CH391L/S13/In vitro Selection of FNAs

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==Introduction==
==Introduction==
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Although the vast majority of such ligand binding activities or enzymatic activities known are performed by proteins a secondary subset of these fall under the category  of Functional nucleic acids (FNAs). FNAs are RNA, ssDNA, 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 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. Still, 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 (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.<cite>Silverman2012</cite> 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 development of new methodologies, such as the SELEX <cite>Stozack1990, Gold1990 </cite> or ''In vitro'' selection, as we strive to expand their potential both as tools for exploring biology and solving real world problem solving.
 +
 
 +
[[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>. ]]
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Note to self: mention RNA World
 
==Functional Nucleic Acids==
==Functional Nucleic Acids==
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[[Image:1st-Ribozymes-discovered2.png|600px|]]
 
-
 
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<cite>Cech1982</cite>,<cite>Altman1983</cite>,<cite>Ellington1990</cite>,<cite>Gold1990</cite><cite>BreakerJoyce1994</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 ligation reaction. In the case of ''in vitro'' selected ribozymes their function has been expanded to include .  It was in such attempts that [http://2011.igem.org/Team:Peking_R/Project/RNAToolkit1 TPP ribozyme biobrick] was made from both by combining an aptamer and a ribozyme for regulatory purposes. Most of other focus also on the regulation of an specific part, the self-modification of the part itself or both. 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>.
+
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.<cite>Cech2002</cite>
-
Natural Ribozymes <cite>Patel2007</cite>
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Examples of Natural Ribozymes
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*Cleaving Ribozymes
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*[http://en.wikipedia.org/wiki/Hepatitis_delta_virus_ribozyme HDV ribozyme]
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**#1 Self-cleaving  i.e. The Hammerhead family (satellite RNA)
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*[http://en.wikipedia.org/wiki/Group_I_catalytic_intron Group I intron]
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**#2 Trans-cleaving i.e. RNase P  
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*[http://en.wikipedia.org/wiki/RNase_P RNase P]
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*Splicing Ribozymes
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*[http://en.wikipedia.org/wiki/Hammerhead_ribozyme Hammerhead Ribozyme]
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**#3 Group I       
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-
**#4 Group II
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*Riboswitches
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**#5 Thermosensors
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-
**#6 sRNAs
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-
**#7 T-Boxes
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-
**#8 Metabolites
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**#9 Magnesium
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===Deoxyribozymes===
===Deoxyribozymes===
 +
[[Image:8-17OP.png|right|thumb|300px|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. <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>
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>
-
 
-
[[Image:8-17OP.png]]c
 
-
 
===Aptamers and Riboswitches===
===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.<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.
+
The word aptamer from the latin ''aptus'' and translates as the past participle of "to fit" were originally identified by employing the protocol SELEX <cite>Stozack1990, Gold1990</cite>. 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 [http://en.wikipedia.org/wiki/Riboswitch riboswitch] <cite>Winkler2002</cite>.
==In vitro Selection of Functional Nucleic Acids==
==In vitro Selection of Functional Nucleic Acids==
-
[[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> 
+
[[Image:In-vitro-selection.png| SELEX or In vivo Selection Experiment |right|thumb|200px]]
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===New Methods that assit ''In vitro'' selections===
+
A SELEX or ''In vitro'' Selection Experiment , describes the basic method for performing a SELEX or  uses single stranded nucleic acids that are [http://en.wikipedia.org/wiki/Oligonucleotide_synthesis chemically synthesized],with a constant region (CR) and a fixed random region of length ''n''. The random region of length ''n'' has only 4 nucleotides available, so the size of sequence space tested increases as 4<sup>n</sup>. Since only 10<sup>14</sup> molecules can be sample in any given experiment, a point where the sequence space is larger, we can only sample sparse regions of this sequence space. Case in point, for a pool where "n" = 60 the actual sequence space is 10<sup>36</sup> molecules.
 +
Important considerations of an ''In vitro'' selection:
 +
# Random Region Length: Shorter N<sub>x</sub> regions allow greater coverage.
 +
# Potential Structural Complexity: Longer N<sub>x</sub> regions allow greater complexity of secondary and tertiary structure.
 +
# Potential to misfold: Longer N<sub>x</sub> regions tend to misfold much more than short N<sub>x</sub> regions. Therefore it's important to asses the emergent catalytic activities vs. random region length.
 +
<cite>Silverman2012</cite>
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====Computational Methods====
 
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*Mfold - originally developed as a GUI or graphical user interface server for the prediction of the secondary structure of single stranded nucleic acids. The core algorithm predicts structures based on minimum free energies necessary to fold a structure. This algorithm can be used initially as a step to predict possible secondary structures of active sequences <cite>Zucker2003</cite>.
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===The ''In vitro'' selection experiment===
-
 
+
-
====FRET====
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*Fluorescent analogues
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====Next Generation Sequencing====
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The first step is subjecting the population of single stranded nucleic acids or "pools" 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 regenerate the single 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. The reason why several rounds of selection are required is that we want to enrich the portion of the population with our desired function from the background noise, cheater sequences (non-function), that exist at the beginning of the experiment.
 +
 
 +
==Examples of Functional Nucleic Acids==
 +
===DNA-catalyzed DNA cleavage===
 +
DNA cleavage by a deoxyribozyme uses to main mechanisms, either they cleaved their DNA substrate by hydrolysis or via deglycosylation, a carbohydrate (sugar) is detached from the DNA subtrate which is the followed by two β-eliminations. Examples of such Deoxyribozyme include 8VA4 or 8VA10 respectively<cite>Silverman2012</cite>.
 +
 +
 
 +
 
 +
===Deoxyribozyme-Catalyzed RNA labeling ===
 +
Deoxyribozyme-catalyze labeling (DECAL), by 10DM24 deozyribozyme, "approach for site-specific internal RNA modification" that is attached to a labelled target RNA.<cite>Silverman2007</cite>
 +
In a similar manner 7TQ20, can form a nucleopetide linkage to 5' pppRNA, provided an anchored small tripeptide is provided, in this case Cys-Tyr-Ala. FOr this deoxyribozyme to form the nucleopeptide linkage the Tyr -OH plays an important role<cite>Silverman2012</cite>.
 +
 
 +
 
 +
===The Flexizyme===
 +
One of the most interesting FNAs now available to new tool are flexizymes that perform a ''“de novo”'' self-aminoacylating ribozyme. The ribozyme was generated in an “''in vitro'' selection” of a tRNA with a N<sub>70</sub> region, that replaces the aromatic moeity of a tRNA, and that can add both natural and non-natural amino acids by reprogramming genetic code. According to the authors the “Flexizyme system [has] virtually no limitation in the use of amino acid donors and tRNAs. It is also gives “flexibility in the choice of acids” <cite>GoTo2006</cite>.
 +
 
 +
==iGEM Link==
 +
 
 +
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 of making a genetic rheostat. A genetic rheostat would be a device that modulates or regulates precisely the expression in response to a wide range of ligand concentration. Furthermore, it would only  respond to an orthogonal ligand to the host cell.
==References==
==References==
Line 63: Line 68:
#Breaker2002 pmid=12022469
#Breaker2002 pmid=12022469
#Silverman2009 isbn=978-0-387-73711-9
#Silverman2009 isbn=978-0-387-73711-9
-
#Wilson1999 pmid=10872462
 
#Ellington1990 pmid=1697402
#Ellington1990 pmid=1697402
#Gold1990 pmid=2200121
#Gold1990 pmid=2200121
#Winkler2002 pmid=12410317
#Winkler2002 pmid=12410317
-
#Zucker2003 pmid=12824337
 
#GoTo2006 pmid=17150804
#GoTo2006 pmid=17150804
-
#Patel2007 pmid=17846637
 
#Breaker2000 pmid=11187837
#Breaker2000 pmid=11187837
 +
#Cech2002 pmid=12110898
 +
#Silverman2012 pmid=23088677
 +
#Silverman2007 pmid=17394278
 +
<\biblio>
<\biblio>

Current revision


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.[1] 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 development of new methodologies, such as the SELEX [2, 3] or In vitro selection, as we strive to expand their potential both as tools for exploring biology and solving real world problem solving.

Functional Nucleic Acid Chronology [2, 3],[4],[5],[6],[7],[8].
Functional Nucleic Acid Chronology [2, 3],[4],[5],[6],[7],[8].

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.[9]

Examples of Natural Ribozymes

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. [7] 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 [10]

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 [2, 3]. 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 [11, 12]. Yet, it would take several years until the discovery of the first in vivo aptamer or riboswitch [13].

In vitro Selection of Functional Nucleic Acids

SELEX or In vivo Selection Experiment
SELEX or In vivo Selection Experiment

A SELEX or In vitro Selection Experiment , describes the basic method for performing a SELEX or uses single stranded nucleic acids that are chemically synthesized,with a constant region (CR) and a fixed random region of length n. The random region of length n has only 4 nucleotides available, so the size of sequence space tested increases as 4n. Since only 1014 molecules can be sample in any given experiment, a point where the sequence space is larger, we can only sample sparse regions of this sequence space. Case in point, for a pool where "n" = 60 the actual sequence space is 1036 molecules. Important considerations of an In vitro selection:

  1. Random Region Length: Shorter Nx regions allow greater coverage.
  2. Potential Structural Complexity: Longer Nx regions allow greater complexity of secondary and tertiary structure.
  3. Potential to misfold: Longer Nx regions tend to misfold much more than short Nx regions. Therefore it's important to asses the emergent catalytic activities vs. random region length.

[1]


The In vitro selection experiment

The first step is subjecting the population of single stranded nucleic acids or "pools" 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 regenerate the single 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. The reason why several rounds of selection are required is that we want to enrich the portion of the population with our desired function from the background noise, cheater sequences (non-function), that exist at the beginning of the experiment.

Examples of Functional Nucleic Acids

DNA-catalyzed DNA cleavage

DNA cleavage by a deoxyribozyme uses to main mechanisms, either they cleaved their DNA substrate by hydrolysis or via deglycosylation, a carbohydrate (sugar) is detached from the DNA subtrate which is the followed by two β-eliminations. Examples of such Deoxyribozyme include 8VA4 or 8VA10 respectively[1].


Deoxyribozyme-Catalyzed RNA labeling

Deoxyribozyme-catalyze labeling (DECAL), by 10DM24 deozyribozyme, "approach for site-specific internal RNA modification" that is attached to a labelled target RNA.[14] In a similar manner 7TQ20, can form a nucleopetide linkage to 5' pppRNA, provided an anchored small tripeptide is provided, in this case Cys-Tyr-Ala. FOr this deoxyribozyme to form the nucleopeptide linkage the Tyr -OH plays an important role[1].


The Flexizyme

One of the most interesting FNAs now available to new tool are flexizymes that perform a “de novo” self-aminoacylating ribozyme. The ribozyme was generated in an “in vitro selection” of a tRNA with a N70 region, that replaces the aromatic moeity of a tRNA, and that can add both natural and non-natural amino acids by reprogramming genetic code. According to the authors the “Flexizyme system [has] virtually no limitation in the use of amino acid donors and tRNAs. It is also gives “flexibility in the choice of acids” [15].

iGEM Link

TheTPP ribozyme biobrick made by the Pekin 2011 team combines both an aptamer and a ribozyme for regulatory purposes of making a genetic rheostat. A genetic rheostat would be a device that modulates or regulates precisely the expression in response to a wide range of ligand concentration. Furthermore, it would only respond to an orthogonal ligand to the host cell.

References

  1. Velez TE, Singh J, Xiao Y, Allen EC, Wong OY, Chandra M, Kwon SC, and Silverman SK. . pmid:23088677. PubMed HubMed [Silverman2012]
  2. Tuerk C and Gold L. . pmid:2200121. PubMed HubMed [Gold1990]
  3. Kruger K, Grabowski PJ, Zaug AJ, Sands J, Gottschling DE, and Cech TR. . pmid:6297745. PubMed HubMed [Cech1982]
  4. Guerrier-Takada C, Gardiner K, Marsh T, Pace N, and Altman S. . pmid:6197186. PubMed HubMed [Altman1983]
  5. Ellington AD and Szostak JW. . pmid:1697402. PubMed HubMed [Ellington1990]
  6. Doudna JA and Cech TR. . pmid:12110898. PubMed HubMed [Cech2002]
  7. Breaker RR. . pmid:11187837. PubMed HubMed [Breaker2000]
  8. isbn:978-0-387-73711-9. [Silverman2009]
  9. Winkler W, Nahvi A, and Breaker RR. . pmid:12410317. PubMed HubMed [Winkler2002]
  10. Baum DA and Silverman SK. . pmid:17394278. PubMed HubMed [Silverman2007]
  11. Murakami H, Ohta A, Goto Y, Sako Y, and Suga H. . pmid:17150804. PubMed HubMed [GoTo2006]
  12. Emilsson GM and Breaker RR. . pmid:12022469. PubMed HubMed [Breaker2002]
All Medline abstracts: PubMed HubMed
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