Donor-acceptor distance distributions in a double-labeled fluorescent oligonucleotide both as a single strand and in duplexes.

A 16-mer deoxyribonucleotide was labeled at the 5'-end with x-rhodamine and at the 3'-end with fluorescein. Förster resonance energy transfer was used to determine the distribution, P(R), of donor-acceptor distances for the oligomer in three duplex structures (hybridized to complementary strands having 10, 16, and 24 bases) and as a single strand; measurements were made in 0.18 M NaCl and 1 M KCl solutions. These distributions were derived from lifetime measurements made in the frequency domain using a multiharmonic frequency phase fluorometer. The fluorescein fluorescence decay for each duplex structure was fit very well with P(R) modeled as a shifted Gaussian. On the basis of the mean donor-acceptor distance, these structures for the 16-mer and the 24-mer were consistent with that of B-DNA. For the 16-mer duplex in 0.18 M NaCl, the distribution was centered at 68.4 A with sigma = 6.4 A. For the 10-mer duplex, the mean donor-acceptor distance, 61.8 A, is much larger than that for a structure with the fluorophores extended perpendicular to the helix axis. For the single-strand data, various high-quality fits yielded physically unreasonable distributions or could not accurately account for the acceptor response. Considered analyses suggested that the single-strand distribution was best represented by a shifted Gaussian, with R = 51.5 A and sigma = 10.0 A in 0.18 M NaCl. In all cases, sigma increased and R decreased in 1 M KCl relative to their values in 0.18 M NaCl, consistent with the increased flexibility of the polymer.