CH391L/S13/In vitro Selection of FNAs

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==Introduction==
==Introduction==
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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.  
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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 1980sthe search for new and better FNAs continues. This has led the discovery of new methods, such as the SELEX <cite>Stozack1990, Gold1990 </cite> or ''In vitro'' selection process, as we strive to their potential both as tools for exploring biology and solving real world problem solving.
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[[Image:FNA Chronology.png|center|thumb|800px|Functional Nucleic Acid Chronology <cite>Stozack1990, Gold1990</cite>,<cite>Cech1982</cite>,<cite>Altman1983</cite>,<cite>Ellington1990</cite>,<cite>BreakerJoyce1994</cite>,<cite>Wrinkler2002</cite>. ]]
==Functional Nucleic Acids==
==Functional Nucleic Acids==
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[[Image:1st-Ribozymes-discovered2.png|600px|]]
 
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<cite>Cech1982</cite>,<cite>Altman1982</cite>
 
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===Ribozymes===
===Ribozymes===
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As previously mentioned ribozymes fall under the category of enzymes. Most of the ribozymes studied up until recently in living organism fall into 9 classes. Of these most perform some type scission and/or ligation reaction. In the case of ''in vitro'' selected ribozymes their function has been expanded to include . 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<cite>GoTo2006</cite>.
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 +
Natural Ribozymes
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*Splicing Ribozymes-
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*Riboswitches - Translational control mechanism found at the mRNA level.
===Deoxyribozymes===
===Deoxyribozymes===
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[[Image:8-17OP.png]]
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[[Image:8-17OP.png|right|thumb|300px|Bimolecular Construct of 8-17 Deoxyribozyme and its Ribose Likage Substrate Cleaving Site]]
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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. <cite>BreakerJoyce1994</cite> 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 <cite>Breaker2000</cite>
===Aptamers and Riboswitches===
===Aptamers and Riboswitches===
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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.<cite>Lu2009Silverman2009, Stostak1999</cite>
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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.<cite>Silverman2009, Wilson1999</cite> Yet, it would take several years until the discovery of the first ''in vivo'' aptamer or riboswitch <cite> Winkler2002 </cite>. See the following page to get a better understanding of aptamers and riboswitches.
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==In vitro Selection of Functional Nucleic Acids==
==In vitro Selection of Functional Nucleic Acids==
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[[Image:In-vitro-selection.png|600px|]]<br>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 [http://en.wikipedia.org/wiki/Oligonucleotide_synthesis chemically synthesized]an 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. <cite>Breaker2002</cite> 
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[[Image:In-vitro-selection.png| SELEX or In vivo Selection Experiment |right|thumb|200px]]
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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 [http://en.wikipedia.org/wiki/Oligonucleotide_synthesis 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.
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==Examples of Functional Nucleic Acids==
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Flexizyme
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===New Methods and Tools that assit ''In vitro'' selections===
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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<cite>GoTo2006</cite>.
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====Computational Methods====
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*Mfold
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====FRET====
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*Fluorescent analogues
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====Next Generation Sequencing====
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==iGEM Link==
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The[http://2011.igem.org/Team:Peking_R/Project/RNAToolkit1 TPP ribozyme biobrick] made by the Pekin 2011 team combines both an aptamer and a ribozyme for regulatory purposes.
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==New Applications of FNAs==
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==References==
<biblio>
<biblio>
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#Silverman2009 isbn=978-0-387-73711-9
#Silverman2009 isbn=978-0-387-73711-9
#Wilson1999 pmid=10872462
#Wilson1999 pmid=10872462
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#Ellington1990 pmid=1697402
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#Gold1990 pmid=2200121
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#Winkler2002 pmid=12410317
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#Zucker2003 pmid=12824337
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#GoTo2006 pmid=17150804
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#Patel2007 pmid=17846637
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#Breaker2000 pmid=11187837
<\biblio>
<\biblio>

Revision as of 18:09, 5 May 2013


Contents

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

Functional Nucleic Acids

Ribozymes

As previously mentioned ribozymes fall under the category of enzymes. Most of the ribozymes studied up until recently in living organism fall into 9 classes. Of these most perform some type scission and/or ligation reaction. In the case of in vitro selected ribozymes their function has been expanded to include . 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[8].

Natural Ribozymes

  • Splicing Ribozymes-
  • Riboswitches - Translational control mechanism found at the mRNA level.


Deoxyribozymes

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


iGEM Link

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

References

  1. Tuerk C and Gold L. . pmid:2200121. PubMed HubMed [Gold1990]
  2. Kruger K, Grabowski PJ, Zaug AJ, Sands J, Gottschling DE, and Cech TR. . pmid:6297745. PubMed HubMed [Cech1982]
  3. Guerrier-Takada C, Gardiner K, Marsh T, Pace N, and Altman S. . pmid:6197186. PubMed HubMed [Altman1983]
  4. Ellington AD and Szostak JW. . pmid:1697402. PubMed HubMed [Ellington1990]
  5. Murakami H, Ohta A, Goto Y, Sako Y, and Suga H. . pmid:17150804. PubMed HubMed [GoTo2006]
  6. Breaker RR. . pmid:11187837. PubMed HubMed [Breaker2000]
  7. isbn:978-0-387-73711-9. [Silverman2009]
  8. Wilson DS and Szostak JW. . pmid:10872462. PubMed HubMed [Wilson1999]
  9. Winkler W, Nahvi A, and Breaker RR. . pmid:12410317. PubMed HubMed [Winkler2002]
  10. Emilsson GM and Breaker RR. . pmid:12022469. PubMed HubMed [Breaker2002]
  11. Zuker M. . pmid:12824337. PubMed HubMed [Zucker2003]
  12. Serganov A and Patel DJ. . pmid:17846637. PubMed HubMed [Patel2007]
All Medline abstracts: PubMed HubMed
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