CH391L/S13/In vitro Selection of FNAs - Revision history

Alvaro E. Rodriguez M.: /* Introduction */

2013-05-06T19:10:30Z

Introduction

←Older revision		Revision as of 19:10, 6 May 2013
Line 3:		Line 3:
	==Introduction==		==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.<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 1980s, the 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.	+	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 1980s, the 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>. ]]		[[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>. ]]

Alvaro E. Rodriguez M.: /* In vitro Selection of Functional Nucleic Acids */

2013-05-06T18:53:48Z

In vitro Selection of Functional Nucleic Acids

←Older revision		Revision as of 18:53, 6 May 2013
Line 30:		Line 30:
	[[Image:In-vitro-selection.png\| SELEX or In vivo Selection Experiment \|right\|thumb\|200px]]		[[Image:In-vitro-selection.png\| SELEX or In vivo Selection Experiment \|right\|thumb\|200px]]

-	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 <cite>Silverman2012</cite>.	+	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>
		+

	===The ''In vitro'' selection experiment===		===The ''In vitro'' selection experiment===

Alvaro E. Rodriguez M.: /* Examples of Functional Nucleic Acids */

2013-05-06T18:43:27Z

Examples of Functional Nucleic Acids

←Older revision		Revision as of 18:43, 6 May 2013
Line 37:		Line 37:

	==Examples of Functional Nucleic Acids==		==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-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>		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>.

Alvaro E. Rodriguez M.: /* iGEM Link */

2013-05-06T14:53:05Z

iGEM Link

←Older revision		Revision as of 14:53, 6 May 2013
Line 46:		Line 46:
	==iGEM Link==		==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.	+	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==

Alvaro E. Rodriguez M.: /* The Flexizyme */

2013-05-06T14:43:00Z

The Flexizyme

←Older revision		Revision as of 14:43, 6 May 2013
Line 42:		Line 42:

	===The Flexizyme===		===The Flexizyme===
-	One of the most interesting FNAs now 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>.	+	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==		==iGEM Link==

Alvaro E. Rodriguez M.: /* Aptamers and Riboswitches */

2013-05-06T14:29:18Z

Aptamers and Riboswitches

←Older revision		Revision as of 14:29, 6 May 2013
Line 24:		Line 24:
	===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<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>.	+	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==

Alvaro E. Rodriguez M.: /* References */

2013-05-06T14:28:15Z

References

←Older revision		Revision as of 14:28, 6 May 2013
Line 56:		Line 56:
	#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

Alvaro E. Rodriguez M.: /* References */

2013-05-06T14:27:27Z

References

←Older revision		Revision as of 14:27, 6 May 2013
Line 60:		Line 60:
	#Gold1990 pmid=2200121		#Gold1990 pmid=2200121
	#Winkler2002 pmid=12410317		#Winkler2002 pmid=12410317
-	~~#Zucker2003 pmid=12824337~~
	#GoTo2006 pmid=17150804		#GoTo2006 pmid=17150804
	#Breaker2000 pmid=11187837		#Breaker2000 pmid=11187837

Alvaro E. Rodriguez M.: /* References */

2013-05-06T14:26:10Z

References

←Older revision		Revision as of 14:26, 6 May 2013
Line 62:		Line 62:
	#Zucker2003 pmid=12824337		#Zucker2003 pmid=12824337
	#GoTo2006 pmid=17150804		#GoTo2006 pmid=17150804
-	~~#Patel2007 pmid=17846637~~
	#Breaker2000 pmid=11187837		#Breaker2000 pmid=11187837
	#Cech2002 pmid=12110898		#Cech2002 pmid=12110898

Alvaro E. Rodriguez M.: /* References */

2013-05-06T14:25:03Z

References

←Older revision		Revision as of 14:25, 6 May 2013
Line 66:		Line 66:
	#Cech2002 pmid=12110898		#Cech2002 pmid=12110898
	#Silverman2012 pmid=23088677		#Silverman2012 pmid=23088677
		+	#Silverman2007 pmid=17394278
		+

	<\biblio>		<\biblio>