[14] In vitro selection of RNA aptamers

Marshall, Kristin A.; Ellington, Andrew D.

doi:10.1016/s0076-6879(00)18053-x

Cited by 73 publications

(44 citation statements)

References 34 publications

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“…Typically, the stringency is progressively increased in the course of a SELEX process. This can be achieved by reducing the target concentration in later SELEX rounds or changing the binding and washing conditions (buffer composition, volume, time) (Marshall and Ellington, 2000).…”

Section: General Principlementioning

confidence: 99%

SELEX—A (r)evolutionary method to generate high-affinity nucleic acid ligands

Stoltenburg

Reinemann

Strehlitz

2007

Biomolecular Engineering

1,260

947

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Section: General Principlementioning

confidence: 99%

SELEX—A (r)evolutionary method to generate high-affinity nucleic acid ligands

Stoltenburg

Reinemann

Strehlitz

2007

Biomolecular Engineering

1,260

947

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“…The SELEX method has permitted the identification of unique RNA/DNA molecules, from very large populations of random sequence oligomers (DNA or RNA libraries), which bind to the target molecule with very high affinity and specificity. Selections are frequently carried out with RNA pools due to the known ability of RNAs to fold into complex structures which can be a source of diversity of RNA function, but single-stranded DNA pools can also yield aptamers (Marshall and Ellington, 2000). Single-stranded DNAs are also known to fold in vitro into structures containing stem-loop, internal loops, etc., even if less stable than the corresponding RNA structures (Harada and Frankel, 1995).…”

Section: Introductionmentioning

confidence: 99%

Analytical applications of aptamers

Tombelli

Minunni

Mascini

2005

Biosensors and Bioelectronics

931

499

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“…in vitro selection ͉ nucleic acid tag ͉ phosphoamide ͉ Tat ͉ TAR I n vitro selection has been used in combination with rational design to identify and optimize numerous ribozymes and binding motifs (1)(2)(3). The results of these experiments continue to give insight into the capacity of RNA and DNA to catalyze a variety of chemical reactions and the ability of nucleic acids to recognize an array of substrates.…”

mentioning

confidence: 99%

A ribozyme that ligates RNA to protein

Baskerville

Bartel

2002

Proc. Natl. Acad. Sci. U.S.A.

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We have used a combination of in vitro selection and rational design to generate ribozymes that form a stable phosphoamide bond between the 5 terminus of an RNA and a specific polypeptide. This reaction differs from that of previously identified ribozymes, although the product is analogous to the enzymenucleotidyl intermediates isolated during the reactions of certain proteinaceous enzymes, such as guanyltransferase, DNA ligase, and RNA ligase. Comparative sequence analysis of the isolated ribozymes revealed that they share a compact secondary structure containing six stems arranged in a four-helix junction and branched pseudoknot. An optimized version of the ribozyme reacts with substrate-fusion proteins, allowing it to be used to attach RNA tags to proteins both in vitro and within bacterial cells, suggesting a simple way to tag a specific protein with amplifiable information.in vitro selection ͉ nucleic acid tag ͉ phosphoamide ͉ Tat ͉ TAR I n vitro selection has been used in combination with rational design to identify and optimize numerous ribozymes and binding motifs (1-3). The results of these experiments continue to give insight into the capacity of RNA and DNA to catalyze a variety of chemical reactions and the ability of nucleic acids to recognize an array of substrates. Some of these catalysts are being developed into effective tools for use in molecular biology. These include allosteric ribozymes, used as detectors of small molecules and proteins, and DNA enzymes that efficiently cleave target sequences (4-6).Here we use in vitro selection coupled with rational design to produce a new ribozyme that ligates RNA to protein through the formation of a phosphoamide bond. This is similar to a reaction found in naturally occurring proteins such as guanyltransferase, and DNA and RNA ligases that form phosphoamide bonds as enzyme-nucleotidyl intermediates during cap formation and ligation events (7-9). The selection of this new ribozyme expands the repertoire of RNA-catalyzed chemistry and further illustrates the ability of RNA to chemically modify a variety of substrates. Moreover, this ribozyme might be used to synthesize oligonucleotide-tagged proteins in vitro and in vivo. These attached oligonucleotides might be useful as affinity tags or could function as amplifiable identifying markers. For example, this ribozyme might be used to attach mRNAs to the proteins that they encode. A diverse library of mRNA-protein fusions could be used in conjunction with in vitro selection strategies to identify previously undescribed proteins with unique or enhanced properties, as has been shown previously by using phage display, ribosome display, and mRNA display (10-13). Materials and MethodsPool Construction. The double-stranded DNA pool was constructed from two 110-nt DNA fragments, each containing 76 completely randomized positions (14). Each fragment was amplified separately in 110 ml PCR reactions and ligated through BanI sites to generate a final pool with the sequence: TTCTAATACGACTCACTATAG-GACAGCTCCGAGCATTCTCGTGT...

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[14] In vitro selection of RNA aptamers

Cited by 73 publications

References 34 publications

SELEX—A (r)evolutionary method to generate high-affinity nucleic acid ligands

SELEX—A (r)evolutionary method to generate high-affinity nucleic acid ligands

Analytical applications of aptamers

A ribozyme that ligates RNA to protein

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