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| Content Provider | PubMed Central |
|---|---|
| Author | Ballin, Jeff D. Prevas, James P. Shashank, Bharill Gryczynski, Ignacy Zygmunt, Gryczynski Wilson, Gerald M. |
| Abstract | Acrylamide quenching is widely used to monitor the solvent exposure of fluorescent probes in vitro. Here, we tested the utility of this technique to discriminate local RNA secondary structures using the fluorescent adenine analogue 2-aminopurine (2-AP). Under native conditions, the solvent accessibilities of most 2-AP-labeled RNA substrates were poorly resolved by classical single population models; rather, a two-state quencher accessibility algorithm was required to model acrylamide-dependent changes in 2-AP fluorescence in structured RNA contexts. Comparing 2-AP quenching parameters between structured and unstructured RNA substrates permitted the effects of local RNA structure on 2-AP solvent exposure to be distinguished from nearest neighbor effects or environmental influences on intrinsic 2-AP photophysics. Using this strategy, the fractional accessibility of 2-AP for acrylamide (fa ) was found to be highly sensitive to local RNA structure. Base-paired 2-AP exhibited relatively poor accessibility, consistent with extensive shielding by adjacent bases. 2-AP in a single base bulge was uniformly accessible to solvent, whereas the fractional accessibility of 2-AP in a hexanucleotide loop was indistinguishable from that of an unstructured RNA. However, these studies also provided evidence that the fa parameter reflects local conformational dynamics in base-paired RNA. Enhanced base pair dynamics at elevated temperatures were accompanied by increased fa values, while restricting local RNA breathing by adding a C-G base pair clamp or positioning 2-AP within extended RNA duplexes significantly decreased this parameter. Together, these studies show that 2-AP quenching studies can reveal local RNA structural and dynamic features beyond those measurable by conventional spectroscopic approaches. |
| Related Links | http://dx.doi.org/10.1021/bi800487c |
| Starting Page | 7043 |
| File Format | |
| ISSN | 15204995 |
| e-ISSN | 15204995 |
| Journal | Biochemistry |
| Issue Number | 27 |
| Volume Number | 47 |
| Language | English |
| Publisher Date | 2008-07-08 |
| Access Restriction | Open |
| Subject Keyword | Research in Higher Education |
| Content Type | Text |
| Resource Type | Article |
| Subject | Biochemistry |
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