Journal Club, Next Meeting November 30

I have pushed back the journal club for November 30 instead of November 23 for two reasons: (1) may decide to let you off the hook for class the week of Thanksgiving and (2) we can talk about an article while enjoying scrumptious desserts for the lab party.

In previous journal clubs, we have focused on therapeutics and seen how our nemesis, the antibody, has captured most of the therapeutics world. We discussed why this is, how antibodies have the advantage in some respects, but how aptamers show great promise with their many benefits. Over the summer, we talked about aptamers involved in drug delivery. If you are interested, those articles were:

• Lupold et al. Identification and characterization of nuclease-stabilized RNA molecules that bind human prostate cancer cells via the prostate-specific membrane antigen. Cancer Res (2002) vol. 62 (14) pp. 4029-33
• Chu et al. Aptamer:toxin conjugates that specifically target prostate tumor cells. Cancer Res (2006) vol. 66 (12) pp. 5989-92

For this month's journal club, I would like to focus on aptamers used for diagnostics.

• Davis et al. Staining of cell surface human CD4 with 2'-F-pyrimidine-containing RNA aptamers for flow cytometry. Nucleic Acids Research (1998) vol. 26 (17) pp. 3915-24

I chose an older paper for a number of reasons:

1. The authors developed the technique in a previous paper circa 1996
2. I would like to familiarize you with the procedures of flow cytometry
3. They selected against a conserved extracellular glycoprotein receptor
4. They showed potential binding sites using known antibodies
5. CD4 is a target of HIV
6. They used modified RNA for selection
7. The company was NeXstar Pharmaceuticals which later became SomaLogic with whom I've previously mentioned.
8. Jayasena, the directing author, published many aptamer related papers in the 1990's.

Questions I would like you to address:

1. Find original paper where aptamers were used in flow cytometry diagnostics (ref in paper). How does flow cytometry work?
2. How many papers has Jayasena published? Can you recreate his career based on publications?
3. What other methods had been developed at the time of writing for diagnostic aptamers? What has been developed since then? (Hint - check out a recent review for a summary)
4. What was novel about this paper?
5. Explain how CD4 is involved T cell recognition in addition to HIV infection (HINT: this will also require outside reading of a review article)
6. The authors utilized 2'-Fluoro modified nucleotides. What other modifications could they have used had they done the selection today? (HINT: look up current selections with modifications and modifications in general such as that discussed in another post on this blog).
7. Which region of the membrane do these aptamers bind to? What evidence shows this?

As always, please read the article and post questions in the comments section. This is especially true for the mentors.

Cheers! - Brad

Journal Club, Next Meeting October 26th

Journal Club this semester will be interesting. We will explore a Nature Proceedings paper about aptamer diagnostics that hasn't been peer reviewed and ask why, a review article about aptamer therapeutics that details many of the current and future problems associated with utilizing aptamers as drugs, and a Nature Biotechnology Letter about the selection of antibodies that is short but has a punch. Each of these are wildly different published presentation mediums so I would like to discuss how they are similar and different. Each paper discusses an important aspect of aptamer utility so I hope that we can also use these papers to spring board a discussion of how others are using aptamers. We will explore one main paper each month with some supporting documents.

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For the first week, I would like to start off with a review of using aptamers as therapeutics since most of your projects deal with this in some way. The review includes a comparison of aptamers to antibodies. There are two main Aptamer companies that hold much of the intellectual property involved with nucleic acid aptamer technologies:

• SomaLogic, lead by Larry Gold, formerly CEO of NeXstar, is headquartered in Boulder, Colorado and is the main aptamer diagnostics company. Dr. Gold also co-authored the "other" original aptamer paper with Craig Tuerk and is the lead author on one of the papers we will read this semester.
• Archemix, currently lead by Ken Bate, started around the turn of the century (2000-ish) by Marty Stanton, David Epstein, Andy and Ron Breaker. Anthony Keefe is currently the senior director at X-chem, but was a senior PI at Archemix prior to this and is the first author of the review we will read this month.

Here is the reference for the paper for Journal Club September 28th:

Keefe et al. Aptamers as therapeutics. Nature Reviews Drug Discovery (2010) vol. 9 (7) pp. 537-50. You can find the PDF here.

When reading reviews, I first read the title/abstract and then make an outline of the review with the main headings, subheadings and figure/descriptions. Please start by doing this and then work your way through the review. Skip over the "Aptamers in the Clinic" section during your first read through. I will try to get one of the authors, Supriya Pai, to join us during lecture so think up some good questions for her also.

Some initial questions:

1. Name some advantages/limitations of aptamers and antibodies. Do you feel one is superior than the other for therapeutics or are they each suited for particular tasks? Explain.
2. What is a Spiegelmer and how can it prevent nuclease degradation? What other aptamer modifications can prevent degradation? Is this more important for therapeutics than diagnostics? Explain
   
3. What is meant by pharmacokinetics and why should drug designers be concerned with therapeutic potency in vivo? You may need to look this word up elsewhere and see how it applies in the review.

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Here is the reference for the paper for Journal Club October 26th:

Reddy et al. Monoclonal antibodies isolated without screening by analyzing the variable-gene repertoire of plasma cells. Nature Biotechnology (2010) vol. 28 pp. 965-969. You can find the PDF here.

Nature Biotechnology is probably my favorite journal and one of the reasons I'm having you read an article published therein. When reading a letter, appreciate that the authors published fast with only some preliminary ground breaking data. The work was done in 2009/early 2010 and the manuscript was received June 11. It is now in publication (less than a year). This is a short paper with only 2 figures and a table. Since it is short, I also want you to look over the Supplemental Data as the authors discuss this data. Again, it is another one partially out of the Ellington Lab. Due to this, I hope to have one of the authors come (in addition to Dr. Supriya Pai from last month).

When reading this article I want you to delve into more than just the data, focus on these questions:

1. How is this article different from the review?
2. What is the scope of this Journal or why did the authors choose this journal to publish in? Can you think of any other journals that may be appropriate (based on other journals referenced in the bibliography).
3. How do you think the authors chose to put certain information in the supplemental? Why do you think they did this? (hint, check out the author guidelines for letters - found on page 3).
4. Is there supplemental data that should have been included in the main article or vise versa? WhFind the limitations for
5. What does it mean for the future of aptamers?
6. How can these methods be easier or harder than selecting aptamers?
7. Are the figures visually appealing?

I came upon this on accident:

I was looking through CNN and this was in today's headlines, I though some of you might find it interesting. :)

http://pagingdrgupta.blogs.cnn.com/2010/09/01/new-clue-may-lead-to-possible-cure-for-alzheimers-one-day/?hpt=T2

For those of you who work/worked with Angiotensin II...

MicroRNA-155 Regulates Human Angiotensin II Type 1 Receptor Expression in Fibroblasts

Mickey M. Martin, Eun Joo Lee, Jessica A. Buckenberger, Thomas D. Schmittgen and Terry S. Elton

http://www.jbc.org/content/281/27/18277.long

Study where miRNA binds to and regulates mRNA responsible for translational expression of human angiotensin II type 1 receptor (hAT1R).

Interesting article if you want to learn more about miRNA.

Keeping up with the Jones

Keeping up with current literature is difficult at best. I will present this fall on ways to keep up with literature searches, automating the process and writing about the literature you find. The problems are muli-fold:

• finding relevant literature
• preparing to read and reread
• storing and accessing articles
• organizing knowledge

To "keep up with the Jones's", I suggest three simple tasks to get you started:

1.) Create a pubcrawler account and let it do the walking. Pubcrawler is like an RSS feed for pubmed. It repeatedly searches pubmed for terms and e-mails you the results daily/weekly. Consider the following search terms to get you started:

• Keyword based - selection [All Fields] AND nucleic acid [All Fields] AND aptamer [All Fields]
• Common author based - Ellington AD [Author] OR Yingfu Li [Author] OR Sullenger BA [Author] OR Gorenstein DG [Author] OR Keefe AD [Author] OR Famulok M [Author] OR Mayer G [Author] OR Blind M [Author] OR Rossi J [Author]

2.) Get a PDF organizer and use it. PDFs are the future. Organization and recall is the key. Some common ones are:

• Mendeley - cross platform, free, performs reference formatting and inline citing
• Papers -Mac only, $, has plugin to send web pdf files directly to Papers
• Zotero - cross platform browser based (firefox), free, but can perform like Endnote/Mendeley
• Endnote - cross platform, performs cite while you write, $$$

3.) Read and keep notes. Use Google docs, Google notebook or this blog to keep yourself organized with the relevant literature you are finding.

Modified Nucleotides

Larry Gold et al, just published an article about a single selection against 800 protein targets for the purposes of proteomic diagnostics. Pretty interesting paper that we will read for journal club this semester. The cool figure for our stream though is plastered in Table 1 of the Supplementary data. In summary, they attempted selection against 14 targets with 4 different base modifications and showed the general success (with binding affinity) for each of the selections. The conclusion is that regular dT only produced viable aptamers for 2 of the 14 targets. 5-benzylaminocarbonyl-dU (BndU) and 5-isobutylaminocarbonyl-dU (iBudU) worked for about 50% of the targets. However, the real news is 5-tryptaminocarbonyl-dU (TrpdU) worked for all of the targets attempted. Figure below from the paper:

Figure legend from the paper: Nucleotide triphosphate analogs modified at the 5-position (R) of uridine (dUTP). The 5-position modifications include: (A) 5-benzylaminocarbonyl-dU (BndU); (B) 5-naphthylmethylaminocarbonyl-dU (NapdU): (C) 5-tryptaminocarbonyl-dU (TrpdU); and (D) 5-isobutylaminocarbonyl-dU (iBudU).

Let me reiterate.

In our lab we have a lot of failures. Maybe we could increase our hit rate by using modified nucleotides. Now, the thoughts that should be floating in your mind are: great(!) but how do I get TrpdU and how do I incorporate it into my pool and where does it get incorporated and...does this mean I'm not a failure?

TrpdU must be made in house and is not particularly easy. I don't think anyone is selling it currently, but you should do a thorough search at Tri-Link and other nucleotide suppliers. Shawn, our friendly organic chemist may be able to help out, or we may be able to ask one of the Organic streams to help out. The paper describing the method is by Vaught et al. The compound we want is 6d in figure 1 and the method for producing it is in Schema 1 (shown below). Bruce Eaton has also published two papers explaining alternate synthesis of these modifications. "The Joys of in vitro selection..." and "New uridine derivatives for systematic evolution...".

Primer extension efficiency is around 83% with KOD XL. We will likely want to amplify

template with normal nucleotides and then peform one primer extension with the following mixture: (nucleotide incorporation and amplification) they used 0.5 mM each dNTP (dATP, dGTP, 5'-methyl-dCTP, 5'-tryptaminocarbonyl-dU), 4.8nmol of forward primer, 4nmol template with 1X Thermococcus kodakaraensis (KOD) XL DNA Polymerase Buffer (EMD) and 0.125U/uL KOD enzyme.

During the selection, they also used dextran sulfate (a polyanion) as a nonspecific inhibitor.

Capillary Electrophoresis

A recent paper was published on the use of capillary electrophoresis to select DNA aptamers for lysozyme by Tran et al in Molecules: http://www.mdpi.com/1420-3049/15/3/1127/  This is nothing new as capillary electrophoresis has been used for a number of years to select aptamers, first published by Krylov's lab in Canada.  For a review of the methods and current papers, check out pubmed, wikipedia, and this figure from Andrei Drabovich:

The selection method sounds very appealing for a number of reasons:

• Fast selection since fewer rounds are required - 3-5 vs 8-12
  
• Little protein necessary - 0.1pmol or less compared to 200pmol/round
• Binding assays can be performed with the CE

However, there are also drawbacks:

• Collection times/volumes
• Buffer incompatibility - rumors you can't use divalent ions because of sticking to the tube walls
• Experience required to use the machine effectively
• The pool or primer set must be ordered with fluorescence for detection on the CE
  

If someone would like to read up on these methods and work to implement them for a selection, I would approve it.  I would work with you because I would like to become familiar with the machine.  But we only have one CE and it can only handle one sample at a time, so it may be problematic for more than one student to learn.  However, Arshia (former mentor) is currently working with the machine and so, you would have help in that regard.