Structure and Dynamics of a Fluorescent DNA Oligomer Containing the EcoRI Recognition Sequence: Fluorescence, Molecular Dynamics, and NMR Studies

Academic Article

Abstract

  • The self-complementary DNA decamer duplex d(CTGAATTCAG)2 and its modified counterpart d(CTGA[2AP]TTCAG)2, where the innermost adenine (6-aminopurine) has been replaced with the fluorescent analogue 2-aminopurine (2AP), have been studied by fluorescence and NMR spectroscopy and simulated by molecular dynamics. Both decamers are recognized and cleaved by the EcoRl restriction endonuclease. 2D NMR results show that both decamers have a standard B-type conformation below 20 °C, though a disturbance exists to the 5' side of the 2AP site which may originate from increased local mobility. The fluorescence and fluorescence anisotropy decays of both decamers, as well as the one containing 2AP in only one chain, were studied as a function of temperature. The data show that the 2AP base exists in a temperature-dependent distribution of states and shows rapid motions, suggesting interconversion among these states on a time scale of about 10-10s. The integrated fluorescence of the decamer with 2AP in both chains shows a large increase around the helix melting temperature whereas the decamer with one 2AP shows only a mild increase, showing that the mixed helix has a different structural transition as sensed by the 2AP base. The data suggest a model of conformational states which have distinct fluorescence decay times. The various states may differ in the degree of base stacking. Fluctuations in the degree of stacking of the A or 2AP base are supported by molecular dynamics simulations, which additionally show that the 2AP-T or A-T base pair hydrogen bonds remain intact during these large motions. © 1989, American Chemical Society. All rights reserved.
  • Authors

    Published In

  • Biochemistry  Journal
  • Digital Object Identifier (doi)

    Start Page

  • 9095
  • End Page

  • 9103
  • Volume

  • 28
  • Issue

  • 23