CH391L/S13/In vitro Selection of FNAs

Introduction

Functional nucleic acids (FNAs, as described by Dr. S.K. Silverman, are DNA and RNA aptamers that bind targets, or they are deoxyribozymes (single stranded DNA) and ribozymes (RNA) that have catalytic activity. Aptamers, Ribozymes, and Deoxyribozymes are grouped into three main categories that are further classified into either natural or artificial depending on their origin. The exception being Deoxyribozymes as they have yet to be discovered in a living organism. Although the first ribozyme was discovered only in the 1980s, the search for new and better FNAs continues. This has led the discovery of new methods, such as the SELEX [1, 2] or In vitro selection process, as we strive to their potential both as tools for exploring biology and solving real world problem solving.

Functional Nucleic Acid Chronology [1, 2],[3],[4],[5],[6],[7].

Functional Nucleic Acids

Ribozymes

Most natural ribozymes, RNA catalysts, catalyze mostly some type of scission or ligation that involves some type of phosphodiester chemistry. In vitro selected ribozymes are displaying a versatile level of sophistication of the sort required for cellular metabolism.[8]

Examples of Natural Ribozymes

• HDV ribozyme
• Group I intron
• RNase P
• Hammerhead Ribozyme

Deoxyribozymes

Bimolecular Construct of 8-17 Deoxyribozyme and its Ribose Likage Substrate Cleaving Site

An interesting discovery was made in the early 1990s, when for the first time DNA was shown that, besides being a genetic information storage molecule, it could also be both an enzyme and an aptamer. In the figure below you can observe 8-17, an RNA cleaving DNA enzyme. This molecule with 10-23 were the first to be described and tested in vivo as potential new therapies for cleaving the expressed mRNA of a virus. [6] Although proteins offer a larger diversity chemistries, depending on amino acids vs. both kinds of nucleic acids, as the latter ones depend on a limited array of nucleotides. Until know around a dozen distinct types of reactions. These include the following activities such as self-phosphorylation, RNA labeling, depurination,etc [9]

Aptamers and Riboswitches

The word aptamer from the latin aptus and translates as the past participle of to fit were originally identified by employing the protocol SELEX. Therefore the word Aptamer describes their basic function as RNA or single stranded DNA (ssDNA)that can bind a ligand by assuming an specific structure.[10, 11] Yet, it would take several years until the discovery of the first in vivo aptamer or riboswitch [12]. See the following page to get a better understanding of aptamers and riboswitches.

In vitro Selection of Functional Nucleic Acids

SELEX or In vivo Selection Experiment

The figure to the right, SELEX or In vivo Selection Experiment , describes the basic method for performing a SELEX or In vivo selection experiment using single stranded nucleic acids that are chemically synthesized,with a constant region (CR) and a random region. The first step is subjecting the population of single stranded nucleic acids to specific selective condition in which function is possible. Then a (2) diverse subset of the population will perform the desired function and will be then (3) PCR (Polymerase Chain Reaction) amplified to generate double stranded nucleic acids making use of CR introduced previously. The previous step is necessary for the selection's continuation into the next selection round, while at the same time a sample is obtained and can be sequenced.

Examples of Functional Nucleic Acids

Flexizyme

Finally, one of the newest tools available to new tool are flexizymes that perform a self-aminoacylating reaction on an in vitro selected tRNA with a N70 region and that can add nonnatural amino acids by reprogramming genetic code[13].

iGEM Link

TheTPP ribozyme biobrick made by the Pekin 2011 team combines both an aptamer and a ribozyme for regulatory purposes.

References

<biblio>

1. Cech1982 pmid=6297745
2. Altman1983 pmid=6197186
3. Breaker2002 pmid=12022469
4. Silverman2009 isbn=978-0-387-73711-9
5. Wilson1999 pmid=10872462
6. Ellington1990 pmid=1697402
7. Gold1990 pmid=2200121
8. Winkler2002 pmid=12410317
9. Zucker2003 pmid=12824337
10. GoTo2006 pmid=17150804
11. Patel2007 pmid=17846637
12. Breaker2000 pmid=11187837
13. Cech2002 pmid=12110898

<\biblio>