have you considered this target?

USP14 "a deubiquitinating enzyme (DUB) that interferes with the breakdown of misfolded proteins. Since misfolded protein accumulation is thought to play a role in neurodegenerative diseases, control over their degradation could lead to future therapeutics for conditions such as Alzheimer’s disease" (BioTechniques article, link below). If we inhibited this enzyme with an aptamer, then goodbye protein accumulation (or something like that) & cure for disease ...

http://www.biotechniques.com/news/biotechniquesNews/biotechniques-305005.html?utm_source=BioTechniques+Newsletters+%26+e-Alerts&utm_campaign=611317cb48-BioTechniques_Daily&utm_medium=email

Aliquots

There are new aliquots of N34 forward and reverse primers!

FRI External Open House

The FRI program is growing and we are in the process of sustainability. Therefore, over the next year there will be a big push to export and advertise ourselves. We are revising our core mission to cross the chassim from "Can we do this" to "Let's keep doing this". To this end, the FRI program is hosting an external open house on Thursday, October 28th from 1-4pm. The Open House is a very important event and we need the help of students just like you.

There will be a common welcome and poster session in the Texas Union Welcome Room from 1pm-3pm. In addition, I will host a number of lab tours with your help.

Attending:

• Companies with substantial College of Natural Sciences (CNS) undergraduate research (UGR) relationship
• Companies with current CNS relationships that we hope to nurture
• Educational, Medical and Graduate Programs
• Other colleges, UT Administration
• FRI Advisory Council (and associates)

We need help in two major areas:

A.) Lab Enthusiasm

These visitors want to talk to you – not me. Therefore, I want enthusiastic students to be engaged in research and available to chat with our visitors about what we do and why it matters. I need all levels of students and mentors and alumni. Please reply below (comment) if you are willing and able. As the event approaches, I will "groom" you on the tenants of the FRI and what to say (like I did with the poster), but I really want your enthusiasm to show through. Be ready to talk about YOUR project, why you chose it, what your target does in the body or pathway and what aptamers could do against your target.

B.) Walking tour guides (get people from lab to lab).

If interested – please reply to this poll: Survey Monkey. You should dress nicely for this as industry notices at that kind of thing.

Above all, never underestimate the power of networking. Consider this an opportunity to network with very focused individuals who are excited about what you do. I've never gotten a job by applying with resumes. They have all been due to networking and communication at the human level.

- Brad

Mentor Application Due NOVEMBER 5

Hello Future FRI Mentors,

I want to invite you to apply to be an FRI Peer Mentor to new FRI students beginning in the Spring 2011 Semester. The online application is available, and will be available until Friday, November 5. As you probably already know, mentors play a vital role in the FRI experience, and being a Mentor is a great opportunity for professional development.

The application for Spring 2010 FRI Peer Mentors is now available. In order to serve as an FRI Mentor, you MUST attend Peer Leader Academy training in January if you have not previously attended the August or January 2010 training sessions. We anticipate the training will be an all day session meeting on Thursday or Friday, January 13 or 14, 2011. You are expected to return to campus in time to participate in this training. If you cannot, you should not apply.

I apologize in advance if the application is confusing, appears to solicit information that seems irrelevant, or asks for the same information, over and over. The application is being used for several positions. If you are not applying for the other positions covered by this application, you need not answer questions 8-14. You may enter N/A in the first box of each reply, or check the "not applicable" box. You will be asked multiple times to indicate you are applying to be an FRI-Peer Mentor. You must check the corresponding box in both questions 15 and 17 and check yes for question 16, to be routed to the actual FRI Mentor Application. You must provide a reference (Question 20) of someone who is familiar with how you function in a research lab setting. Typically this is the Research Educator, or independent research supervisor. Your reference will be contacted to provide a reference for you, but you should contact them ahead of time to ask/give heads up.

Information about Mentoring can be found here:

http://web3.cns.utexas.edu/fri/beyond-freshman-lab/peer-mentoring-program

The application itself can be found here:

https://www.surveymonkey.com/s/CNS_PLA_Application_20109

Note that the application must be completed by November 5. We anticipate that decisions will be made quickly thereafter, and anticipate notifying all applicants by the middle of November.

If you have questions about what is expected of mentors serving in a particular FRI stream, you should contact Brad Hall or Gwen Stovall. Any general questions can be directed to Al MacKrell (mackrell@mail.utexas.edu).

Thanks.

Al MacKrell
FRI Peer Mentoring

SALSA COOKOFF FOR BREAST CANCER EDUCATION AND AWARENESS

Hey guys!

Zeta Tau Alpha (the sorority I'm in) is having a Crown Cookoff, or a Salsa Cookoff, this Friday from 1-4 in the SOUTH MALL. It is going to be so much fun, so it would be great to have all you guys there! The purpose of the cookoff is to raise money for Breast Cancer Awareness and Education. The tickets are 5 dollars each and you can get them from me either tomorrow during meeting or throughout the week in lab. If you are interested, please tell me during meeting tomorrow or text me at 817-287-1648.

PLEASE PLEASE PLEASE think about coming. I really want to introduce all my lab mates to my sorority friends!!!!

HAVE A WONDERFUL WEEK!!!

Crush Soak of PAGE

We are all out of red dot tubes! Fortunately, yellow dot tubes can be used for the same purpose. We will have more red dot tubes shortly!

Hcp1 Binding Assay Update: Corollary to Current Progress

Within the initial post addressing my current progress with Hcp1 SELEX, I concluded with a statement foreshadowing an eventual identification and exposition of binding assay data. Finally, considering all of you are so painstakingly curious, the very forces which undergird your lives pausing briefly upon my every syllable, here it is!

Reproduced below is a composite image, identifying the data yielded by the recent binding assay in three distinct formats representing the analytical progression of the unfashioned radioactive imprints (a) unto the final graphical depiction (c):

Upon first glance, in this case an eminently accurate perspective of the data offered, the binding assay is particularly inconclusive, in may ways highly similar to the initial binding assay casting rounds two, four, six, and eight performed in late June. Like this binding assay, the antecedent assay featured such characteristics as: comparatively uniform degrees of nonspecific vs. specific binding, nonspecific binding superseding that of specific binding on all occasions, and generally, high quantities of both binding types. Unfortunately, this confers virtually no sentiments of success to my attempts over the past several months, as the RNA pool I have evolved via sixteen round of SELEX seeming bears effectually equivalent proportions of nonspecific and specific binding species, differing by an approximate mean of 0.74%.

A potential conclusion which, I feel, may be equitably gleaned from this data, upholds the speculation virtually pervading the progress section of October 11^th; that is, assuming a pool possessing inherently tenacious, sticky specifies, one would anticipate a certain amount of nonspecific filter-binding, in addition to the specific binding observed. Naturally, this postulate fails to address the exact quantitative relationships among the two types of binding or the extent to which nonspecific filter-binding would occur with a pool intentionally engineered for sticky properties.

Extant possibilities for future selection include the following:

1. Reinstatement of early round (potentially R3) coupled with highly stringent selection techniques: i.e. negative selections, wash volumes, etc. which will hopefully function for the elimination of nascent nonspecific binding, after which a greater amount of target-binding sequences will be amplified at a comparatively greater rate

2. Complete selective restart with R0 N34 RNA

o However, featuring the following methodological manipulation: initial round selecting solely for beads, lsPCR amplification of W0 for elimination of initial bead-binding species, R2 initial target round with R1, W0 RNA.

3. Some other outlet suggested by incredibly kind aptamate/Brad/Gwen/mentor… :)

Infomercial...do this!

My name is Ryan Hirsch. I am a member of the Senate of College Council’s Undergraduate Research Committee. This year I am creating an informational video that will educate students on undergraduate research opportunities and encourage them to get involved. I am looking to interview undergraduate students that are involved in research currently across all disciplines. If you are interested, please let me know. I will be conducting interviews over the next few weeks. The interview will not take up a large amount of your time, and it will be essential to the success of this video as a tool for promoting undergraduate research. I can be reached by phone or by e-mail. My contact information is below.

I would greatly appreciate your time. Thank you in advance.

Ryan Hirsch
Undergraduate Research Committee | Senate of College Councils
The University of Texas at Austin
(972) 955-1091 | ryan.hirsch@mail.utexas.edu

Protein target arrival!

Recomb. Hemagglutinin is stored in 4C fridge.

Arrived 10/22/2010

3.8% Agarose

Please make them in the small bottles (0.5L). It takes too long to heat and cool larger volumes of agarose.

Thanks.

New Targets to consider from the conference

Think about these targets:

Most of the Oligonucleotide Therapeutics Society (OTS) 6th annual conference has discussed therapeutic siRNA and RNAi. I'll be "live blogging" :) or rather note-taking on the blog when cool talks come up. Anytime people start talking about these technologies, it is usually to knock down a particular protein (inhibition) which also lends nicely to aptamer related work.

Talk given by Claes Wahlestedt, MD, PhD, Scripps Research Institute talked about BACE1 beta-secretase. Known to be involved in Alzheimers disease, cleaves amyloid beta. Inactivation by siRNA reduces by BACE-1 and provides recovery in diseased mice. Mayank is currently working on APP, with current results.

He also talked about BDNF (Brain-derived neurotrophic factor) for neuronal growth and maturation, synaptic plasticity, Alzheimer's disease, etc. The expression is most active is the Ectx portion of the brain in diseased patients. Knockdown of the antisense mRNA results in outgrowth of hippocampal nerve cells due to increased BDNF production. So, this may not be a good target (since it appears BDNF is good for the body). Recent paper Filion et al, Cell, October 15, 2010.

Jiehua Zhou, PhD, Beckman Research Institute, City of Hope working in John Rossi's lab is looking at aptamer siRNA/dendrimer/aptamer conjugates for gp120 as a therapeutic for HIV. There are a few HIV targets that would also be interesting. Arshia thought about CCR5 previously, It sits with CD4. gp41 and other exterior targets on.

Michael Lahn from Lilly Research suggested Survivin, also called baculoviral inhibitor of apoptosis repeat-containing 5 or BIRC5, may be a good target for cancer therapy. By inhibiting survivin activation pathways apoptosis and decrease in tumour growth are promoted. Survivin is upregulated in most human cancers and not present in normal differentiated cells. Therefore, it makes an ideal diagnostic and therapeutic target. It is also commercially available from ProSpec.

10x PBS vs 1x PBS

There has been some confusion about when to use 10x PBS and when to use 1x PBS. I believe this confusion stems from a misunderstanding of the "10x" or "1x" concentration notation. The working concentration of a buffer or solution is always 1x.

For instance, a Coke in an aluminum can is at a 1x concentration, while the syrup that goes into the soda machine to make the Coke is at a 10x concentration (or something like that). That means, the syrup has to be diluted 10-fold to get to the regular 1x concentration of Coke.

So - if you're trying to figure out what concentration of PBS to use, spend some time thinking about what concentration you want it to be. If you're reconstituting protein target into 100 uL of buffer. You may suspend it in 100 uL of 1x PBS or you may suspend it in 90 uL water + 10 uL of 10x PBS. Does that make sense?

I hope this helps.
:)
Gwen

1027, Sequencing Data, Oct 19, 2010

Katherine,

Can you take a look at the following sequences (the clone check) and decide to re sequence some of them. Look if these have weird bands or look good and also nanodrop to determine concentration. Compare good sequence data to failed sequence data. Use the PCR product. Dilute the product 1:30 and resubmit 1ul of dilution + 10ul di H2O. Use the M13R (-24) primer for sequencing. I think there was a problem.

Good

16, 16, 11, 14, 16, 17, 24, 25, 29, 30, 33, 34, 37, 38, 39, 9, 8

Failed

15, 18, 26, 28, 32, 36, 1, 2, 4,

Let me know if the above observations show some reason why sequencing failed/worked

Targets

When you receive a target please mention where you placed it! Also, if your target has arrived please come in to aliquot it as soon as possible or at least place it in the right conditions. The person receiving it is not aware of your protein sensitivity and may place it at 4 degrees when -80 degrees is required!

How well does ccPCR indicate the presence of background binders?

I started carrying both + and - protein selected RNA to ccPCR at R3. The ccPCR gel images can be seen here. R3 showed nearly identical amplification between + and - protein. R4 seemed to show a reduction in background binders through the later amplification of - protein selected DNA. Finally, R5 showed nearly identical amplification of + and - protein again. I was wondering how clear of an indicator this is as to the current composition of the selected pool. If we are still performing binding assays after R6, I am not to far from determining the real binding of my pool, but I was wondering if the evidence from the ccPCR gels is enough to go ahead and split of another selection from R4, the round that seemed to show a reduction in background binding.

Desalting Protocol

A follow up on Mimi's post regarding functionalizing your protein, this is the desalting protocol you will use! Note there are two sizes of columns make sure to use 0.5 mL columns!

Desalting Procedure
For maximal protein recovery, add 3 μl of buffer or water above the sample as a stacker. This stacker may be omitted if desired; however, a reduction in protein recovery will result.
A.
Additional Materials Required
•
Variable-speed bench-top microcentrifuge
•
1.5 microcentrifuge collection tubes
B.
Spin Column Preparation
1.
Remove column’s bottom closure and loosen cap (do not remove cap).
2.
Place column in a 1.5-2.0 ml microcentrifuge collection tube and centrifuge at 1,000 × g for 1 minute to remove storage solution.
3.
Blot bottom of column to remove excess liquid.
C.
Sample Loading
1.
Place column in a new collection tube, remove cap and apply 2-12 μl of sample to the top of the compact resin bed.
2.
Apply a 3 μl stacker of ultrapure water or buffer to the top of the resin bed after the sample has absorbed.
3.
Centrifuge at 1,000 × g for 2 minutes to collect the desalted sample. Discard desalting column after use.

Out of Nickel Beads?

Does anyone know if we are out of nickel beads?...if not, do you know where they are? I cannot find them in the fridge.
Thanks,
Austin

More targets have arrived!

10-15-2010

EphA2

Thrombin

Factor 1Xa

Biotinylation of Targets

For those who need to biotinylate your proteins, here's the protocol. Gwen will have it on Blackboard as well.

Biotinylation of Protein

The N-hydroxysulfosuccinimide (NHS) ester group on this reagent reacts with the e -amine of lysine residues to produce a stable product. Although a -amine groups present on the N-termini of peptides react with NHS esters, a -amines on proteins are seldom accessible for conjugation.

NHS esters react with deprotonated primary amines, therefore, the reaction requires neutral to basic pH values to proceed. Primary amines react with NHS esters by nucleophilic attack and NHS is released as a byproduct. Hydrolysis of the NHS-ester competes with the reaction in aqueous solution and increases with increasing pH.

With large proteins, labeling of several lysine residues and the N-terminus does not usually harm protein function or binding properties. With short peptides, however, the random biotinylation of the ε-amino groups of lysine residues is much more likely to block binding sites whose function is necessary for downstream applications of the biotinylated peptide. Labeling of lysine residues in peptides, especially short ones, may alter their functional properties and/or immunoreactivity.

Preparing the Target

1. Find out the molecular weight of your target. Most range between 2000 and 70,000 Daltons. A Dalton is a measure of grams/mol or ug/umol.
1. If your target is more than 1mg in the vial, then also find out the extinction coefficient.
2. If it is under 1mg (say 100ug?), then determine if it is already resuspended in a buffer.

i. If it is in solution determine what the solution is. Tris buffers have primary amines and react with the biotinylation reagent.

2. Calculate the molar concentration or quantity of your target.
1. i.e. if you have 100ug of target and it is 34kDa, then (100ug / 34000ug/umol) = 0.00294umol or 2.94nmol or 2940pmol total.
2. If the above protein was in 100ul it would be at a concentration of 2940pmol/100ul = 29pmol/ul or 29uM.
3. If your protein is in powder form (desicated), then resuspend it in a suitable volume of buffer.
1. Typically, you should consider between 20 and 200uM concentration. You can certainly go higher, but try not to go much lower.
2. 100 to 200ul of a 1X PBS buffer or 50mM HEPES buffer work well. Try to use the buffer (not with salts added) that you plan to perform the selection with.
3. DO NOT use Tris buffer.
4. Calculate the volume of target to biotinylate.
1. Typically you should biotinylate between 1000 and 4000pmol protein total. However, please don’t biotinylate all of your protein in case something goes horribly wrong. In the above example, I would suggest biotinylating 1500pmol. 1500pmol / 29pmol/ul = ~51ul or half the total volume.
5. Aliquot any remaining protein in easy use volumes in 0.2ml tubes to prevent freeze thawing multiple times. Label the tube with the protein name.
1. Typically aliquots will be the same volume as used for step 4.
6. Label a sticky note with the target name, supplier, stock number, lot number, concentration and volume per tube. Also put your name and the date it was first aliquoted.
7. Place aliquots in the -80°C freezer.
1. Mark the empty supplier tube with an X on the cap and place it in the freezer.
2. In addition, put the sticky note in the box.

Preparing the Biotinylation Reagent

8. Remove EZ-Link Sulfo-NHS-LC-Biotin (Pierce product 21335) from the desiccator (blue saucer) in the -20 °C freezer box and allow it to warm to room temperature (5 minutes).
9. Add 2 mg of EZ-Link Sulfo-NHS-LC-Biotin to ~200 uL of 50mM HEPES, pH 7.5.
1. MW of Biotin is 556 g/mol. 2 mg gives 3.6 umole reagent. If mixed in 200ul buffer, the concentration is 18nmol/ul.
10. You should add between 5x and 10x EZ-Link reagent to target. Because the reagent starts to hydrolyze once its dissolved, it is active for only a short period of time.

Biotinylating the Target.

• The reagent will attack primary amines on the protein (or buffer if using Tris). These are found at the N-terminus (NH₂) or lysine residues.
• If you use too much reagent, the protein will denature when immobilized to the bead.
• If you use too little reagent, some protein molecules may not be biotinylated and therefore may be lost during washes.
• It may be necessary to choose multiple molar excesses and test each for protein function/binding after biotinylation with some known property.

11. Add X ul of step 4 to Y ul EZ-Link reagent from step 6. This is your total Z ul volume.
1. In our example, we should add 51ul protein (volume X) to 1ul EZ-link reagent (volume Y) for 9x molar excess.
2. The unused Biotin solution should be discarded. Try to biotinylate with others as this reagent is expensive and much is wasted.
12. Refrigerate the reaction at 4 C for >2 hours – overnight.
1. Shaking or agitation during incubation is prefered but not required.

Purifying the target.

• It is important to remove any unbound biotin from the reaction. Therefore a desalt column should be used with a molecular weight cutoff below your target.

Zebra Colum Sizes and recommended sample volumes
Column Size	Resin Bed	Sample Size
Micro	75ul	2-12ul
0.5ml	0.5ml	30-130ul
2ml	2ml	200-700ul
5ml	5ml	500-2000ul
10ml	10ml	1500-4000ul

• The NHS-Biotin reagent has a molecular weight of » 600 Da. The Zebra spin columns have a MWCO of » 7000 Da (anything smaller will be trapped).
• Sephadex can also be used. G-10 has a MWCO of ~ 700Da. G-25 has a MWCO of ~ 5000Da, G-50 has a MWCO of 30kDa and G-100 has a MWCO of 100kDa.
• Use the proper column volume for your total reaction (see table).

13. Measure the absorbance of reaction on the nanodrop using the “Protein A280” button. Print out the graph.
14. Follow the desalting protocol on Blackboard with half your sample.
15. Remeasure the absorbance and compare the A280 measurements before and after.
16. If the second measurement is within 50% of the first, desalt the remaining protein over the same column.
17. Discard the desalting column after use.

Prepping the Target for Immobilization.

• Provided sufficient labeling of protein, you should have recovered it all in the volume of buffer your initial reaction (Z ul, step 8).

18. Calculate the concentration of your protein. You can do this spectrophometrically if you know the extinction coefficient, or assume a rough concentration.

1. Your concentration of protein should be Q pmol used for for biotinylation (from step 4) / Z ul biotinylation reaction.
2. From the example above, we had 1500pmol in a volume of 51ul. 1500pmol / 51ul » 30 pmol/ul or uM.

19. If you lost some sample in the desalt procedure (your final A280 measurement is significantly less than your initial A280 reading) then you must adjust by first calculating the difference and multipling through by that factor.
1. For instance, if the initial measurement was 22.5 and the last measurement is 18.7, then you recovered 18.7/22.5 or 83%. Assuming you initially biotinylated 1500 pmol then you recovered .83*1500 or roughly 1250 pmol.
20. Aliquot your protein into tubes for long term storage. Label the tube with the protein name-Biotin.

3. Aliquot 100pmol target per tube.
4. In our example we would use ~ 4ul per tube or 12 tubes.

21. Label a sticky note with the target name-Biotin, date the reaction was performed, concentration volume and your name.
22. Place aliquots in the -80°C freezer in the same box as the unbiotinylated target.

5. In addition, put the sticky note in the box.
6. Mark where your protein is on the freezer map on the door of the -80 °C.

Summer Fun 2011

I have great news! There are very exciting opportunities out there that will give people an opportunity to do research in other labs across the country for ten weeks throughout the summer. UT is one of the best public research universities in the country and because of this many people think highly of the students.

As a recipient of some of these awards I highly encourage everyone to apply to these programs. They are ten weeks long, for the most part your housing is covered and you are given a stipend for the summer. The application process seems long but it is a great way to keep all of your college accomplishments up to date.

UT Southwestern Medical Center partners with UT to give undergraduates this opportunity. This program runs from June 6, 2011 to August 12, 2011. The application can be found at:

www.utsouthwestern.edu/QP-SURF. .

is due the latest by February 9, 2011. I encourage everyone to look into it whether you are planning to go to medical/dental/graduate (basically any kind of professional school). They have more information on the website for you to look at. Please look at this early -- they require transcripts and rec letters (Brad does not want to be bombarded on February 1st). On a general note, you should ask professors for rec letters at least a month in advance!!

There are many programs out there like this and people are willing to give you a chance to work in their lab without knowing you which is a very big deal! You have a chance to meet people in the field and ask questions and develop relationships.

I highly encourage everyone to do it even if it is to live in a new city for free for the summer! If you have any questions on summer programs in general you can ask me at any time. If you have questions about this program in specific I am happy to lead you in the right direction in having the questions answered.

Lab Attendance Hours

I am posting an auto-updating chart for lab hours:

Posters on the Hill 2011

Hi all,

Council on Undergraduate Research (CUR) is hosting its 15th annual undergraduate poster session next spring on Capitol Hill. This is a chance for you to gain more experience telling people, specifically Congress members, more about the research we do here on campus and why it's so important. This is a great opportunity to be recognized for all the work you've put into your projects.

If you're interested, please send Brad an email. It requires you to fill out an application, get letters of recommendation, write an abstract, and provide contact information by November 15th.

For more information, go to: http://www.cur.org/pohcall.html

Current Progress on Aptamer Selection for gag p24

The abstract to this proposal can be found here

Progress, Results, and Discussion

Three rounds of selection using N34 RNA pool, gag p24 as the protein target and PBS SELEX as the buffer solution have been performed.

Perameters for the First Round of Selection:

RNA:Protein- 400pmol:200pmol

Negative Selection- 40ul for 50 minutes

Selection with Protein- 25 minutes

Wash Volume of 1X PBS- 200 ul

Cycle Course on 3.8% Agarose Gel with Ethidium Bromide

Perameters for the Second Round of Selection:

RNA:Protein- 400pmol:200pmol

Negative Selection- 40ul for 32 minutes

Selection with Protein- 23 minutes

Wash Volume of 1X PBS- 200 ul

Cycle Course on 3.8% Agarose Gel with Ethidium Bromide

Parameters for Thrid Round of Selection:

RNA:Protein- 200pmol:100pmol

Negative Selection- 40cl for 32 minutes

Selection with Protein- 15 miuntes

Wash Volume of 1X PBS- 300 ul

Cycle Course on 3.8% Agarose Gel with Ethidium Bromide

There are two remarkable results that can be seen in my first and second cycle course. The first is that despite a lengthy negative selection in the first round, my elution visibly amplified at twelve cycles. Also, in both my first and second rounds, there is no visible amplification in the W3 and a good amount of amplification in the elution, despite the low wash volumes and the early stage of the selection. From the first to second round of selection, I didn't want to change my conditions too much, because I wanted to allow my pool to stabilize. However, when I received the same promising results in the second cycle course as I did in the first cycle course I decided to to increase the stringency of selection. This led to a large amount of RNA in both washes and the elution, which is at first glance odd, because I increased my wash volume. However, I postulate that the increase of RNA in my third wash is due to the decrease in incubation time and possibly due to the decrease in protein. Next round, I plan to perform a +/- selection to see if the large amount of RNA in my elution is due to protein binding or bead binding. I am also going to drastically increase my wash volume to 500ul per wash.

Three more rounds of selection using N34 RNA pool, gag p24 as the protein target and PBS SELEX as the buffer solution have been performed, since the last update.

Parameters for the Fourth Round of Selection (+/-):

RNA:Protein- 200pmol:100pmol

Negative Selection- 40ul for 33 minutes

Selection with Protein- 15 minutes

Wash Volume of 1X PBS- 500 ul

Cycle Course on 3.8% Agarose Gel with Ethidium Bromide

Parameters for the Fifth Round of Selection:

RNA:Protein- 200pmol:100pmol

Negative Selection- 80ul for 39 minutes

Selection with Protein- 12 minutes

Wash Volume of 1X PBS- 500 ul

Cycle Course on 3.8% Agarose Gel with Ethidium Bromide

Parameters for the Sixth Round of Selection:

RNA:Protein- 150pmol:100pmol

Negative Selection- 80ul for 38 minutes

Selection with Protein- 10 minutes

Wash Volume of 1X PBS- 500 ul

Cycle Course on 3.8% Agarose Gel with Ethidium Bromide

The most important thing to note about these cycle courses is that in the R4 +/- selection the E4(-) amplifies earlier than the E4(+), so there may be substantial nonspecific binding. A filter based binding assay was performed after the sixth round of selection, there was no relevant binding. The results of the binding assay can be seen below. It should however be noted that the binding assay was done on a filter, where as the selection was performed on magnetic beads. Also, tRNA was used in the binding assay and not in the selection. Both of these factors could contribute to the low amount of binding.

Progress on INF-g (Ashley Dawson)

Abstract for this target can be found here
The target protein (INF-g) arrived Friday, 10/1 and selection began the following Monday.

Round One Conditions
Target: IFN-g with 6X His-tag
Beads: Nickel NTA
Pool: N34
Ratio of Pool to Target: 200:200 pmol
Buffer: 1X PBS with MgCl2
Incubation Time/Temp: 30 min at 37C
Wash Volume/Number: 3 washes, 2 volumes

A PCR cycle course was performed to determine the optimal number of cycles for a large scale PCR of the sequences that were eluted off the beads (theoretically the strongest binders).



This shows amplification very late for the elution (optimal is showing around 18-20 cycles) and very early for W0, indicating that not very many sequences bound tightly to the protein. While the early amplification for W0 indicates many weak binders, the lack of amplification in W3 implies that the wash process is possibly too stringent for the first round.

Large scale will be performed for eighteen cycles and the DNA will then be transcribed and purified for the next round if there is a high enough ending concentration.

Current Progress: Hcp1 SELEX

As-opposed to providing a mind-numbingly exhaustive account of the current target progress I have protracted throughout the summer unto now, I find it more apt to refer all to the “Experimental Design and Current Progress” section of my complete proposal for a necessary and succinct review of pre-fall semester progress (in an effort to both legitimate my laziness and obviate encumbering most of this post with gratuitous data). Thus, below is a brief exposition of the current state of my research with Hcp1.

Initially, upon returning to lab in early September, a round of selection (specifically, Round 15) was performed in order to assess the resultant R14 RNA pool for latent degradation. Following successful completion, in preparation for a binding assay, a (+)/(-) protein selection was performed for the determination of the degree of specific (i.e. target-specific) and nonspecific binding (i.e. bead-specific binding).

Referring directly to Figure 6 of the complete proposal, and its apparent signification therein of increasingly specific binding independently of stringency manipulations, three conclusions were reached, and are as follows: specific binding, unwarranted nonspecific binding, and inherent pool “stickiness.” Specific binding, obviously the most desirable, could be examined against nonspecific binding via a simple (+)/(-) selection as mentioned before. In the context of pool tenacity however, a separate negative selection was performed on streptavidin beads. Now, the N34 pool is not intrinsically sticky, however, it is an extant possibility that, through various round of selection, strands featuring high concentrations of inherently sticky adenine/thymine bases may have been evolved. Each of these potentialities, and the extent to which they applied to the pool, were to be completely elucidated via the composite gel, depicted below.

As a pretext for a binding assay, this (+)/(-) selection was rather inconclusive, is it confers equivalent binding capacity for both (+) target and (-) target selection, decisively contradicting the definition of aptamer, i.e. specific nucleic acid binding agent. However, despite this ostensible failure, I have proceeded with the binding assay, as it may more conclusively establish the nature of select RNA pools derived via past rounds of selection, namely 8, 12, and 16. Additionally, assuming the pool primarily features nonspecifically tenacious or Ni-NTA-binding sequences, isolating them would offer at least one respectable use, reverse engineering them out of R0 pools for the prevention of bead-binding background.

At the moment, the binding assay is in progress, and should afford results at the end of the week, prompting a forthcoming addition to this report.

Undergraduate Research Journal

We have a great opportunity coming up. The undergraduate research journal is having an information session OCTOBER 12, 2010 from 5;00 - 6:30 pm at UTC 3.124

As you are all aware it is very important to get your name out there and this is an awesome way to do so and you can use the work you have been doing in the FRI. The undergraduate research journal has requirements that are very similar to a journal submission. As an extra incentive there is a cash priz that is awarded if your submission is selected.

I would highly recommend this opportunity to everyone because it exposes you to the process beyond the paper writing.

Good Luck!!

Progress Report- 1027

Binding assay data here

Cloning and sequencing started here

After a small scale clone check, a large scale amplification of 12 colonies per plate was performed and it was ascertained that from Round 2, all of the clones were sequenced, from Round 7A, clone 1 was omitted due to ambiguity of purity of sequences, from Round 7B, clones 2, 6, 8, 9 were omitted due to failure of amplification, and from Round 10B, everything was sequenced.

Following sequencing, in Round 2, it was complete randomness of sequences. In 7A, there was also randomness in the sequences, but in 7B, clones 1, 7, and 12 were almost identical in sequence, leading to the assumption that this particular sequence should have a higher binding affinity for the protein 1027. There was then an attempt to assemble the sequences together to establish similarities.

These are the three sequences from R7B that were identical. The primer regions are identified by the green arrows, while the space inbetween identifies the random oligonucleotide that is used for selection and binding to the target protein.

In round 7A, we were unable to find most of the primer regions for both the reverse and forward pool primers. The sequences were not purified enough perhaps, but we were unable to tell what the sequences of the oligonucleotides were.

In Round 10, there were primer dimer formations that are both bad and inexplicable, other than an annealing temperature that was too low at the time of amplification.

As shown, the primers line up against one another creating artifacts that amplify faster than other actual sequences. This shows an assembly of 7 of the 12 clones from round 10B that were sequenced. Unfortunately, artifacts cannot be removed, so if selection were to be resumed, it would have to be resumed at a round before 10.

The clones from R7B and R7A were grown up in LB + Amp broth overnight and then regrown to plasmid prep for cleaned plasmids with inserts. Unfortunately, after the second day, only 1, 10, and 11 grew in addition to 7 from R7A. They were plasmid prepped and sequenced with primers added by the sequencing core.

As shown in the image above, once again only amplification artifacts were shown. Troubleshooting for the cause of this is still in process.

After this, a second cloning event was performed. The ligation and cloning were performed per invitrogen and TA cloning manual instructions, respectively. These techniques were first introduced in Spring 2010. The cloning yielded both blue and white colonies as shown below:

The numbered colonies were the white ones selected for further analysis. The clone check for colonies 1, 7, 14 and 24 are shown below:

All but clone 14 worked, which was enough to do amplification on all of the clones and do a largescale clone check, which will be done later. Clone 1 from the previous cloning will be amplified and sequenced again.

Things to continue doing: Ligating sequences from R7B into plasmids and cloning them to establish a complete library of sequences in R7B and analyze for similarities that may aid in binding affinity, sequencing the plasmids from the clones, analyzing the sequences and possibly assembling together the clones with similar sequences.

Ashley Dawson, the things I do for you.

Hey Ashley,

Your second fish is dead. Sorry. It's been a bad week for fish. I just stuck my arm into the tank and with my fingers, excised your fish skeleton to bring to petsmart for a refund/rebuy of fish. I'll hopefully bring the skeleton back to you so you can make your mini funeral pyre.

Katherine.

P.S. Brad, why are her fish dying?

Progress Report One for Selection Against Amyloid Precursor Protein (APP)

Full abstract is here

Beads: Streptavidin
Selection Buffer: TRIS (10x diluted to 1x) pH 7.3
Incubation time/temperature: 45min/37*C
Washes/volume: 3 washes and 1 volume
Pool: RNA N34

Round 1 of selection against APP, pool N34, was started on the 20th of September 2010. A majority of the first week was spent Biotinlyating and re-suspending the target (in 1X PBS), after which the APP was aliquot into 20 tubes of 800pmol and 20 tubes of 400pmol. APP is a miniscule protein, 3717.1 kDal, which is why the two concentrations are necessary. R1 is being done using 400pmol, but in case the R1 binding assay doesn’t yield a high enough concentration, the round will have to be repeated using the 800pmol aliquot. This idea is based on the notion that a more concentrated target will result in a more concentrated binding assay.The rest of the time was spent on performing normal SELEX procedure. Cycle course PCR, however, had to be performed twice:



Figure 1: ccPCR, APP, N34, 9.28.10


As seen in Figure 1, the first attempt at PCR resulted in an overrun gel due to an overcharged and overexposed current, 115V for 35 minutes. The E1 samples ran over the W3 wells and resulted in an unreadable gel. The second run, performed under 90V for 25 minutes, provided much better results.

Figure 2: ccPCR R1, APP, N34, 10.8.10.


As seen in Figure 2, this PCR showed a good amount of amplification in the 9th cycle for E1. The over-amplified W3 was cause for some concern; it could be attributed to increased primer activity.
Large scale will be continued using 9 cycles, and a more concrete answer for the W3 over-amplification shall be determined by running a W3 cycle for 9 cycles. Running another gel, with this W3 sample, will determine if the primer is to blame for the results seen.
Problems Encountered:
The first PCR agarose gel was overexposed, both in time and current, making the end result unreadable. This setback was expensive to the experiment in terms of time. Were it not for this mistake, two rounds of selection might have been possible by now. More care must be taken while determining gel conditions in the future. The second gel showed excessive amplification on the third wash. This could have been due to excessive primer activity, but the true root of this problem has yet to be determined; the end of this round should locate an answer.

Conclusion and Future Work:
This reporting period saw half of a round of selection; the lack of pace is mainly attributed to the mistake on the PCR gel. Although the pace is slow, there seems to be potential with target, especially with the amplification seen on the ninth cycle of E1 in Figure 2. With the selection moving forward, a cause for the W3 over-amplification will also be determined. Future work should also see the pace of selection pick up, leading to at least two more rounds and concrete binding assays (with concentration) by the next reporting period.

Progress Report of mTagBFP RNA Bead-Based Selection

To gain a general understanding as to the purpose and direction of this selection, here is the target abstract.

Thus far, two rounds of RNA bead-based selection have been performed on mTagBFP, a fluorescent protein emitting a blue color. Overall, the selection is progressing well, however personal errors have presented some hindrance.
Initial Conditions for selection were as follows:

Target: mTagBFP
Beads to Use: Nickel-NTA
Pool: N34
Incubation Time and Temperature: 25 minutes at 37 0C
Buffer and pH: PBS SELEX, pH 7.4
Ratio: 400 pmol RNA: 400 pmol mTagBFP
Wash Volume and Number: 2 volumes (200uL), 3 washes

Note: These conditions are similar to those of Alec Rezigh as our research is partnered.

For round 1, cycle course PCR (ccPCR) was performed on the reverse transcription products of wash zero (W0), wash three (W3), and elution (E1): samples acquired during selection. As can be seen from Figure 1 below, elution sample was slightly overamplified at cycle twelve, but under amplified at cycle nine. Therefore, eleven cycles were chosen for the amplification of the elution ssDNA during large scale PCR (lsPCR).



Promising in the gel was the fact that no samples showed up in the W3 portion of the gel. This could indicate either significant protein binding or immense background binding. However, this cannot be determined until a binding assay is performed in a few more rounds.

In the hopes of eliminating background binding, the wash volume for round 2 was increased from two (200uL) to four (400uL) volumes. After leaving out my RNA yield from the previous round for four days, reverse transcription of this product, along with W0, W3, and E1, was performed. After conducting ccPCR on all four products, the hope was that the round 1 RNA product would amplify far before the elution, illustrating that the RNA was not degraded. The ccPCR results were as follows:




As can be seen, W0 and W3 samples are missing for the large 3.8% agarose gel. Not shown as well are the 24 samples below the ones presented as they belonged to another student and were not visible either. Since one of the 100 base pair ladders showed up and one of them did not, it was concluded, with the help of Nia, that either there was not enough ethidium bromide present in the gel, or that there was enough, but it was not adequately dispersed throughout the gel to be able to visualize all of the samples. However, through a stroke of luck, the samples that showed up were the ones necessary to continue with the round. By looking at the gel, the RNA product was greatly overamplified at cycle six, demonstrating that the round 1 RNA yield was not degraded. Additionally, the elution sample at cycle twelve was adequately amplified, and thus was chosen for lsPCR.

The concentrations yielded from round one and two were 56.22uM and 58.92 uM, respectively. Due to the similarity between the two yields, further rounds of selection will have to be completed to make any conclusion as to whether mTagBFP aptamers are being amplified.

Overall, the selection process against mTagBFP has been satisfactory. I hope to eliminate small as well as large personal errors to increase the speed and accuracy of each round. In next round, a negative selection will be performed and a decrease in incubation time from twenty-five to twenty minutes will be implemented in the hope of again eliminating bead/background binders as well as weak binders. In the future, other initial conditions will be modified to help isolate the best aptamer.

mCherry Fluorescent Protein Progress Report

See the abstract, outlining the purpose of this selection, here.

Three rounds of N34 RNA bead-based selection have been performed against mCherry, a monomeric fluorescent protein. The results so far have been promising, encountering no problems thus far.

Initial Conditions:

Target: mCherry
Beads to Use: Nickel-NTA
Pool: N34
Incubation Time and Temperature: 25 minutes at 37 0C
Buffer and pH: PBS SELEX, pH 7.4
RNA:Protein Ratio: 400:400
Wash Volume and Number: 3 washes, 2 volumes

Cycle course PCR was performed to determine the appropriate number of amplification cycles to be utilized in Large scale PCR (lsPCR). For Round One, the DNA amplified around the same time in both wash three (W3) and the elution (E1), as seen in Figure 1. Amplified well, but not over-amplified, fifteen cycles was determined for utilization in lsPCR.

To combat this excess non-specifc DNA, the wash volume was increased from 200 uL to 400 uL. Upon visualization of the gel, the DNA in wash three was reduced significantly, while the DNA present in the elution increased. This could indicate augmented protein binding, but could also signify increased background binding. There is no evidence to support either, however. The cycle course from Round Two can be seen in Figure 2.

To continue reducing background binding, negative selection was performed in Round Three. The incubation time was also reduced from twenty-five to twenty minutes, hopefully weeding out any weak or non-specific binding. The results yielded a decrease in wash volume DNA and an increase in elution DNA, hopefully indicating an increase in protein binding. The gel can be seen in Figure 3.

The concentrations gained from Rounds One, Two, and Three, were as follows: 84.17 uM, 102.34 uM, and 107.15 uM. Ideally, this increase in concentration signifies the increased amplification of mCherry specific RNA. A binding assay after further rounds of selection will be utilized to determine this.

In Round 4, plus and minus selection will be performed to hopefully determine if an increase in protein binding is present. Other conditions, such as incubation time, RNA:protein ratio, and wash volume will be adjusted in future rounds to potentially isolate strong protein binding aptamers. A binding assay will be performed, after Round Five or Six, to check the progress and success of the selection.

EphA2

Brad, I need more EphA2!

Aptamer Selection for Alpha-Synuclein: Progress Report #1

Aptamer Project Site: RNA Aptamer Selection Against Alpha-Synuclein As a Diagnostic Tool For Parkinson’s Disease

Progress, Results, and Discussion

The first round of aptamer selection for alpha-synuclein (asynuc) from the n34 RNA pool was completed on October 5, 2010. Despite a few minor setbacks, the first round showed promising results, and the second round of selection is already under way. Being a novice to filter selection, I expected to have some problems at first, but I’m confident that the subsequent rounds will go faster and have fewer problems.
Cycle course PCR was used in order to find the optimum number of cycles for large scale PCR. PCR product was taken out at the end of the certain cycles, and then the products were run down a 3.8% agarose gel to separate them, Figure 1. As you can see, not even the positive control, washes from round 1 (Wr1), showed up in the gel. This indicates that amplification of the DNA during PCR was unsuccessful. This might be due to bad PCR reagents. To test whether or not the reagents used during cycle course PCR were working properly, a positive control with a DNA sample that had worked previously was performed. The control DNA, Wr1, and E1 were put through 9 cycles of PCR with the same reagents used in the previous cycle course PCR and then ran down a gel, Figure 2. The control DNA and Wr1 showed up, but E1 did not. This indicates the PCR reagents are working properly, but E1 hadn’t been amplified enough to show up in the gel yet.

Wr1 and E1 samples that had already been through 9 rounds of PCR were put through 11 more rounds of PCR, and samples were again taken during various cycles, Figure 3. This was done to see the optimum number of rounds for large scale PCR, and to make sure that something hadn’t gone terribly wrong with E1 in a previous step. As you can see, E1 only showed up on the 20th cycle of PCR, indicating that the product just hadn’t been amplified enough yet. Because of this, 20 cycles of PCR were used for large scale PCR.

Problems Encountered

The main problem faced in round 1 was the failed cycle course PCR, described above. Also, the lines in the agarose gels were all very faint so next time I will use more Ethidium bromide so they show up better under the UV light. I had trouble getting a pellet during the last ethanol precipitation as well. This is because I used an eluant with 3M NaOAc already in it to elute the RNA from the PAGE gel chunk, and then accidentally added more 3M NaOAc during the ethanol precipitation. This altered the delicate concentration ratios in the mixture and did not allow the RNA to precipitate. To fix this, I corrected the concentrations by adding 3ml of water and 8.25ml 100% ethanol. This time it worked, and a pellet was formed. For this ethanol precipitation I put the entire reaction in a 15ml conical. Next time I will divide the reaction up into 1.5ml tubes so that it will be easier to form a pellet.

Conclusion and Future Work

For the first round of RNA aptamer selection for asynuc, I got a final concentration of 1399.6 ng/ul of RNA from the n34 pool. I learned a lot about filter selection during this round and I also learned some problem solving techniques for cycle course PCR and ethanol precipitation. My first cycle course PCR yielded an unsuccessful gel. This problem was correct by repeating the cycle course after a control verified that the PCR reagents were working properly. By the next progress report I plan on completing at least 2-3 more rounds of selection.