L. Dedionisio et Sm. Gryaznov, ANALYSIS OF A RIBONUCLEASE-H DIGESTION OF N3'-]P5' PHOSPHORAMIDATE RNA DUPLEXES BY CAPILLARY GEL-ELECTROPHORESIS, Journal of chromatography B. Biomedical applications, 669(1), 1995, pp. 125-131
Citations number
17
Categorie Soggetti
Chemistry Analytical
Journal title
Journal of chromatography B. Biomedical applications
Phosphodiester oligonucleotides (ODNs) and their analogs are presently
being investigated as potential antisense therapeutics in the treatme
nt of viral infections and various forms of cancer. Here, we would lik
e to report results from an investigation of activity for a ribonuclea
se H (RNase Hi) mediated RNA digestion assay in the duplexes formed by
an ODN or the ODN analog, N3'-->P5' phosphoramidate (3'-phosphoramida
te), and complimentary RNA strands. Capillary gel electrophoresis (CGE
) proved to be an effective method for determining RNA hydrolysis in t
he presence of RNase H. RNA and an ODN or RNA and a 3'-phosphoramidate
were hybridized in a Tris-HCl, MgCl2 buffer at room temperature (RT)
and incubated with RNase H. Digestions were carried out at RT or at 37
degrees C. Control samples were unhybridized RNA with RNase H, RNA wi
thout RNase H, and duplexes (RNA-ODN or 3'-phosphoramidate) without RN
ase H. All controls were incubated in Tris-HCl, MgCl2 buffer, and samp
le aliquots were analyzed at various time intervals. A homodecamer, (d
T)(10), was used as an internal standard to determine the relative mig
ration time of the RNA strand. The final digestion products for the du
plexes and the various controls were monitored by CGE. In addition, po
lyacrylamide gel electrophoresis (PAGE) was used in conjunction with S
tains-All (staining) and a densitometric analysis to verify CGE result
s.