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 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.
Levy
Matthew
M
Institute for Cellular and Molecular Biology, University of Texas, Austin, TX 78751, USA.
Griswold
Karl E
KE
Ellington
Andrew D
AD
D020029:Base Pairing; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry; D004655:Emulsions; D005434:Flow Cytometry; D019793:Fluorescein; D005456:Fluorescent Dyes; D014644:Genetic Variation; D006834:Hydrazines; D066298:In Vitro Techniques; D007700:Kinetics; D008025:Ligases / Q000378:metabolism*; D008863:Microspheres; D008956:Models, Chemical; D021141:Nucleic Acid Amplification Techniques; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D017423:Sequence Analysis, RNA; D013379:Substrate Specificity; D014158:Transcription, Genetic
C417561:Alexa 488 hydrazide; D004655:Emulsions; D005456:Fluorescent Dyes; D006834:Hydrazines; D016337:RNA, Catalytic; D004247:DNA; D008025:Ligases; D019793:Fluorescein