CH391L/S13/In vitro Selection of FNAs

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(Aptamers and Riboswitches)
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#Altman1983 pmid=6197186
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#Breaker2002 pmid=12022469
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#Silverman2009 isbn=978-0-387-73711-9
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Revision as of 08:22, 11 February 2013


Contents

Introduction

Functional nucleic acids (FNAs) are RNA, DNA, or XNA(nucleic acid analogues) that perform an activity such as binding or catalyzing a reaction. FNAs are grouped into three main categories Aptamers, Ribozymes, and Deoxyribozymes that are subdivided into either natural or artificial depending on their origin; the exception being Deoxyribozymes as they have yet to be discovered in a living organism. It was only in the 1980s that the 1st ribozyme was discovered that we started to study FNAs and have allowed for the discovery of new methods, such as the SELEX or In vitro selection process that we are expanding their potential both as tools for exploring biology and real world problem solving.


Functional Nucleic Acids

[1],[2]

Ribozymes

Deoxyribozymes

Image:8-17OP.png


Aptamers and Riboswitches

The word aptamer is derived from the latin aptus that translates as fit or fitted were originally identified by SELEX. This name describes their basic function as RNA or single stranded DNA (ssDNA)that can bind a ligand by assuming an specific structure.[3]

In vitro Selection of Functional Nucleic Acids


The image presented describes the basic method for performing a SELEX or In vivo selection experiment using single stranded nucleic acids (RNA,ssDNA,XNA) that are chemically synthesizedan have a constant region (CR) and a random region. Having the CR allows later amplification using PCR. 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 amplified to make double stranded nucleic acids with the use of the CR introduced previously. Therefore the selection can continue to a following round, while at the same time a sample is obtained and can be sequenced. [4]

New Methods and Tools that assit In vitro selections

Computational Methods

  • Mfold

FRET

  • Fluorescent analogues

Next Generation Sequencing

New Applications of FNAs

  1. Kruger K, Grabowski PJ, Zaug AJ, Sands J, Gottschling DE, and Cech TR. . pmid:6297745. PubMed HubMed [Cech1982]
  2. Emilsson GM and Breaker RR. . pmid:12022469. PubMed HubMed [Breaker2002]
  3. Guerrier-Takada C, Gardiner K, Marsh T, Pace N, and Altman S. . pmid:6197186. PubMed HubMed [Altman1983]
  4. isbn:978-0-387-73711-9. [Silverman2009]
All Medline abstracts: PubMed HubMed
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