Direct selection of trans-acting ligase ribozymes by in vitro compartmentalization

We have used a compartmentalized in vitro selection method to directly select for ligase ribozymes that are capable of acting on and turning over separable oligonucleotide substrates. Starting from a degenerate pool, we selected a trans-acting variant of the Bartel class I ligase which statistically...

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Bibliographic Details
Published in:RNA (Cambridge) 2005-10, Vol.11 (10), p.1555-1562
Main Authors: Levy, Matthew, Griswold, Karl E, Ellington, Andrew D
Format: Article
Language:English
Subjects:
Base Pairing
Base Sequence
Cloning, Molecular
DNA - chemistry
Emulsions
Flow Cytometry
Fluorescein
Fluorescent Dyes
Genetic Variation
Hydrazines
In Vitro Techniques
Kinetics
Ligases - metabolism
Microspheres
Models, Chemical
Nucleic Acid Amplification Techniques
Nucleic Acid Conformation
RNA, Catalytic - chemistry
RNA, Catalytic - genetics
RNA, Catalytic - isolation & purification
RNA, Catalytic - metabolism
Sequence Analysis, RNA
Substrate Specificity
Transcription, Genetic
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Summary:We have used a compartmentalized in vitro selection method to directly select for ligase ribozymes that are capable of acting on and turning over separable oligonucleotide substrates. Starting from a degenerate pool, we selected a trans-acting variant of the Bartel class I ligase which statistically may have been the only active variant in the starting pool. The isolation of this sequence from the population suggests that this selection method is extremely robust at selecting optimal ribozymes and should, therefore, prove useful for the selection and optimization of other trans-acting nucleic acid catalysts capable of multiple turnover catalysis.
ISSN:1355-8382
1469-9001
DOI:10.1261/rna.2121705