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 language | English (US) |
|---|---|
| Pages (from-to) | 4268-4277 |
| Number of pages | 10 |
| Journal | Biochemistry |
| Volume | 30 |
| Issue number | 17 |
| State | Published - 1991 |
| Externally published | Yes |
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- Biochemistry
Cite this
Electrophoretic and hydrodynamic properties of duplex ribonucleic acid molecules transcribed in vitro : Evidence that a-tracts do not generate curvature in RNA. / Hagerman, Paul J.
In: Biochemistry, Vol. 30, No. 17, 1991, p. 4268-4277.Research output: Contribution to journal › Article
}
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
Y1 - 1991
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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M3 - Article
VL - 30
SP - 4268
EP - 4277
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 17
ER -