Site-specific variations in RNA folding thermodynamics visualized by 2-aminopurine fluorescence

Jeff D. Ballin, Shashank Bharill, Elizabeth J. Fialcowitz-White, Ignacy Gryczynski, Zygmunt Gryczynski, Gerald M. Wilson

Research output: Contribution to journalArticle

32 Citations (Scopus)

Abstract

The fluorescent base analogue 2-aminopurine (2-AP) is commonly used to study specific conformational and protein binding events involving nucleic acids. Here, combinations of steady-state and time-resolved fluorescence spectroscopy of 2-AP were employed to monitor conformational transitions within a model hairpin RNA from diverse structural perspectives. RNA substrates adopting stable, unambiguous secondary structures were labeled with 2-AP at an unpaired base, within the loop, or inside the base-paired stem. Steady-state fluorescence was monitored as the RNA hairpins made the transitions between folded and unfolded conformations using thermal denaturation, urea titration, and cation-mediated folding. Unstructured control RNA substrates permitted the effects of higher-order RNA structures on 2-AP fluorescence to be distinguished from stimulus-dependent changes in intrinsic 2-AP photophysics and/or interactions with adjacent residues. Thermodynamic parameters describing local conformational changes were thus resolved from multiple perspectives within the model RNA hairpin. These data provided energetic bases for construction of folding mechanisms, which varied among different folding - unfolding stimuli. Time-resolved fluorescence studies further revealed that 2-AP exhibits characteristic signatures of component fluorescence lifetimes and respective fractional contributions in different RNA structural contexts. Together, these studies demonstrate localized conformational events contributing to RNA folding and unfolding that could not be observed by approaches monitoring only global structural transitions.

Original languageEnglish
Pages (from-to)13948-13960
Number of pages13
JournalBiochemistry
Volume46
Issue number49
DOIs
StatePublished - 11 Dec 2007

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2-Aminopurine
RNA Folding
Thermodynamics
Fluorescence
RNA
Fluorescence Spectrometry
Denaturation
Protein Binding
Fluorescence spectroscopy
Nucleic Acids
Substrates
Urea
Cations
Titration
Hot Temperature
Conformations

Cite this

Ballin, Jeff D. ; Bharill, Shashank ; Fialcowitz-White, Elizabeth J. ; Gryczynski, Ignacy ; Gryczynski, Zygmunt ; Wilson, Gerald M. / Site-specific variations in RNA folding thermodynamics visualized by 2-aminopurine fluorescence. In: Biochemistry. 2007 ; Vol. 46, No. 49. pp. 13948-13960.
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abstract = "The fluorescent base analogue 2-aminopurine (2-AP) is commonly used to study specific conformational and protein binding events involving nucleic acids. Here, combinations of steady-state and time-resolved fluorescence spectroscopy of 2-AP were employed to monitor conformational transitions within a model hairpin RNA from diverse structural perspectives. RNA substrates adopting stable, unambiguous secondary structures were labeled with 2-AP at an unpaired base, within the loop, or inside the base-paired stem. Steady-state fluorescence was monitored as the RNA hairpins made the transitions between folded and unfolded conformations using thermal denaturation, urea titration, and cation-mediated folding. Unstructured control RNA substrates permitted the effects of higher-order RNA structures on 2-AP fluorescence to be distinguished from stimulus-dependent changes in intrinsic 2-AP photophysics and/or interactions with adjacent residues. Thermodynamic parameters describing local conformational changes were thus resolved from multiple perspectives within the model RNA hairpin. These data provided energetic bases for construction of folding mechanisms, which varied among different folding - unfolding stimuli. Time-resolved fluorescence studies further revealed that 2-AP exhibits characteristic signatures of component fluorescence lifetimes and respective fractional contributions in different RNA structural contexts. Together, these studies demonstrate localized conformational events contributing to RNA folding and unfolding that could not be observed by approaches monitoring only global structural transitions.",
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Site-specific variations in RNA folding thermodynamics visualized by 2-aminopurine fluorescence. / Ballin, Jeff D.; Bharill, Shashank; Fialcowitz-White, Elizabeth J.; Gryczynski, Ignacy; Gryczynski, Zygmunt; Wilson, Gerald M.

In: Biochemistry, Vol. 46, No. 49, 11.12.2007, p. 13948-13960.

Research output: Contribution to journalArticle

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