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rdf:type |
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lifeskim:mentions |
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pubmed:issue |
3
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pubmed:dateCreated |
2000-3-6
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pubmed:abstractText |
Naturally occurring hammerhead ribozymes are produced by rolling circle replication followed by self-cleavage. This results in monomer-length catalytic RNAs which have self-complementary sequences that can occupy their trans -binding domains and potentially block their ability to cleave other RNA strands. Here we show, using small self-processed ribozymes, that this self-binding does not necessarily inhibit trans -cleavage and can result in greatly elevated discrimination against mismatches. We utilized a designed 63 nt circular DNA to encode the synthesis of a self-processed ribozyme, MDR63. Rolling circle transcription followed by self-processing produced the desired 63 nt ribozyme, which potentially can bind mdr-1 RNA with 9+9 nt of complementarity or bind itself with 4+5 nt of self-complementarity by folding back its ends to form hairpins. Kinetics of trans -cleavage of short complementary and mismatched RNAs were measured under multiple turnover conditions, in comparison to a standard 40 nt ribozyme (MDR40) that lacks the self-complementary ends. The results show that MDR63 cleaves an mdr-1 RNA target with a k (cat)/ K (m)almost the same as MDR40, but with discrimination against mismatches up to 20 times greater. Based on folding predictions, a second self-processed ribozyme (UG63) having a single point mutation was synthesized; this displays even higher specificity (up to 100-fold) against mismatches. The results suggest that self-binding ends may be generally useful for increasing sequence specificity of ribozymes.
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pubmed:grant |
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pubmed:commentsCorrections |
http://linkedlifedata.com/resource/pubmed/commentcorrection/10637330-10051564,
http://linkedlifedata.com/resource/pubmed/commentcorrection/10637330-10329189,
http://linkedlifedata.com/resource/pubmed/commentcorrection/10637330-10421762,
http://linkedlifedata.com/resource/pubmed/commentcorrection/10637330-10438603,
http://linkedlifedata.com/resource/pubmed/commentcorrection/10637330-1280996,
http://linkedlifedata.com/resource/pubmed/commentcorrection/10637330-1871108,
http://linkedlifedata.com/resource/pubmed/commentcorrection/10637330-1946351,
http://linkedlifedata.com/resource/pubmed/commentcorrection/10637330-2271667,
http://linkedlifedata.com/resource/pubmed/commentcorrection/10637330-2436805,
http://linkedlifedata.com/resource/pubmed/commentcorrection/10637330-2441261,
http://linkedlifedata.com/resource/pubmed/commentcorrection/10637330-2456074,
http://linkedlifedata.com/resource/pubmed/commentcorrection/10637330-2457170,
http://linkedlifedata.com/resource/pubmed/commentcorrection/10637330-3684574,
http://linkedlifedata.com/resource/pubmed/commentcorrection/10637330-3714492,
http://linkedlifedata.com/resource/pubmed/commentcorrection/10637330-7610038,
http://linkedlifedata.com/resource/pubmed/commentcorrection/10637330-8118816,
http://linkedlifedata.com/resource/pubmed/commentcorrection/10637330-8136375,
http://linkedlifedata.com/resource/pubmed/commentcorrection/10637330-8538457,
http://linkedlifedata.com/resource/pubmed/commentcorrection/10637330-8670879,
http://linkedlifedata.com/resource/pubmed/commentcorrection/10637330-9062929,
http://linkedlifedata.com/resource/pubmed/commentcorrection/10637330-9238005,
http://linkedlifedata.com/resource/pubmed/commentcorrection/10637330-9521704,
http://linkedlifedata.com/resource/pubmed/commentcorrection/10637330-9628924,
http://linkedlifedata.com/resource/pubmed/commentcorrection/10637330-9701280,
http://linkedlifedata.com/resource/pubmed/commentcorrection/10637330-9778347,
http://linkedlifedata.com/resource/pubmed/commentcorrection/10637330-9836592
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pubmed:language |
eng
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pubmed:journal |
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pubmed:citationSubset |
IM
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pubmed:chemical |
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pubmed:status |
MEDLINE
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pubmed:month |
Feb
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pubmed:issn |
1362-4962
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pubmed:author |
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pubmed:issnType |
Electronic
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pubmed:day |
1
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pubmed:volume |
28
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
776-83
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pubmed:dateRevised |
2010-9-13
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pubmed:meshHeading |
pubmed-meshheading:10637330-Base Pair Mismatch,
pubmed-meshheading:10637330-Base Pairing,
pubmed-meshheading:10637330-Base Sequence,
pubmed-meshheading:10637330-DNA, Circular,
pubmed-meshheading:10637330-Genes, MDR,
pubmed-meshheading:10637330-Genetic Engineering,
pubmed-meshheading:10637330-Genetic Vectors,
pubmed-meshheading:10637330-Kinetics,
pubmed-meshheading:10637330-Models, Chemical,
pubmed-meshheading:10637330-Molecular Weight,
pubmed-meshheading:10637330-Point Mutation,
pubmed-meshheading:10637330-RNA,
pubmed-meshheading:10637330-RNA, Catalytic,
pubmed-meshheading:10637330-RNA Processing, Post-Transcriptional,
pubmed-meshheading:10637330-Substrate Specificity,
pubmed-meshheading:10637330-Thermodynamics,
pubmed-meshheading:10637330-Transcription, Genetic
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pubmed:year |
2000
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pubmed:articleTitle |
The virtues of self-binding: high sequence specificity for RNA cleavage by self-processed hammerhead ribozymes.
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pubmed:affiliation |
Department of Chemistry, University of Rochester, Rochester, NY 14627, USA.
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pubmed:publicationType |
Journal Article,
Research Support, U.S. Gov't, P.H.S.,
Research Support, U.S. Gov't, Non-P.H.S.
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