Donor—Acceptor Distance Distributions in a Double-Labeled Fluorescent Oligonucleotide Both as a Single Strand and in Duplexes

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Abstract

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 Å with σ = 6.4 Å. For the 10-mer duplex, the mean donor-acceptor distance, 61.8 Å, 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 Å and σ = 10.0 Å in 0.18 M NaCl. In all cases, σ 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.

Original languageEnglish (US)
Pages (from-to)293-300
Number of pages8
JournalBiochemistry
Volume34
Issue number1
DOIs
StatePublished - Jan 1 1995

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Fluorescein
Oligonucleotides
Fluorometers
Deoxyribonucleotides
B-Form DNA
Rhodamines
Fluorophores
Energy Transfer
Oligomers
Energy transfer
Polymers
Fluorescence

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Donor—Acceptor Distance Distributions in a Double-Labeled Fluorescent Oligonucleotide Both as a Single Strand and in Duplexes",
abstract = "A 16-mer deoxyribonucleotide was labeled at the 5′-end with x-rhodamine and at the 3′-end with fluorescein. F{\"o}rster 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 {\AA} with σ = 6.4 {\AA}. For the 10-mer duplex, the mean donor-acceptor distance, 61.8 {\AA}, 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 {\AA} and σ = 10.0 {\AA} in 0.18 M NaCl. In all cases, σ 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.",
author = "Parkhurst, {Lawrence J}",
year = "1995",
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doi = "10.1021/bi00001a036",
language = "English (US)",
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N2 - 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 Å with σ = 6.4 Å. For the 10-mer duplex, the mean donor-acceptor distance, 61.8 Å, 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 Å and σ = 10.0 Å in 0.18 M NaCl. In all cases, σ 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.

AB - 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 Å with σ = 6.4 Å. For the 10-mer duplex, the mean donor-acceptor distance, 61.8 Å, 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 Å and σ = 10.0 Å in 0.18 M NaCl. In all cases, σ 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.

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