Zinaida A. Sergueeva

High potency silencing by single-stranded boranophosphate siRNA

In RNA interference (RNAi), double-stranded short interfering RNA (ds-siRNA) inhibits expression from complementary mRNAs. Recently, it was demonstrated that short, single-stranded antisense RNA (ss-siRNA) can also induce RNAi. While ss-siRNA may offer several advantages in both clinical and research applications, its overall poor activity compared with ds-siRNA has prevented its widespread use. In contrast to the poor gene silencing activity of native ss-siRNA, we found that the silencing activity of boranophosphate-modified ss-siRNA is comparable with that of unmodified ds-siRNA. Boranophosphate ss-siRNA has excellent maximum silencing activity and is highly effective at low concentrations. The silencing activity of boranophosphate ss-siRNA is also durable, with significant silencing up to 1 week after transfection. Thus, we have demonstrated that boranophosphate-modified ss-siRNA can silence gene expression as well as native ds-siRNA, suggesting that boranophosphate-modified ss-siRNAs should be investigated as a potential new class of therapeutic agents.

Reading, Writing, and Modulating Genetic Information with Boranophosphate Mimics of Nucleotides, DNA, and RNA

Abstract: The P‐boranophosphates are efficient and near perfect mimics of natural nucleic acids in permitting reading and writing of genetic information with high yield and accuracy. Substitution of a borane (−BH3) group for oxygen in the phosphate ester bond creates an isoelectronic and isosteric mimic of natural nucleotide phosphate esters found in mononucleotides, i.e., AMP and ATP, and in RNA and DNA polynucleotides. Compared to natural nucleic acids, the boranophosphate RNA and DNA analogs demonstrate increased lipophilicity and resistance to endo‐ and exonucleases, yet they retain negative charge and similar spatial geometry. Borane groups can readily be introduced into the NTP and dNTP nucleic acid monomer precursors to produce α‐P‐borano nucleoside triphosphate analogs (e.g., NTPαB and dNTPαB). The NTPαB and dNTPαB are, in fact, good to excellent substrates for RNA and DNA polymerases, respectively, and allow ready enzymatic synthesis of RNA and DNA with P‐boranophosphate linkages. Further, boranophosphate polymer products are good templates for replication, transcription, and gene expression; boronated RNA products are also suitable for reverse transcription to cDNA. Fully substituted boranophosphate DNA can activate the RNase H cleavage of RNA in RNA:DNA hybrids. Moreover, certain dideoxy‐NTPαB analogs appear to be better substrates for viral reverse transcriptases than the regular ddNTPs, and may offer promising prodrug alternatives in antiviral therapy. These properties make boranophosphates promising candidates for diagnostics; aptamer selection; gene therapy; and antiviral, antisense, and RNAi therapeutics. The boranophosphates constitute a versatile family of phosphate mimics for processing genetic information and modulating gene function.

Boranophosphate backbone: a mimic of phosphodiesters, phosphorothioates, and methyl phosphonates.

Nucleoside boranophosphates are distinctive in that one of the non-bridging oxygens in the phosphate diester 1 is replaced by a borane moiety (BH3). Although they retain the same net charge, BH3(-)-ODN have unique chemical and biochemical characteristics relative to other analogs. The change in polarity, lipophilicity, nuclease resistance, and the activation of RNase H cleavage of RNA in RNA: boranophosphate hybrids make boranophosphates very attractive for applications in enzymology and molecular biology and as potential antisense agents.

BORANE-AMINE COMPLEXES - VERSATILE REAGENTS IN THE CHEMISTRY OF NUCLEIC ACIDS AND THEIR ANALOGS

A new method for synthesis of N-alkylated nucleosides was developed. Exceptionally mild and selective conversion of N-acyl to the corresponding N-alkyl nucleosides was achieved by reduction with borane-amine complexes. The borane-amine complexes were also used as efficient scavengers of a 4,4′-dimethoxytrityl (DMT) cation. Neutralization of the cation eliminated the boranophosphate group degradation during acidic DMT deprotection and allowed milder acidic conditions for the deprotection.

Synthesis of dithymidine boranophosphates via stereospecific boronation of H-phosphonate diesters and assignment of their configuration

Abstract The absolute configurations of the two dithymidine boranophosphate isomers were established by chemical correlation with the H-phosphonate isomers. All chemical transformation leading from H-phosphonate diesters to boranophosphate diesters were found to be stereospecific with retention of configuration around phosphorus. The data verify our previous assignment of Rp and Sp isomers of dithymidine boranophosphate made on the basis of NMR and enzymatic hydrolysis.

Incorporation of (α-P-Borano)-2′,3′-dideoxycytidine 5′-Triphosphate into DNA by Drug-Resistant MMLV Reverse Transcriptase and Taq DNA Polymerase

Abstract The Rp-stereoisomer of 5′-(α-P-borano)triphosphates of 2′-deoxycytidine (Rp-dCTPαB) and 2′,3′-dideoxycytidine (Rp-ddCTPαB) were synthesized. Their steady-state kinetics of incorporation by ddNTP-resistant enzymes, e.g., MMLV reverse transcriptase (RT) and Taq DNA polymerase, were investigated and compared with incorporation of dCTP and ddCTP. The α-boranophosphate substitution in ddCTP results in a 28-fold increase in efficiency of incorporation of the Rp-ddCTPαB isomer by MMLV RT, yet has minimal effect on the efficiency of incorporation by Taq DNA polymerase.

Rapid and Selective Reduction of Amide Group by Borane-Amine Complexes in Acyl Protected Nucleosides

Abstract Borane-amine complexes provide an unusually fast and selective reduction of a deoxynucleoside N-acyl group to a corresponding N-alkyl group. Three different nucleosides (dG, dA, and dC) each having one of three N-protecting groups (benzoyl, isobutyryl, or acetyl) were used to prepare N-alkylated nucleosides in good yields under mild conditions. Deoxyribose O-acyl protecting groups remain intact at the conditions of N-acyl group reduction.

SYNTHESIS OF DIURIDINE 3', 5'-BORANOPHOSPHATE : H-PHOSPHONATE APPROACH

Abstract Diuriine 3′,5′-boranophosphate, the RNA analogue of boranophosphate nucleic acids, was synthesized by a new approach via the H-phosphonate. Two diastereomers of diuridine 3′,5′-boranophosphate were separated by reverse phase HPLC.

SYNTHESIS OF OLIGONUCLEOSIDE BORANOPHOSPHATES VIA AN H-PHOSPHONATE METHOD WITHOUT NUCLEOBASE PROTECTION

Short oligonucleoside boranophosphates containing all four nucleosides were synthesized on solid support using base-unprotected nucleoside H-phosphonate monomers. This strategy avoided irreversible base modifications during the boronation procedure. Structures of the boranophosphate oligomers were confirmed by 1H, 31P, 10B NMR and MS analysis as well as by enzymatic hydrolysis.