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New J Phys. 2013 Feb 1;15. doi: 10.1088/1367-2630/15/2/025028.

Solution conformation of 2-aminopurine (2-AP) dinucleotide determined by ultraviolet 2D fluorescence spectroscopy (UV-2D FS).

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  • 1Oregon Center for Optics and Department of Chemistry, University of Oregon, Eugene, OR 97403, USA ; Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, OR 97403, USA.

Abstract

We have observed the conformation-dependent electronic coupling between the monomeric subunits of a dinucleotide of 2-aminopurine (2-AP), a fluorescent analog of the nucleic acid base adenine. This was accomplished by extending two-dimensional fluorescence spectroscopy (2D FS) - a fluorescence-detected variation of 2D electronic spectroscopy - to excite molecular transitions in the ultraviolet (UV) regime. A collinear sequence of four ultrafast laser pulses centered at 323 nm was used to resonantly excite the coupled transitions of 2-AP dinucleotide. The phases of the optical pulses were continuously swept at kilohertz frequencies, and the ensuing nonlinear fluorescence was phase-synchronously detected at 370 nm. Upon optimization of a point-dipole coupling model to our data, we found that in aqueous buffer the 2-AP dinucleotide adopts an average conformation in which the purine bases are non-helically stacked (center-to-center distance R 12 = 3.5 Å ± 0.5 Å, twist angle θ 12 = 5° ± 5°), which differs from the conformation of such adjacent bases in duplex DNA. These experiments establish UV-2D FS as a method for examining the local conformations of an adjacent pair of fluorescent nucleotides substituted into specific DNA or RNA constructs, which will serve as a powerful probe to interpret, in structural terms, biologically significant local conformational changes within the nucleic acid framework of protein-nucleic acid complexes.

PMID:
24223491
[PubMed]
PMCID:
PMC3819147
Free PMC Article
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