Fluorescence intensity decays of 2-aminopurine solutions: Lifetime distribution approach

Shashank Bharill, Pabak Sarkar, Jeff D. Ballin, Ignacy Gryczynski, Gerald M. Wilson, Zygmunt Gryczynski

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

The fluorescent adenine analog 2-aminopurine (2AP) has been used extensively to monitor conformational changes and macromolecular binding events involving nucleic acids because its fluorescence properties are highly sensitive to changes in chemical environment. Furthermore, site-specific incorporation of 2AP permits local DNA and RNA conformational events to be discriminated from the global structural changes monitored by UV-Vis spectroscopy and circular dichroism. However, although the steady-state fluorescence properties of 2AP have been well defined in diverse settings, interpretation of 2AP fluorescence lifetime parameters has been hampered by the heterogeneous nature of multiexponential 2AP intensity decays observed across populations of microenvironments. To resolve this problem, we tested the utility of multiexponential versus continuous Lorentzian lifetime distribution models to describe fluorescence intensity decays from 2AP in diverse chemical backgrounds and within the context of RNA. Heterogeneity was introduced into 2AP intensity decays by mixing solvents of differing polarities or by adding quenchers under high viscosity to evaluate the transient effect. Heterogeneity of 2AP fluorescence within the context of a synthetic RNA hairpin was introduced by structural remodeling using a magnesium salt. In each case except folded RNA (which required a bimodal distribution), 2AP lifetime properties were well described by single Lorentzian distribution functions, abrogating the need to introduce additional discrete lifetime subpopulations. Rather, heterogeneity in fluorescence decay processes was accommodated by the breadth of each distribution. This approach also permitted solvent relaxation effects on 2AP emission to be assessed by comparing lifetime distributions at multiple wavelengths. Together, these studies provide a new perspective for the interpretation of 2AP fluorescence lifetime properties that will further the utility of this fluorophore in analyses of the complex and heterogeneous structural microenvironments associated with nucleic acids.

Original languageEnglish
Pages (from-to)141-149
Number of pages9
JournalAnalytical Biochemistry
Volume377
Issue number2
DOIs
StatePublished - 15 Jun 2008

Fingerprint

2-Aminopurine
Fluorescence
RNA
Nucleic Acids
Fluorophores
Adenine
Circular Dichroism
Ultraviolet spectroscopy
Viscosity
Magnesium
Distribution functions
Spectrum Analysis

Keywords

  • 2-Aminopurine
  • Lifetime distribution
  • Nucleic acids
  • Solvent relaxation
  • Transient effect

Cite this

Bharill, Shashank ; Sarkar, Pabak ; Ballin, Jeff D. ; Gryczynski, Ignacy ; Wilson, Gerald M. ; Gryczynski, Zygmunt. / Fluorescence intensity decays of 2-aminopurine solutions : Lifetime distribution approach. In: Analytical Biochemistry. 2008 ; Vol. 377, No. 2. pp. 141-149.
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Fluorescence intensity decays of 2-aminopurine solutions : Lifetime distribution approach. / Bharill, Shashank; Sarkar, Pabak; Ballin, Jeff D.; Gryczynski, Ignacy; Wilson, Gerald M.; Gryczynski, Zygmunt.

In: Analytical Biochemistry, Vol. 377, No. 2, 15.06.2008, p. 141-149.

Research output: Contribution to journalArticle

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T1 - Fluorescence intensity decays of 2-aminopurine solutions

T2 - Lifetime distribution approach

AU - Bharill, Shashank

AU - Sarkar, Pabak

AU - Ballin, Jeff D.

AU - Gryczynski, Ignacy

AU - Wilson, Gerald M.

AU - Gryczynski, Zygmunt

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N2 - The fluorescent adenine analog 2-aminopurine (2AP) has been used extensively to monitor conformational changes and macromolecular binding events involving nucleic acids because its fluorescence properties are highly sensitive to changes in chemical environment. Furthermore, site-specific incorporation of 2AP permits local DNA and RNA conformational events to be discriminated from the global structural changes monitored by UV-Vis spectroscopy and circular dichroism. However, although the steady-state fluorescence properties of 2AP have been well defined in diverse settings, interpretation of 2AP fluorescence lifetime parameters has been hampered by the heterogeneous nature of multiexponential 2AP intensity decays observed across populations of microenvironments. To resolve this problem, we tested the utility of multiexponential versus continuous Lorentzian lifetime distribution models to describe fluorescence intensity decays from 2AP in diverse chemical backgrounds and within the context of RNA. Heterogeneity was introduced into 2AP intensity decays by mixing solvents of differing polarities or by adding quenchers under high viscosity to evaluate the transient effect. Heterogeneity of 2AP fluorescence within the context of a synthetic RNA hairpin was introduced by structural remodeling using a magnesium salt. In each case except folded RNA (which required a bimodal distribution), 2AP lifetime properties were well described by single Lorentzian distribution functions, abrogating the need to introduce additional discrete lifetime subpopulations. Rather, heterogeneity in fluorescence decay processes was accommodated by the breadth of each distribution. This approach also permitted solvent relaxation effects on 2AP emission to be assessed by comparing lifetime distributions at multiple wavelengths. Together, these studies provide a new perspective for the interpretation of 2AP fluorescence lifetime properties that will further the utility of this fluorophore in analyses of the complex and heterogeneous structural microenvironments associated with nucleic acids.

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