Identification of antisense nucleic acid hybridization sites in mRNA molecules with self-quenching fluorescent reporter molecules
| Autor: | Lesbeth C. Rodriguez, Kathy Vernovsky, Joanna B. Opalinska, Ponzy Lu, Bao T. Do, Anna Kalota, Paul Greenham, David Jordan, Lida K. Gifford, Vikram Pattanayak, Alan M. Gewirtz, Michelle Robbins |
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| Rok vydání: | 2005 |
| Předmět: |
Fluorophore
Transcription Genetic Base pair Biology Cell Line Oligodeoxyribonucleotides Antisense Antisense nucleic acid Proto-Oncogene Proteins c-myb chemistry.chemical_compound Transcription (biology) Cricetinae Proto-Oncogene Proteins Genetics Animals Humans RNA Messenger Fluorescent Dyes Quenching (fluorescence) Nucleic Acid Hybridization RNA Molecular biology Fluorescence DNA-Binding Proteins Proto-Oncogene Proteins c-kit chemistry Protein Biosynthesis Proto-Oncogene Proteins c-bcl-6 Nucleic acid Biophysics Methods Online Rabbits Transcription Factors |
| Zdroj: | Nucleic Acids Research |
| ISSN: | 1362-4962 |
| DOI: | 10.1093/nar/gni024 |
| Popis: | We describe a physical mRNA mapping strategy employing fluorescent self-quenching reporter molecules (SQRMs) that facilitates the identification of mRNA sequence accessible for hybridization with antisense nucleic acids in vitro and in vivo, real time. SQRMs are 20–30 base oligodeoxynucleotides with 5–6 bp complementary ends to which a 5′ fluorophore and 3′ quenching group are attached. Alone, the SQRM complementary ends form a stem that holds the fluorophore and quencher in contact. When the SQRM forms base pairs with its target, the structure separates the fluorophore from the quencher. This event can be reported by fluorescence emission when the fluorophore is excited. The stem–loop of the SQRM suggests that SQRM be made to target natural stem–loop structures formed during mRNA synthesis. The general utility of this method is demonstrated by SQRM identification of targetable sequence within c-myb and bcl-6 mRNA. Corresponding antisense oligonucleotides reduce these gene products in cells. |
| Databáze: | OpenAIRE |
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