No artifact in the N50 pool.

This is the R1 ccPCR for my target protein H1N1 Hemagglutinin and I have found nothing amplified in the NTC. And as a side note, the 100 bp ladders turned out to be very faint and I'm not the only person whose had really faint ladders.

FliC Monomeric Protein N59 Pool Progress Report I - Jake Van Fleet

Jake Van Fleet

Monomeric FliC Target

N59 RNA Pool

10/18/11

Progress Report I

Before you begin reading this, I could not find a way to transfer the labels and formatting on my gel into this blog, so here is a link to my first progress report. For background information, here is the link to my abstract.

Here is the text without the images.

Progress, Results and Discussion:

Selection for the monomeric FliC protein has taken place using the streptavidin-biotin complex that can be stabilized on magnetic beads. The reactions take place in 1X PBS buffer and at room temperature, the standard conditions for the protein of Burkholderia pseudomallei. The streptavidin beads were bound to 200pmol of the biotinylated protein that makes the initial complex. Then, following denaturation, 200pmol of R0 N59 RNA nucleic acid were incubated at room temperature for 30 minutes. The respective RNA washes are removed from the solution, allowing the bound FliC ligands to be eluted out of the target-pool complex.

An ethanol precipitation was carried out to purify and visualize the eluted RNA samples, since the RNA is transparent and often has beads and other contaminants in solution beforehand. Reverse transcription is proceeded next, transcribing the RNA into and amplifiable DNA pool. This was done with the initial removal of solution from the beads (W0), the third wash (W3), and the elution (E1). Then a cycle course reaction was run in order to test the proper amount of amplification to be done in large scale PCR. Without the proper amount of nucleic acid, one either may not have enough to continue on with the ext binding and selection round, or will have extraneous strands of varying lengths that will skew data. Gel electrophoresis using 3.8% agarose gel at 100V for 30 minutes was run in order to see the amplification of the DNA. The Figure 1 shows the gel for the second cycle course reaction run.

The cycle course PCR shown was the second attempt at cycle course for R1. The initial gel only showed the base pair ladders. Since only the ladders were showing, either the cycle course did not amplify the DNA, or the RT failed and there was no DNA to be amplified. In order to save from possibly having to do three first round cycle course reactions, RT was rerun. The second cycle course (as shown in Figure 1) had limited amplification of W1 as compared to W3. This could be due to variability in incubation time and movement of the beads between the two washes. A light band can be seen in the E1 that signifies overamplification in cycle 15. The proper amplification would reside at approximately 12 rounds, since this is the darkest band without a wide band that shows the DNA polymerase to be acting on too many strands of DNA. However, a no template negative control showed a band, which indicates a problem. Since the NTC did not show any species in the initial cycle course reaction, there are two possibilities. One possibility is the contaminant not being present in the first cycle course due to the PCR not being run properly (yet RT was run correctly). The second possibility is that RT did not run properly the first time, yet on the second it was run correctly, and the species was somehow picked up during the transition or during contamination of the cycle course. Since the second could have been a possibility, the binding and selection was rerun in order to eliminate the contamination from the picture.

Problems Encountered:

The problems encountered mainly arose with the NTC of the second cycle course reaction. This presents a huge problem, since it is impossible to tell exactly what this species is. Since it is about the same length as the pool (59 base pairs) it can be assumed that some species was transferred into the PCR tube when addition of reagents (i.e. not switching a tip, touching a tip to the lab table, etc.). Since these species may or may not be potential binding ligands, they must be completely limited from the reactions. Another problem was my initial cycle course not showing any species. Attention to detail and additions and not leaving enzymes out of ice in reactions before commencing the reactions could help eliminate these errors.

Conclusions and Future Work:

The future steps of the reaction will be to continue on with the preliminary steps of R1 reaction in order to proceed into the later steps and R2. Currently the rerun of the round has gotten to RT. After cycle course, lsPCR will amplify the DNA in order to proceed into transcription (TNX) to obtain RNA to be used in R2. A PAGE gel is used in order to purify the RNA from the TNX, with than is ethanol precipitated out to visualize the RNA and isolate it completely from the gel.

So far the reaction has carried out fairly well, considering that at the moment, besides the positive NTC, the selection is possible and can produce viable results. Continuing on with the rounds should be able to produce an applicable ligand. The conditions for the first two rounds of selection can be seen in Figure 2. The rounds needed for PCR could possibly be less than 12 if the ideal amount of RNA is recovered from TNX. This would mean that the binding species would be fairly complementary to the target FliC protein. Given this, much of the pool should be able to bind, and thus will have less washed away by the protein, since most of that was removed in the R1 washes.

Tau MAP Progress Report 1 - Stephanie Tutak

Stephanie Tutak – st22642

October 18, 2011

Fall 2011

N35 RNA

Tau Microtubule Protein

For a clear understanding of the purpose for this selection, please reference the abstract and proposal, found here.

Here is my final report, detailing the progress of my selection for this semester.

One round of N35 RNA bead-based selection has been performed against Tau, a microtubule-associated protein linked to symptoms unique to Alzheimer’s disease.

Round One Conditions:

Target: Tau Microtubule Associated Protein (6X-His expressed protein)

Beads: Nickel NTA

Pool: N35 RNA*

Pool:Target Ratio: 200:200 pmol

Buffer: 10X PBS (pH 7.4)

Incubation Time/ Temperature: 25 degrees Celsius, 25 minutes

Wash Number/Volume: 3 washes, 1 volume (100 ul)

*Note that N34 RNA was used for the first attempt at selection, but due to a positive reading for the no-template control during Cycle Course PCR, the starting Round was repeated using the N35 pool to avoid artifact interference. See Figure 1 and Problems Encountered for more detail.

Progress, Results, and Discussion

A Nickel bead-based selection was performed against Tau protein because of its classification as a 6X-His expressed protein. The protein was previously stored in 10X PBS buffer such that it was already sequestered into 200 pmol aliquots. In preparation for the start of selection, Ni-NTA beads were washed, and incubated with Tau protein target for thirty minutes at 25 degrees Celsius in 400 ul 1X PBS solution. The supernatant was decanted, and the beads were washed three times with 400 ul of the 1X PBS Solution. While the protein was incubating, N35 RNA was denatured at 65 degrees Celsius for five minutes, and cooled down to 25 degrees Celsius, thereby removing tertiary structures. The beads were exposed to the RNA pool, where Tau protein, Nickel beads, and the N35 RNA pool was incubated for 25 minutes at 25 degrees Celsius as described initially in the round conditions above. Supernatant was removed to represent the wash 0 binders. The beads were then washed in 100 ul 1X Binding Buffer for three more times, and the supernatant was removed in separate quantities. The bound species was then eluted from the beads using 400 ul of 80 degrees Celsius diH2O. This step was repeated two more times, each time removing supernatant to be used later. Water was added to each wash volume to reach 400 ul, and ethanol precipitation was performed to concentrate the RNA. Wash 0, Wash 3, and the Eluted reactions were precipitated and re-suspended in 10 ul diH2O.

Reverse transcription was carried out with 7 ul of the resuspended RNA, 20 uM of N35 reverse primer, and 500 uM of dNTPs. After heat denaturing the reaction for 5 minutes at 65°C, 1X First Strand Buffer, 10 mM DTT, and 1 ul of SuperScript II Reverse Transcriptase enzyme was added. The final solution was subjected to 42°C for 50 minutes, and 70°C for 15 minutes, providing optimal conditions for Reverse transcription.

PCR reactions allowed for the amplification of the N35 pool. Cycle course PCR reactions were prepared, using 1X PCR buffer (lab stock), 200 uM dNTP, 400 nM N35 T7 forward primer, 400 nM of N35 reverse primer, 2 ul of the ssDNA from reverse transcription, and 2.5 U of Taq DNA polymerase. As Figure Two exemplifies, the optimal number of cycles for PCR was 15 cycles. Recall the temperature conditions per cycle of PCR involves initial exposure at 94°C for two minutes, denaturing at 92°C for 45 seconds, annealing at 54 degrees Celsius for 45 seconds, and elongation at 72°C for 60 seconds. A large scale PCR reaction was carried out for 15 cycles with 1X PCR buffer (lab stock), 200 uM dNTP, 500 nM N35 T7 forward primer, 500 nM N35 reverse primer, 2 ul of the ssDNA obtained from reverse transcription, and 2.5 U of Taq DNA polymerase. Ethanol precipitation concentrated the PCR product. A transcription reaction was prepared using 1X Ampliscribe Transcription Buffer, 10 mM DTT, 7.5 mM of each NTP, 5 ul of dsDNA from lsPCR, and 2 ul of T7 enzyme solution. The reaction was incubated at 42°C for approximately 3 hours before adding 1 ul DNase I at 37°C. After pouring, loading, and visualizing the PAGE gel with the RNA product from Transcription, RNA was visualized and eluted from the gel using the RNA Crashing Method.

Problems Enountered

As mentioned earlier, the original Cycle Course PCR gel showed a positive “No Template Control”. This implies that there is an artifact within one of the reagents, possibly 10X PCR Buffer, or one of the primers specific to the N34 pool. It was decided to restart the round using the N35 RNA pool instead to prevent the risk of contamination.

Additional Work

The RNA crashing method was used to recover the RNA from the PAGE gel and elute it in a concentrated solution suitable for starting the next round of selection. This new technique was introduced during the summer and mastered this semester. In addition, I learned how to create fresh 3.8% Agarose gel for the benefit of the entire stream. A significant amount of time was spent making personal aliquots of primers, deionized water, and a stock solution of 10X TE.

Conclusion and Future Steps

As shown below in Table 1, the initial concentration of RNA was 3679.4 ng/ul. Such a large concentration indicates the presence of background binders interfering with the beads of the tube’s surface. To confirm that this concentration was valid, the concentration of diluted RNA was recorded. This value (280.6 ng/ul) was used to calculate a final concentration of 109.9 uM, confirming that conditions for the next round will need to be altered to allow for more stringent binding. For Round 2 of selection, the wash volume will be doubled to remove any non-competitive binders . If successful, then the concentration of RNA yielded from the previous round will be significantly higher, proving the background binders were eliminated.

Table 1: Current Progress for Tau Aptamer Bead Based Selection with N35 Pool
Round	LsPCR Cycles	Quantity of NA used	Wash # and volume	Concentration (ng/ul)	Concentration (recovered in uM)
1	12	200pmol	3w/1v	3679.4 ng/ul	109.9 uM
2	Pending	400pmol	3w/2v	Pending	Pending

Nucleic Acid Aptamer Selection Against Hemagglutinin for the Inhibition of Influenza Binding Capabilities to Human Cells - Shaan Patel

Shaan Patel

November 30, 2011

Fall 2011

N50 RNA - Hemagglutinin

Nucleic Acid Aptamer Selection Against Hemagglutinin for the Inhibition of Influenza Binding Capabilities to Human Cells

The H1N1 Influenza virus, along with many other flu strains, is very easily transmissible and presents a range of symptoms that includes fever, mild respiratory infections, and some severe respiratory complications such as pneumonia that can lead to death (3). Along with seasonal epidemics in several countries, a few of the strains have created pandemics such as that of the H1N1, killing some 20 million people in 1918 (1). This RNA virus replicates itself in the human body by first binding to sialic acid sites on erythrocytes and epithelial cells, causing the host cells to agglutinate (4).

The Hemagglutinin (HA) Influenza protein is the surface-binding protein that the virus uses in attaching to host cells, and is dubbed after its mechanism in agglutinating red blood cells into visible clumps (1/2). HA is made up of two different types of chains, one that detects sialic acid on human cell-surface glycoproteins, and another that facilitates in the attack and agglutination of cells. The severity of each strain of influenza is based on the slightly different structures of the HA protein, however in general, the HA protein is the only method by which the flu virus can bind to host cells and begin replication and agglutination (4).

The HA binding protein is essential for the influenza virus, and without it, there is no host cell for which the virus can tether itself to for mass RNA replication (1). By understanding the binding properties of Hemagglutinin proteins, a nucleic aptamer can be developed in order to inhibit the HA protein from binding to host cells. Such a therapeutic aptamer can be used to contain seasonal epidemics and worldwide pandemics, thereby lifting public health standards and possibly pave the way for the inhibition of future mutated strains of HA.

Specific Aim 1: Selection of RNA Aptamers against Hemagglutinin.

As stated, HA is a crucial surface-binding protein for the influenza virus to attack the cells of the human body and propagate the replication of the virus throughout. The protein has sequences that make up the head, which fits into sialic acid sequences of human cells like a lock and key (2). In this case, an aptamer can be selected in order to alter the confirmation of the HA protein, or attach to the fusion peptides of HA, thereby preventing sailic acid from binding to the protein (1). As displayed in Figure 1, once the virus enters the body, its hemagglutinin proteins will bind to human cells via sialic acid sites. The specific aim is to inhibit the function of the protein, thereby impeding the virus’s ability to use the host cell for infection and mass replication.

The H1 strain of the hemagglutinin protein can be purchased through Immune Tech Corp (http://immune-tech.com/) for the price of $349 per 100 ug. The catalog number for the order is IT-003-00101∆TMp.

-------------------

The information above is my original abstract.

Here's the link to my Final Manuscript

Here's the link to my Progress Report 1

Here's the link to my Hemagglutinin Proposal

Thanks and Enjoy,

Shaan Patel

Juan A. Herrejon September 16, 2011 Fall 2011 Pool N34, RNA, Target CCR5

Juan A. Herrejon
September 16, 2011
Fall 2011
Pool N34, RNA, Target CCR5


Here is a link to my proposal

https://docs.google.com/document/d/1ieL2RrZwu9tX-kk_NCue7Wuq2FvsybXMMRhjQlhyBIM/edit?hl=en_US

*Note: Formatting on google docs altered original format and lengthened total page count to 9. Although all formatting guidelines were followed, the google doc does not look identical to a word document.

Abstract into Proposal Formatting

So technically in the rubric, it didn't say anything about line spacing for the abstract, just that it had to be exactly 1 page. However, in the guidelines for the full proposal, it says 1.5 line spacing. So when I go back and make my abstract 1.5 spacing, it will definitely be more than one page (and this is the one that I had uploaded on the aptamer blog) Should I try to condense it? Or not worry about it? Or can you allow the abstract to take more than one page on the proposal? The latter will definitely be helpful in reaching the page limit criteria! :)

RNA Selection Against Burkholderia pseudomallei (BPM) to Accelerate Burkholderia Diagnostics and Aid in Treating Melioidosis

By: Arsany Gadallah

Melioidosis is a commonly misdiagnosed infection that can be presented as a fatal case of pneumonia or sepsis. The presence of melioidosis has been proven to be related to rainfall and extreme weather cases such as in the tropics (Baker and Tahani D, 2011). It has been spreading across the globe from northern Australia, several African areas and Asian countries to neighboring countries within the Americas at a fast rate. The causative agent of melioidosis is Burkholderia pseudomallei (BPM), which is a motile soil-borne bacterium that infects humans and animals, inducing symptoms such as chest pain and engendering cases of sepsis (Cheng and Currie, 2005). The disease has a mortality rate of 20-68% (White NJ and Dance DA, 1989), ranging from acute cases to severe and blood-borne cases. Both forms of the disease, acute and chronic forms, have called for attention from public health institutions, pointing out melioidosis as an endemic. Regarding the slow response to the current treatment of the disease, global researchers have been looking for faster, more responsive therapeutics to minimize the morbidity of the disease.

A vaccine, however, is being developed through extravagant, costly techniques, which would cause economic hardships to many infected areas (Cheng and Currie, 2005). Therefore, institutions are looking for cheaper and faster methods to treat melioidosis. One way to carry out such a task is to utilize nucleic acid oligonucleotides. Through the usage of aptamers, identification of the bacterium is possible. BimA, a trimetric Autotransporter/ macromolecule secreted by BMP (Brown and Iverson, 2011), which is an integral protein on the outer surface of B. pseudomallei, is functionalized with enough Histidine tags and actin-nucleating factors for a SELEX process using Nickel-NTA beads (Lazar and Stevens, 2011). Through selection, a fast therapeutic use of aptamers can help in treating the infection with accurate resources.

Specific aim 1: An aptamer, a precise nucleic acid sequence, is needed for selection against BMP through Nickel-NTA beads. The beads should help the protein and the RNA to meet and bind. The RNA sequence has to bind Burkholderia pseudomallei at the BPS site in order to activate a signal transduction that would aid in indentifying the bacteria and in accelerating the diagnosis and treatment of the infection (melioidosis). This therapeutic advantage of aptamers would be a congenial treatment, considering the precision and efficiency of SELEX rounds. If the selection is successful and the goals are achieved, inhibition of the Burkholderia bacteria would be easier and faster. Similarly, the same process can be applied onto Burkholderia mallei targets and similar results can be expected in treating glanders, since both B. pseudomallei and B. mallei are closely related.

Figure 1. An aptamer is needed to bind the Burkholderia pseudomallei (BPM) bacterium at the surface (BimA) for identification, which would later help identifying the bacterium and avoiding the enhancement of melioidosis.

***The target is on the “Recommended and Available” list of targets provided by Dr. Katy Brown, found in the -80⁰C freezer. The protein is sold by the company ATCC (ATCC.org) for $215 per 5 ug genomic DNA. Catalog # BAA-244D-5***

References:

1. “Groundwater Seeps Facilitate Exposure to Burkholderia pseudomallei.” Appl Environ Microbiol. 2011 Aug 26, Baker A, Tahani D, Gardiner C, Bristow KL, Greenhill AR, Warner J.

2. PLoS Negl Trop Dis. 2010 Nov 30;4(11):e900: “The epidemiology and clinical spectrum of melioidosis: 540 cases from the 20 year Darwin prospective study. Currie BJ, Ward L, Cheng AC.

3. J Infect Dis. 1989 May;159(5):890-9. "Melioidosis: a major cause of community-acquired septicemia in northeastern Thailand." Chaowagul W, White NJ, Dance DA, Wattanagoon Y, Naigowit P, Davis TM, Looareesuwan S, Pitakwatchara N.

4. Faraday Discuss. 2011;149:23-36; discussion 63-77. "Development of reagents and assays for the detection of pathogenic Burkholderia species."Qazi O, Rani M, Gnanam AJ, Cullen TW, Stead CM, Kensing H, McCaul K, Ngugi S, Prior JL, Lipka A, Nagy JM, Gregory CW,Judy BM, Harding SV, Titball RW, Sidhu SS, Trent MS, Kitto GB, Torres A, Estes DM, Iverson B, Georgiou G, Brown KA.

5. Front Microbiol. 2011;2:151. Epub 2011 Jul 15. "Autotransporters and Their Role in the Virulence of Burkholderia pseudomallei and Burkholderia mallei. Lazar Adler NR, Stevens JM, Stevens MP, Galyov EE.

LINK TO PROPOSAL: http://dl.dropbox.com/u/42009520/Proposal%202.docx

LINK TO PROGRESS REPORT 1: http://aptamerstream.blogspot.com/2011/10/progress-discussion-problems-and.html

LINK TO FINAL MANUSCRIPT: http://dl.dropbox.com/u/42009520/Final%20Manuscript.docx

Touria Rguig

08/30/2011

Tr7356

N59, Hemoglobin S

Nucleic Acid Selection against Hemoglobin S to Prevent the Aggregation of Red Blood Cells in Sickle Cell Anemic Patients

Hemoglobin S differs from normal adult hemoglobin called hemoglobin A only by a single amino acid substitution; a valine replacing a glutamine in the 6th position of the beta chain of globin [1]. This single amino acid substitution causes a genetically inherited disease called sickle cell anemia. This disease results in sickle-shaped red blood cells that deliver less oxygen to the body's tissues. These abnormal cells can also get stuck more easily in small blood vessels, and break into pieces that interrupt healthy blood flow.

Valine is a less polar amino acid than glutamate and thus favors hydrophobic interactions between each strand and its neighboring amino acids such as leucine and Histidine [1]. These types of interactions cause the red blood cells to aggregate and become sickled shaped preventing regular blood flow through capillaries and dramatically decreasing the hemoglobin’s high oxygen-binding affinity [1].

Selecting an aptamer against hemoglobin S to inhibit the polymerization of red blood cells caused by valine’s reactivity can prevent the sickling of blood cells. Also, it can potentially restore the high oxygen binding affinity of hemoglobin. Although there are drugs such as “Hydroxyurea” that enhance the quality of life of sickle cell anemic patient, this aptamer could be very specific and causing no detrimental side effects[2].

Specific aim: Synthesis of a specific aptamer that inhibits the aggregation of red blood cells in sickle cell anemic patients.

Hemoglobin can be purchased through Sigma-Aldrich for $204 per 25 mg. The catalog number is H0392-25MG .

References:

1- Lanzkron S, Strouse JJ, Wilson R, et al (June 2008). "Systematic review: Hydroxyurea for the treatment of adults with sickle cell disease". Annals of Internal Medicine 148 (12): 939–55.
2- (2008) Hydroxyurea for Sickle Cell Anemia. New England Journal of Medicine 359:1, 98-99

Here is the link to my proposal: https://docs.google.com/document/d/1so6mZ7vjYMlsYqiFvoKlozjp6_6ktJcX73dZ-vkq9xM/edit?hl=en_US

Here is the link to my progress report: http://aptamerstream.blogspot.com/2011/10/v-behaviorurldefaultvml-o_18.html
Here is a link to my new manuscript: https://docs.google.com/open?id=0By1AdiE2epJ_ZDQzN2E2MTEtZjc1Yy00OTZhLWI1NTgtMjYxY2EzMWU0Mzdl