Local RNA conformational dynamics revealed by 2-aminopurine solvent accessibility

Jeff D. Ballin, James P. Prevas, Shashank Bharill, Ignacy Gryczynski, Zygmunt Gryczynski, Gerald M. Wilson

Research output: Contribution to journalArticle

19 Citations (Scopus)

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 (f a) 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 that can be measured by conventional spectroscopic approaches.

Original languageEnglish
Pages (from-to)7043-7052
Number of pages10
JournalBiochemistry
Volume47
Issue number27
DOIs
StatePublished - 8 Jul 2008

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2-Aminopurine
RNA
Acrylamide
Quenching
Base Pairing
Clamping devices
Adenine
Substrates
Fluorescent Dyes
Shielding

Cite this

Ballin, Jeff D. ; Prevas, James P. ; Bharill, Shashank ; Gryczynski, Ignacy ; Gryczynski, Zygmunt ; Wilson, Gerald M. / Local RNA conformational dynamics revealed by 2-aminopurine solvent accessibility. In: Biochemistry. 2008 ; Vol. 47, No. 27. pp. 7043-7052.
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Local RNA conformational dynamics revealed by 2-aminopurine solvent accessibility. / Ballin, Jeff D.; Prevas, James P.; Bharill, Shashank; Gryczynski, Ignacy; Gryczynski, Zygmunt; Wilson, Gerald M.

In: Biochemistry, Vol. 47, No. 27, 08.07.2008, p. 7043-7052.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Local RNA conformational dynamics revealed by 2-aminopurine solvent accessibility

AU - Ballin, Jeff D.

AU - Prevas, James P.

AU - Bharill, Shashank

AU - Gryczynski, Ignacy

AU - Gryczynski, Zygmunt

AU - Wilson, Gerald M.

PY - 2008/7/8

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N2 - 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 (f a) 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 that can be measured by conventional spectroscopic approaches.

AB - 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 (f a) 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 that can be measured by conventional spectroscopic approaches.

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