002). This mutation causes immunity towards HIV

because its absence in the membrane does not allow the formation of the fusion complex (see Figure 4). CCR5 therefore plays

a crucial role in HIV pathogenesis, for without its functional existence, HIV cannot

infect its target cells.

A multistep approach is proposed that seeks to prevent the binding of HIV strains to the coreceptor CCR5, thus preventing HIV

infection and pathogenesis. Aptamers, RNA molecules developed to bind specific

targets, have many diagnostic and therapeutic uses and are a promising means of

inhibiting the binding of HIV to CCR5. Much like an allosteric or competitive

inhibitor, an aptamer that binds tightly to CCR5 will alter its three-dimensional

configuration and thus impede it from acting as a functional coreceptor to HIV.

Developing and isolating an aptamer against CCR5 will provide an alternative to

existing receptor antagonists and provide a novel way of preventing and treating

infections caused by M-tropic strains of HIV.

Specific Aim 1: Selection of RNA Aptamers against CCR5

CCR5 is a coreceptor crucial to HIV pathogenesis. It has been shown that a mutation in the CCR5 gene results in HIV

immunity (Tang & Kaslow, 2003) and that using CCR5 receptor antagonists

prevent the binding of HIV to CCR5, inhibiting HIV infection (Pulley, 2007).

Thus, the development of nucleic aptamers against CCR5 will serve as another means of preventing the binding of HIV to the

crucial protein CCR5 and inhibit infection altogether by acting as an entry inhibitor.

CCR5 can be acquired from Abcam (www.abcam.com) for $160 per 100 ug. The catalog number is ab95861.

Introduction and Background

Classified as a pandemic by the World Health Organization, HIV affects the lives of about 35 million people

worldwide, with nearly 1 million cases in the United States (CDC, 2011). Among the many strains of the virus, the M-tropic

strain of HIV-1 accounts for about 90% of all HIV cases. This particular strain requires the primary receptor CD4+ and the

coreceptor CCR5, the core of this research paper, to infect the cells of the immune system. C-C Chemokine Receptor Type 5

(CCR5), depicted in Figure 2, is a plasma membrane protein encoded by the CCR5 gene, located on the short arm at position

21 on chromosome number three (Samson et al, 1996). It is the main coreceptor protein used by M-tropic strains of the HIV

type 1 (HIV-1) and type 2 (HIV-2) virus. It is a member of the beta chemokine receptor family, a group of small proteins


involved in the communication of the immune system (Samson et al, 1996).

CCR5 is expressed predominantly by T cells, including the helper T cells that

serve as the target of HIV to suppress the immune system, macrophages, and

dendritic cells of humans and most other mammals. Its functional structure as a

trans-membrane protein in humans has been shown to play a crucial role in the

entry of HIV to host cells (Vazques-Salat et al, 2007). The protein is

approximately 40.5 KDa in size and is stable at a pH of 7.4, in proximity to

normal physiological pH. An interesting and sought-after region of binding on

the three-dimensional protein is the N-terminus, which is the binding site of gp120 (Huang et al, 2007). The amino acid

sequence to the protein from the protein database on the NCBI website (http://www.ncbi.nlm.nih.gov/sites/entrez?db=protein)

is as follows:

1 epcqkinvkq iaarllpply slvfifgfvg nmlvililin ckrlksmtdi yllnlaisdl 61 fflltvpfwa hyaaaqwdfg ntmcqlltgl yfigffsgif fiilltidry lavvhavfal 121 kartvtfgvv tsvitwvvav faslpgiift rsqkeglhyt csshfpFor the purposes of this selection against CCR5, the full-length protein will not be used. Instead, a short polypeptide sequence

of the protein will be used as the target of this selection. The twenty-amino-acid-long peptide weighs 2,374 grams/mole, has a

pI of 6.20, an absorbance of 0.144, and an extinction coefficient of 3960 cm-1m-1. This peptide has the following sequence of

amino acids:

Ser-Pro-Ile-Tyr-Asp-Ile-Asn-Tyr-Tyr-Thr-Ser-Glu-Pro-Cys-Hyp-Lys-Ile-Asn-Val-Lys

To understand the exact role of CCR5 in HIV’s entry into host cells, the pathogenesis of HIV must first be defined.

Macrophage-tropic strains of HIV-1 need the presence of the primary receptor CD4+ and at least one coreceptor to successfully

enter the host cell. R5 M-tropic strains of HIV utilize CCR5 as a coreceptor, and thus need functional CD4+ and CCR5 to fuse

with the plasma membrane of host cells (Alkhatib, 2009; Moore et al, 2004; Weinstein et al, 2010). As depicted in Figure 3, the

gp120 protein on the surface of HIV-1 binds to the surface protein CD4+ on host cells. This initial interaction between gp120

and CD4+ changes the configuration of gp120 to increase its affinity towards CCR5 (Lu et al, 1997). Once HIV binds to

functional CCR5, fusion is complete and HIV can gain entry into host cells.

Experiments have shown that the presence of coreceptors is crucial to the pathogenesis of HIV. The sole presence of

CD4+, the primary receptor, is not enough for fusion to occur. Experiments

have revealed that certain strains of HIV can enter a host cell without the need

of CD4+, using only coreceptors such as CCR5 (Reeves et al, 1999). In

addition, other studies have shown that CCR5 is crucial for HIV entry,

demonstrating that HIV pathogenesis cannot occur without the presence of

functional CCR5 in the membrane (Bhattacharya et al, 2003). A special case

study revealed that humans with a mutation in the CCR5 coding region of the

gene, dubbed the delta-32 mutation, exhibit resistance to HIV (Samson et al,

1996). The delta-32 gene mutation provides evidence for the importance of

CCR5, as a non-functional protein on the surface of host cells inhibits the

fusion of HIV despite its binding to CD4+ (Alkhatib, 2009; Moore et al, 2004; Weinstein et al, 2010). The reason is that HIV


cannot enter the cell unless it binds its gp120 envelope glycoprotein to both CD4+ and CCR5 (Huang et al, 2007). Humans

who possess this mutation are immune to HIV-1 strains that require CCR5 as a coreceptor, suggesting that CCR5 plays a more

important role in HIV. As a result of these findings, CCR5 antagonists have been targeted as a means of blocking HIV from

entry into host cells.

While there have not been aptamers developed against CCR5, a

number of therapeutic agents—named CCR5 antagonists, such as Maraviroc

(depicted in Figure 4)—that bind to CCR5 in an attempt to prevent the binding

of gp120 and CCR5 have been developed (Emmelkamp & Rockstroh, 2007).

An aptamer could be more medically useful because the aptamer would be

designed in vitro under physiological conditions to bind solely to CCR5 with a

high affinity. An aptamer can also be produced at a lower price, quickly and

efficiently, and are capable of higher affinitiy and specificity than antibodies

(Ellington and Szostak, 1990). Its mechanism would be to bind to CCR5 to

alter its three-dimensional configuration. HIV will not recognize a functional CCR5 and will be unable to bind it, preventing

fusion and further pathogenesis altogether.

This paper introduces an alternative to existing therapeutic drugs that combat HIV pathogenesis by preventing HIV’s

fusion to the cell membrane of CD4+ cells (i.e. helper T-cells, macrophages, and dendritic cells). The method proposed to

achieve this endeavor is to develop an aptamer, RNA molecules that bind with a high affinity to specific targets (Ellington and

Szostak, 1990), against CCR5. These RNA molecules exhibit high binding affinity towards targets and can be used for

therapeutic and diagnostic purposes. The selection protocol, SELEX (Stoltenburg et al, 2007), was followed in this experiment

to develop an aptamer molecule whose high affinity for CCR5 allows it to bind tightly to the protein. To develop an aptamer

against CCR5, variants from the RNA pool N50 were introduced to CCR5 and were allowed to bind to the target in a binding

reaction. After multiple washes, the final elution retrieves

the variants of RNA that bind tightly to the target.

Summarized briefly in Figure 5 and explained further in the

next section, the method to produce an aptamer RNA

revolves around the concept of molecular recognition and

intermolecular attraction.

Materials and Methods

In devising the experimental design for the selection, several important conditions were established. The storage

buffer in which CCR5 was shipped contained 0.001% Tween 20, 30 mM HEPES, 2 mM EDTA, and 150 mM sodium chloride

at pH 6.75. It was shipped at 4°C and will required storage at -80°C. Because CCR5 is known to be stable around physiological

pH, specifically 7.4 (Moore et al, 2004), PBS selection buffer (1X PBS) at pH 7.4 was used in binding reactions. At 1X, PBS

contains 137mM NaCl, 10 mM Phosphate, 2.7 mM KCl, and 5 mM MgCl2. The isoelectric point of CCR5 is 8.99 and the

estimated charge at pH 7 is 5.8 according to the protein calculator (http://www.scripps.edu/~cdputnam/protcalc.html). The

incubation time was established at 30 minutes, to allow maximum binding time, and carried out at 37°C to mimic the

physiological temperature. Three washes were done to maximize the elimination of poor binders. Since the protein was


functionalized with biotin, streptavidin beads were used to immobilize the target. The starting ratio of nucleic acid to protein

will be 1:2, or 200 pmol: 400 pmol. The following is the general selection protocol, using the conditions established.

The selection against CCR5 began after successfully biotinylating the protein with biotin. The N34 RNA pool was

introduced to the protein in a binding reaction. After a 30 minute incubation period, the RNA species with poor affinity toward

the protein were discarded through a series of washes. After performing three washes, an ultimate elution was performed to

retrieve the variants that adhered more tightly to CCR5. After executing an ethanol precipitation, the eluted RNA was reverse

transcribed to obtain ssDNA. A cycle course was then run to determine how many cycles of PCR would amplify the DNA the

best, running a no-template sample as a negative control. After the cycle course, an agarose gel was run using the samples

retrieved and were observed under ultraviolet light.

Another ethanol precipitation was carried out and the DNA was resuspended in 20ul diH2O. After resuspension, the

DNA was subject to a transcription reaction to obtain RNA of the variants that adhered tightly to the protein. After removing

the DNA using DNAse I, a PAGE gel was run to view and purify the RNA. After purifying the RNA via an ethanol

precipitation, the concentration was determined using a nanodrop. The final eluted RNA was reintroduced to the target and the

selection process was repeated, reducing the amount of target every other round. Negative selections were performed every

other round by allowing the RNA retrieved at the end of a round to react in a binding reaction with streptavidin beads without

the presence of protein. This process identifies background binders, species that bind the beads and not the protein itself, and

eliminates them altogether.

Budget

To calculate the budget for the development of an aptamer against CCR5, several factors must be considered. First, as

a benefit of working in a molecular biology lab at the University of Texas at Austin, the primers, enzymes, and buffers needed

for the selection (i.e. DNA polymerase, dNTPs, HEPES buffer, etc.) are readily available. The only materials that would need

to be accounted for are the protein (CCR5) and a kit to biotinylate the protein.

Human CCR5 can be purchased from Abcam (www.abcam.com) for $160 per 100ug (catalog number ab95861;

telephone number 1-888-772-2226). The molecular weight of CCR5 is 40,524 g/mol. If 200 pmol of protein are used per

round, then 8.2ug of protein will be needed, which is equivalent to paying $13.12 per round. Lastly, in order to work with the

protein, a kit will be needed to tag CCR5 with biotin. To do this, the EZ-Link NHS-Biotin will be purchased from Pierce

Protein Scientific Products (www.piercenet.com) for $115 for a package size of 100mg. The catalog number for the reaction kit

is 20217 and the phone number for the company is 1-800-874-3723. If the cost of the bitotin kit is divided over 25 rounds, then

the total cost per round of selection totals $17.72.

Results and Discussion

The first round of selection began after a successful second attempt at

functionalizing the CCR5 peptide with biotin. In a pool binding reaction,

400 pmol of the biotinylated CCR5 were introduced to 200 pmol N34 RNA

and were allowed to bind for 40 minutes at 37˚C. After eluting the RNA,

reverse transcribing it into ssDNA, and subjecting it to cycle course PCR,

the DNA was run through a 3.8% agarose gel and visualized under UV


light. Figure 6 represents the gel for the first round. Although faint, a band did appear at cycles 15 and 20, so 18 cycles was

chosen for large scale PCR. After transcription, a PAGE gel was run to purify and visualize the RNA recovered after

transcription. The thick white band observed suggested an ample amount of RNA was retrieved and indicated the round was

proceeding successfully. After eluting the RNA from the gel and performing another ethanol precipitation, the RNA was

spec’d and a final concentration of 43.8 ng/uL (or 6.57 uM) was retrieved.

The first round of selection against CCR5 yielded a very low concentration of 6.57 uM. Several attempts were made

to re-spec the RNA to eliminate an error on behalf of the nanodrop. However, the yield remained constant; the peak at 260 nm

suggested the concentration was the result of RNA, so the problem must have occurred during transcription or the elution of

the RNA after PAGE. Further analysis of the results indicated that the problem may have originated during cycle course PCR.

After reevaluating the agarose gel, a faint band was noticed in the no template control, which suggested the presence of an

artifact in the N34 RNA pool. Additionally, since no bands showed up before cycle 15, and the bands that did show up were

relatively faint, it could have been the case that a small amount of DNA was produced using 18 cycles in large scale, which

produced a low yield of RNA after transcription. It must also be noted that even after adding 3 mL of 1X TE to the PAGE gel

containing the RNA band, only 2 mL of buffer were eluted after the overnight incubation, which could have influenced the low

final concentration. The round itself didn’t necessarily fail, but at such low concentration, there was not enough RNA to

continue to a second round. It was therefore decided to start over using N50 RNA to eliminate the possibility of encountering

artifacts later in selection.

The next round of selection began with the introduction of 200 pmol

N50 RNA to 400 pmol of CCR5 protein in a pool binding reaction. After a 30

minute incubation at 37˚C, a series of washes were done to weed out the bound

specimens from the unbound. The ultimate elution, E1, contained the RNA that

adhered most tightly to CCR5—this is the RNA of interest. The eluted RNA

was then subject to ethanol precipitation. The point of this is to force the

precipitation of the RNA from solution. This occurs by adding salt (sodium

acetate) to neutralize the charge of the phosphate backbone of the RNA.

Because the charge is

neutralized, the RNA is

forced to precipitate out

of solution, since it can

no longer dissociate because its neutral charge alienates it from the rest

of the solution.

After reverse transcription and cycle course PCR and gel

electrophoresis, the gel in Figure 7 was observed under UV light. Based

on the results of the gel, the best band without over-amplification was

cycle 9. However, because it displayed a small amount of over-

amplification, cycle 8 was chosen to be the best cycle for large scale PCR. LsPCR was executed to produce thousands of

copies of the ssDNA that were produced in reverse transcription After transcription, the image in Figure 8 was observed after

running a PAGE gel and visualizing it under UV light. As seen in the representation, a considerably faint band was observed,


indicating a low yield of RNA would be expected. The RNA was nonetheless eluted and ethanol precipitated, and then

resuspended in 30 ul 1X HEPES buffer. The results of the nanodrop are

shown in Figure 9. For reasons discussed later, transcription was redone to

try to achieve better results. After performing another PAGE gel, the band

observed under UV light was considerably darker than the one in figure 7

but still relatively faint. RNA crashing was performed instead of the

overnight elution due to time constraints, and the eluted RNA was spec’d

once again. The results in Figure 10 show the solution contained RNA, but

at a very low concentration of 53 ng/uL. Due primarily to time constraints and to the fact that not enough RNA was retrieved at

the end of the round, the selection against CCR5 was halted here.

The round of selection using N50 RNA proved to be problematic. Because the results of the first round using N34

were questionable, it was restarted using a different pool of RNA. The agarose gel of the cycle course PCR seemed promising,

as the bands were much more discernible than the ones of the first round. There was a noticeable difference in the amplification

of Wash 0 (W0) and Wash 3 (W3), with W3 displaying no bands for cycles 6 and 9 and little amplification in the rest of the

bands compared to W0. These results are desired because W3 should contain less RNA species than the first wash. The

difference in amplification in the two washes indicated that the washes

were performed correctly. The bands for the elution were also promising,

as 8 cycles seemed to be the best number to perform large scale with. In

addition, the no template control showed no band, indicating no artifacts

were present in the reaction. The first nanodrop results showed a peak at

around 225 nm and had a relatively small peak at 260 nm, suggesting

there was a high salt concentration and a significantly small amount of

RNA. To eliminate the presence of salts, RNA crashing was carried out

after transcription. After running it through another PAGE, a darker band was observed, indicating more RNA was collected.

As seen in figure x, the concentration of RNA was not at all higher, but the more prominent peak at 260 nm suggested more

RNA was retrieved in comparison to the first gel. However, there was still a noticeable peak at 225 nm, signifying there was

still some artifact in the solution (more than likely salts) that was causing such results.

Overall Problems Encountered

Over the course of the selection against CCR5, many problems were encountered that slowed down the progress of the

selection rounds. The first major problem encountered occurred before the first round during the biotinylation of CCR5. The

results of the first attempt at biotinylation exhibited an unusual phenomenon in which the washes of the biotinylation reaction

expressed a higher protein concentration than the elution that was supposed to contain all of the protein. Tests were carried out

to eliminate potential sources of error, and the identified cause was the use of the wrong spin column. After the correct spin

column was acquired, the biotinylation reaction was repeated and the results suggested it was successful.

After spending several days solving the biotinylation issues, the actual selection began using the N34 RNA pool. The

first gel (Figure 6) showed up faintly under UV light. This was probably due to the fact that the diluted base pair ladder was

used instead of the stock ladder, and because the dye that was used was not properly aliquoted. It was observed in other


student’s gels that adding more dye (2 ul instead of 1 ul for instance) and using the stock ladders resulted in brighter,

discernible gels. The presence of the artifact, although faint, was due to contamination in the lab setting. Due to the low yield at

the end of the round and the presence of the artifact, it was decided best to restart using a different pool.

The round using N50 RNA began positively, with a better cycle course gel and a thicker PAGE gel band. However,

the major issue occurred at the end when the eluted RNA was specd in the nanodrop. The peak before 260 nm and the odd

shape suggested there were salts present in the solution. Because everything seemed to be working properly up to the point of

large scale, transcription was repeated in an attempt to eliminate the salt concentration in the final yield. When PAGE was done

for the second time, the thicker band proved promising. After RNA crashing and re-specing, however, the yield remained low

despite the larger peak at 260 nm. The round didn’t fail, but the low concentration would require the next round to be done

with 100 pmol RNA versus 200 pmol. However, because of the lack of time, the selection stopped at this point.

Conclusion and Future Work

CCR5 was successfully functionalized with biotin after a second attempt using the appropriate spin column. The first

round carried out against CCR5, using N34 RNA yielded a final concentration of 43.8 ng/uL, or 6.57 uM. The low

concentration and artifact present in cycle course led me to start over using N50 RNA. This round yielded a final concentration

of 52.4 ng/uL or 7.86 uM. Because of the low concentration, however, transcription was repeated to attempt to get a better

concentration. To my surprise, the yield for the second final RNA elution was the same, at 52.4 ng/uL or 7.86 uM. Below is a

table that summarizes the rounds performed in this experiment:

Table 1: Summary of round results and data. N34/50 RNA, CCR5. Juan Herrejon.

Cycle Course Absorbance Quantity Nuc. Acid used Nucleic Acid Recovered

Round 1 18 0.864 400 pmol 6.57 pmol/ul (uM)

Round 2 8 1.447 400 pmol 7.86 pmol/ul (uM)

In the selection against CCR5, time only allowed for two complete rounds to be performed altogether. If I could

continue this research project, I would restart a fresh round using N50 RNA and apply all the knowledge I gained this semester

to prevent any error. This project has great potential, and I would definitely like to continue it at some point in time.

Student Stories

As a pre-med student, it was never my desire to pursue research as a career. I thought the life of a scientist was not for

me, but the Aptamer Stream truly made me consider research as a profession, or at least a back-up plan if medical school does

not work out. After my experiences in the stream, I am still set on pursuing medicine, but I have grown interested in engaging

in research simultaneously.

Among the things I will take away from this course, I have learned to accept failure and use it as a learning

experience. At first, when my reactions failed I took it as a personal failure and credited it to my personal error. However, as I

became more immersed in the practice of research, rather than waste time believing I had created the error, I began to

troubleshoot to identify the source of the error. My numerous failures this semester excited me to ask questions and formulate

hypotheses as well as ways to test them. Because I do not like to fail, I became determined to overcome every bump on the


road and ensure it did not happen again. In retrospect, it was failure that forced me to think about what exactly I was doing and

is the reason I truly learned troubleshooting methods and the theory behind selections. Of course, the numerous successes I

experienced were always rewarding and fueled my desire to keep moving forward with the research, but in the long run, I

learned more from my failures than from my success.

Overall, the FRI has made my first three semesters at UT quite unforgettable. From the first exposure to research

methods during my first semester, to my current experiences as an undergraduate researcher, this program has changed the way

I think about science. I have learned that science is a dynamic process that not only requires knowledge about procedures and

the theory behind them, but also the ability to apply the knowledge and use it in innovative ways to achieve a goal. For the

most part, a researcher guides the path of his/her research, but to a certain extent, the researcher must also allow the research to

guide him/her.

In my opinion, the FRI and Aptamer Stream are well structured and efficiently coordinated. The only thing I would

suggest would be to cut down the hour requirement during the second semester of research down to about 7 or 8 hours instead

of 10 weekly hours. I would not change any other aspect!

References

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