Electrophoretic and hydrodynamic properties of duplex ribonucleic acid molecules transcribed in vitro: Evidence that a-tracts do not generate curvature in RNA

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Abstract

We have developed a T7 RNA polymerase based transcription system for the production of fully complementary RNA molecules (i.e., molecules capable of forming blunt-ended duplex species) as the direct products of transcription, thus rendering unnecessary the enzymatic removal of single-stranded ends. A combined gel electrophoretic and hydrodynamic analysis of a 180 bp double-stranded (ds) RNA molecule containing four A5-tracts in approximate phase coherence with the helix repeat provides no indication that the helix axis is curved, in sharp contrast to DNA molecules containing phased A-tracts. The electrophoretic behavior of dsRNA molecules reveals that their mobilities in nondenaturing acrylamide gels are approximately 10-20% lower than the corresponding mobilities of duplex DNA, in accord with earlier observations in the literature. Furthermore, the relative mobilities are only slightly modulated by gel concentration, the concentration of monovalent salt, or the presence of spermidine and/or Mg2+. The reduced mobilities are not caused by increased contour length, since direct hydrodynamic measurements using transient electric birefringence indicate that the average helix rise, h, of the dsRNA molecules examined in the current study is 2.8 ± 0.1 Å/bp. The reduced electrophoretic mobilities, extrapolated to zero acrylamide concentration, are consistent with the lower residual charge predicted for dsRNA by counterion condensation theory. Finally, birefringence measurements indicate that dsRNA is only marginally stiffer than DNA, with a persistence length of ca. 500-700 Å.

Original languageEnglish (US)
Pages (from-to)4268-4277
Number of pages10
JournalBiochemistry
Volume30
Issue number17
StatePublished - 1991
Externally publishedYes

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Hydrodynamics
Birefringence
Acrylamide
Gels
RNA
Molecules
DNA
Complementary RNA
Spermidine
Double-Stranded RNA
Transcription
Salts
Electrophoretic mobility
In Vitro Techniques
Condensation

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Electrophoretic and hydrodynamic properties of duplex ribonucleic acid molecules transcribed in vitro: Evidence that a-tracts do not generate curvature in RNA",
abstract = "We have developed a T7 RNA polymerase based transcription system for the production of fully complementary RNA molecules (i.e., molecules capable of forming blunt-ended duplex species) as the direct products of transcription, thus rendering unnecessary the enzymatic removal of single-stranded ends. A combined gel electrophoretic and hydrodynamic analysis of a 180 bp double-stranded (ds) RNA molecule containing four A5-tracts in approximate phase coherence with the helix repeat provides no indication that the helix axis is curved, in sharp contrast to DNA molecules containing phased A-tracts. The electrophoretic behavior of dsRNA molecules reveals that their mobilities in nondenaturing acrylamide gels are approximately 10-20{\%} lower than the corresponding mobilities of duplex DNA, in accord with earlier observations in the literature. Furthermore, the relative mobilities are only slightly modulated by gel concentration, the concentration of monovalent salt, or the presence of spermidine and/or Mg2+. The reduced mobilities are not caused by increased contour length, since direct hydrodynamic measurements using transient electric birefringence indicate that the average helix rise, h, of the dsRNA molecules examined in the current study is 2.8 ± 0.1 {\AA}/bp. The reduced electrophoretic mobilities, extrapolated to zero acrylamide concentration, are consistent with the lower residual charge predicted for dsRNA by counterion condensation theory. Finally, birefringence measurements indicate that dsRNA is only marginally stiffer than DNA, with a persistence length of ca. 500-700 {\AA}.",
author = "Hagerman, {Paul J}",
year = "1991",
language = "English (US)",
volume = "30",
pages = "4268--4277",
journal = "Biochemistry",
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publisher = "American Chemical Society",
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TY - JOUR

T1 - Electrophoretic and hydrodynamic properties of duplex ribonucleic acid molecules transcribed in vitro

T2 - Evidence that a-tracts do not generate curvature in RNA

AU - Hagerman, Paul J

PY - 1991

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N2 - We have developed a T7 RNA polymerase based transcription system for the production of fully complementary RNA molecules (i.e., molecules capable of forming blunt-ended duplex species) as the direct products of transcription, thus rendering unnecessary the enzymatic removal of single-stranded ends. A combined gel electrophoretic and hydrodynamic analysis of a 180 bp double-stranded (ds) RNA molecule containing four A5-tracts in approximate phase coherence with the helix repeat provides no indication that the helix axis is curved, in sharp contrast to DNA molecules containing phased A-tracts. The electrophoretic behavior of dsRNA molecules reveals that their mobilities in nondenaturing acrylamide gels are approximately 10-20% lower than the corresponding mobilities of duplex DNA, in accord with earlier observations in the literature. Furthermore, the relative mobilities are only slightly modulated by gel concentration, the concentration of monovalent salt, or the presence of spermidine and/or Mg2+. The reduced mobilities are not caused by increased contour length, since direct hydrodynamic measurements using transient electric birefringence indicate that the average helix rise, h, of the dsRNA molecules examined in the current study is 2.8 ± 0.1 Å/bp. The reduced electrophoretic mobilities, extrapolated to zero acrylamide concentration, are consistent with the lower residual charge predicted for dsRNA by counterion condensation theory. Finally, birefringence measurements indicate that dsRNA is only marginally stiffer than DNA, with a persistence length of ca. 500-700 Å.

AB - We have developed a T7 RNA polymerase based transcription system for the production of fully complementary RNA molecules (i.e., molecules capable of forming blunt-ended duplex species) as the direct products of transcription, thus rendering unnecessary the enzymatic removal of single-stranded ends. A combined gel electrophoretic and hydrodynamic analysis of a 180 bp double-stranded (ds) RNA molecule containing four A5-tracts in approximate phase coherence with the helix repeat provides no indication that the helix axis is curved, in sharp contrast to DNA molecules containing phased A-tracts. The electrophoretic behavior of dsRNA molecules reveals that their mobilities in nondenaturing acrylamide gels are approximately 10-20% lower than the corresponding mobilities of duplex DNA, in accord with earlier observations in the literature. Furthermore, the relative mobilities are only slightly modulated by gel concentration, the concentration of monovalent salt, or the presence of spermidine and/or Mg2+. The reduced mobilities are not caused by increased contour length, since direct hydrodynamic measurements using transient electric birefringence indicate that the average helix rise, h, of the dsRNA molecules examined in the current study is 2.8 ± 0.1 Å/bp. The reduced electrophoretic mobilities, extrapolated to zero acrylamide concentration, are consistent with the lower residual charge predicted for dsRNA by counterion condensation theory. Finally, birefringence measurements indicate that dsRNA is only marginally stiffer than DNA, with a persistence length of ca. 500-700 Å.

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