| Efficient RNA 5'-adenylation by T4 DNA ligase to facilitate practical applications. | |
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MedLine Citation:
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PMID: 16618967 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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We describe a simple procedure for RNA 5'-adenylation using T4 DNA ligase. The 5'-monophosphorylated terminus of an RNA substrate is annealed to a complementary DNA strand that has a 3'-overhang of 10 nucleotides. Then, T4 DNA ligase and ATP are used to synthesize 5'-adenylated RNA (5'-AppRNA), which should find use in a variety of practical applications. In the absence of an acceptor nucleic acid strand, the two-step T4 DNA ligase mechanism is successfully interrupted after the adenylation step, providing 40%-80% yield of 5'-AppRNA after PAGE purification with few side products (the yield varies with RNA sequence). Optimized reaction conditions are described for 5'-adenylating RNA substrates of essentially any length including long and structured RNAs, without need for sequestration of the RNA 3'-terminus to avoid circularization. The new procedure is applicable on the preparative nanomole scale. This 5'-adenylation strategy using T4 DNA ligase is a substantial improvement over our recently reported adenylation method that uses T4 RNA ligase, which often leads to substantial amounts of side products and requires careful optimization for each RNA substrate. Efficient synthetic access to 5'-adenylated RNA will facilitate a range of applications by providing substrates for in vitro selection; by establishing a new protocol for RNA 5'-capping; and by providing an alternative approach for labeling RNA with (32)P or biophysical probes at the 5'-terminus. |
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Authors:
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Yangming Wang; Scott K Silverman |
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Publication Detail:
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Type: Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't Date: 2006-04-17 |
Journal Detail:
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Title: RNA (New York, N.Y.) Volume: 12 ISSN: 1355-8382 ISO Abbreviation: RNA Publication Date: 2006 Jun |
Date Detail:
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Created Date: 2006-05-24 Completed Date: 2006-07-12 Revised Date: 2009-11-18 |
Medline Journal Info:
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Nlm Unique ID: 9509184 Medline TA: RNA Country: United States |
Other Details:
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Languages: eng Pagination: 1142-6 Citation Subset: IM |
Affiliation:
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Department of Chemistry, University of Illinois at Urbana-Champaign, 61801, USA. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Adenosine Triphosphate
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metabolism,
pharmacology DNA Ligases / metabolism* DNA, Complementary / metabolism Models, Genetic Oligodeoxyribonucleotides / genetics, metabolism RNA / metabolism* RNA Processing, Post-Transcriptional* Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Substrate Specificity |
| Grant Support | |
ID/Acronym/Agency:
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5-FY02-271//PHS HHS; GM-65966/GM/NIGMS NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/DNA, Complementary; 0/Oligodeoxyribonucleotides; 56-65-5/Adenosine Triphosphate; 63231-63-0/RNA; EC 6.5.1.-/DNA Ligases |
| Comments/Corrections | |
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