Automated RNA Selection

Cox, J. Colin; Rudolph, P.; Ellington, Andrew D.

doi:10.1021/bp980097h

Cited by 145 publications

(96 citation statements)

References 11 publications

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“…Fifteen rounds of selection were undertaken using a degenerate library of z10 15 sequences, consisting of an N 30 random region flanked by primer sequences for amplification and in vitro transcription. The SELEX procedure was carried out on a Biomek 2000 automated work station using protocols similar to those previously described by the Ellington Laboratory (Cox et al 1998;Cox and Ellington 2001). As expected, the selected pool displayed increased affinity for 3Dpol compared with the naive RNA population as assessed by electrophoretic mobility shift assays (data not shown).…”

Section: Aptamers With Similarity To the Viral Genomementioning

confidence: 60%

Selection and characterization of RNA aptamers to the RNA-dependent RNA polymerase from foot-and-mouth disease virus

Ellingham¹,

Bunka²,

Rowlands³

et al. 2006

RNA

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Foot-and-mouth disease virus causes a highly contagious disease of agricultural livestock and is of enormous economic importance. Replication of the RNA genome of the virus, via negative strand intermediates, involves an RNA-dependent RNA polymerase (3Dpol). RNA aptamers specific to this enzyme have been selected and characterized. Some of these molecules inhibit enzymatic activity in vitro, with IC 50 values of <20 nM and K i values of 18-75 nM. Two of these show similarity, both with each other and with regions of the viral genome. Furthermore, truncated versions of one of the aptamers have been used to define the parts of the molecule responsible for its inhibitory activity.

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Section: Aptamers With Similarity To the Viral Genomementioning

confidence: 60%

Selection and characterization of RNA aptamers to the RNA-dependent RNA polymerase from foot-and-mouth disease virus

Ellingham¹,

Bunka²,

Rowlands³

et al. 2006

RNA

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“…Selection experiments were facilitated by the development of protocols for automated in vitro selection (Cox et al 1998(Cox et al , 2002bCox and Ellington 2001). Peptide targets were synthesized with a C-terminal biotin and immobilized on streptavidin beads.…”

Section: In Vitro Selection Of Anti-arm Aptamersmentioning

confidence: 99%

“…Selection was performed on a Beckman Biomek 2000 workstation (Beckman-Coulter), using an automated selection protocol that has been described in detail (Cox et al 1998(Cox et al , 2002bCox and Ellington 2001). Biotinylated peptides were loaded on streptavidin beads (DynaBeads M280; Dynal) at a ratio of 1:10 peptide per streptavidin site (according to the manufacturer's instructions).…”

Section: Automated In Vitro Selectionmentioning

confidence: 99%

Arginine-rich motifs present multiple interfaces for specific binding by RNA

Bayer

Booth

Knudsen

et al. 2005

RNA

110

102

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A number of proteins containing arginine-rich motifs (ARMs) are known to bind RNA and are involved in regulating RNA processing in viruses and cells. Using automated selection methods we have generated a number of aptamers against ARM peptides from various natural proteins. Aptamers bind tightly to their cognate ARMs, with K d values in the nanomolar range, and frequently show no propensity to bind to other ARMs or even to single amino acid variants of the cognate ARM. However, at least some anti-ARM aptamers can cross-recognize a limited set of other ARMs, just as natural RNA-binding sites have been shown to exhibit so-called ''chameleonism.'' We expand upon the number of examples of cross-recognition and, using mutational and circular dichroism (CD) analyses, demonstrate that there are multiple mechanisms by which RNA ligands can crossrecognize ARMs. These studies support a model in which individual arginine residues govern binding to an RNA ligand, and the inherent flexibility of the peptide backbone may make it possible for ''semi-specific'' recognition of a discrete set of RNAs by a discrete set of ARM peptides and proteins.

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“…Aptazymes could also be selected against synthetic peptide targets identified by analyses of proteomics databases. The selection methods used for the generation of aptazymes are similar to those that have been used to generate aptamers, and are amenable to automation (Cox et al 1998;Sooter et al 2001). Moreover, aptazymes have already been adapted to array analysis (Seetharaman et al 2001).…”

Section: Peptide-activated Aptazymesmentioning

confidence: 99%

In vitro selection of ribozymes dependent on peptides for activity

Robertson¹,

Knudsen²,

Ellington

2003

RNA

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A peptide-dependent ribozyme ligase (aptazyme ligase) has been selected from a random sequence population based on the small L1 ligase. The aptazyme ligase is activated > 18,000-fold by its cognate peptide effector, the HIV-1 Rev arginine-rich motif (ARM), and specifically recognizes the Rev ARM relative to other peptides containing arginine-rich motifs. Moreover, the aptazyme ligase can preferentially recognize the Rev ARM in the context of the full-length HIV-1 Rev protein. The only cross-reactivity exhibited by the aptazyme is toward the Tat ARM. Reselection of peptide-and protein-dependent aptazymes from a partially randomized population yielded aptazymes that could readily discriminate against the Tat ARM. These results have important implications for the development of aptazymes that can be used in arrays for the detection and quantitation of multiple cellular proteins (proteome arrays).

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Automated RNA Selection

Cited by 145 publications

References 11 publications

Selection and characterization of RNA aptamers to the RNA-dependent RNA polymerase from foot-and-mouth disease virus

Selection and characterization of RNA aptamers to the RNA-dependent RNA polymerase from foot-and-mouth disease virus

Arginine-rich motifs present multiple interfaces for specific binding by RNA

In vitro selection of ribozymes dependent on peptides for activity

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