Flexibility of single-stranded DNA: use of gapped duplex helices to determine the persistence lengths of Poly(dT) and Poly(dA)

Abstract

The forces responsible for the formation and stabilization of secondary and higher-order nucleic acid structure can be more fully understood once the sequence-dependent properties (e.g. intrinsic rigidity, effective rise) of the component single-stranded species are well-defined. Knowledge of the conformations of the single-stranded polymers is also important for the development of better polyelectrolyte models for various structural or strand-dissociation reactions. However, there is at present little quantitative information regarding the sequence dependence of either rise or rigidity in single-stranded DNA or RNA polymers. To address this issue, we describe a form of transient electric birefringence (TEB) measurement in which the rotational decay times (τgap) of DNA molecules possessing central, single-stranded regions (gaps) are compared with the corresponding times (τdplx) for duplex control molecules of the same length (in nucleotides per strand) as the continuous strand in the “gapped duplex”. For magnesium ion concentrations above 1–2 mM, the τ ratios (τgap/τdplx) for the gapped duplexes reach plateau values, above which no further change in τ ratio is observed; values for the persistence length (P) and internucleotide spacing (h) of the gap sequences are obtained from the experimental τ ratios. For dTn, the permissible ranges of P and h are 20–30 Å and 5–7 Å per nucleotide (nt), respectively, with optimal values of 31 Å (P) and 5.2 Å/nt (h). For dAn, the persistence length for the low temperature (4 °C), stacked form is 78 (±8) Å for a helix rise of 3.2 Å/nt. One significant advantage of the current method over previous approaches is the use of short (80–100 nt) molecules, thus facilitating the production of various gap sequences through synthetic means.