Boranophosphates Support the RNase H Cleavage of Polyribonucleotides

Abstract

Modification of the phosphodiester linkages in DNA by replacing one of the nonbridging oxygens with borane, BH3, produces an isoelectronic mimic of DNA called boranophosphates. Nonstereoregular oligodeoxyribonucleoside all-boranophosphates are shown here for the first time to elicit the RNase H hydrolysis of polyribonucleotides. We compared the ability of three types of dodecamers (dodecathymidine phosphate, phosphorothioate, and boranophosphate) to mediate the cleavage of poly(A) by Escherichia coli RNase H1. The rates of poly(A) hydrolysis induced by boranophosphates were 76-fold (at 20 degrees C) and 18-fold (at 30 degrees C) greater than the rates induced by dodecathymidine phosphate. In conjunction with the measured melting temperatures for each heteroduplex, carried out under the same conditions as the RNAse H cleavage experiments, the data establish an inverse relationship between the heteroduplex thermostability and the rate of poly(A) hydrolysis. Chromatographic analysis revealed another correlation: the higher the heteroduplex Tm, the higher the pApA:pApApA ratio in the corresponding hydrolysates. The specific content of these final products provides insight into the relative contribution of RNase H1 exonucleolytic/endonucleolytic mechanisms, with a low ratio for the lower melting heteroduplexes reflecting more endonucleolytic-type hydrolysis. In total, our data support the concept that antisense molecules with a weakened hybridization potential enhance the rate of hydrolysis of RNA in RNA-DNA hybrids.

New Antisense Strategies: Chemical Synthesis of RNA Oligomers

“…Boranophosphates are highly water-soluble and the boranophosphate linkage is more nuclease-resistant than the natural phosphodiester linkage. Boranophosphate oligodeoxynucleotides support RNase H activity [38], while boranophosphate-modified siRNA is reported to be slightly more active than unmodified siRNA in vitro [39]. Although boranophosphates have not yet found application in the development of oligonucleotide drugs, the potential of this modification continues to be explored.…”

Flexible and Frozen Sugar-Modified Nucleic Acids - Modulation of Biological Activity Through Furanose Ring Dynamics in the Antisense Strand

“…Remarkably, the difficulty with which the properties of the wild type substrates have been duplicated within the latter class has been a primary challenge that has only been met with little success thus far. Among the currently expanding list of modified AONs that retain RNase H-mediated cleavage of the mRNA message are the phosphorothioates, phosphorodithioates [33,34], boranophosphates [35,36] arabino-(ANA) and 2'-deoxy-2'-fluoro-β-D-arabinonucleic acids (2'F-ANA) [37,38], cyclohexene nucleic acids (CeNA) [39] and more recently, α-L-ribo-configured locked nucleic acids (α-L-LNA) [40]. Interestingly, the extent of cleavage by each of these classes varies significantly but all appear to share the same minor groove width dimensions, which is believed to be key to maintaining RNase H activity.…”

DNA and RNA derivatives to optimize distribution and delivery

“…1) are isosteric with methylphosphonate DNAs, but the BH 3 group exhibits a negative charge, isoelectronic with the oxygen of the phosphodiester group [37]. Aside from normal phosphodiester DNA and phosphorothioate DNA, only the boranophosphonate modification also exhibits RNase H activity [99] Further, it increases lipophilicity while maintaining binding to the targeted mRNA and exhibits a relatively low toxicity [37]. A solid phase H-phosphonate synthesis is possible [100], but enzymatic coupling has proved more facile [37].…”