Synthesis and properties of novel triphosphate analogues: ribonucleoside and deoxyribonucleoside (alpha-P-borano, alpha-P-thio)triphosphates.

The first ribo- and deoxyribo-nucleoside (alpha-P-borano, alpha-P-thio)triphosphates have been synthesized. The chemical and biochemical properties of adenosine (alpha-P-borano, alpha-P-thio)triphosphate and thymidine (alpha-P-borano, alpha-P-thio) triphosphate have been investigated.

Direct PCR sequencing with boronated nucleotides.

A method is described to simultaneously amplify and sequence DNA using a new class of nucleotides containing boron. During the polymerase chain reaction, boron-modified nucleotides, i.e. 2'-deoxynucleoside 5'-alpha-[P-borano]-triphosphates, are incorporated into the product DNA. The boranophosphate linkages are resistant to nucleases and thus the positions of the boranophosphates can be revealed by exonuclease digestion, thereby generating a set of fragments that defines the DNA sequence. The boranophosphate method offers an alternative to current PCR sequencing methods. Single-sided primer extension with dideoxynucleotide chain terminators is avoided, with the consequence that the sequencing fragments are derived directly from the original PCR products. Boranophosphate sequencing is demonstrated with the Pharmacia and the Applied Biosystems 373A automatic sequencers, producing data that is comparable with cycle sequencing.

N7-cyanoborane-2'-deoxyguanosine 5'-triphosphate is a good substrate for DNA polymerase.

The 5'-triphosphate of the boronated nucleoside analog N7-cyanoborane-2'-deoxyguanosine (7bdGTP) was synthesized, and a series of experiments was initiated to assess the potential of the compound to serve as a substrate for DNA polymerases. We show here that 7bdGTP can be incorporated into DNA by Sequenase. The resulting hemiboronated extension products are resistant to cleavage by treatment with either DMS and heat or a number of restriction enzymes. Further, in the polymerase chain reaction, 7bdGTP can be utilized as a substrate for Taq polymerase. Finally, by kinetic analysis, we have found that 7bdGTP is a more efficient substrate for exonuclease-free Klenow than normal dGTP. Thus, the introduction of a cyanoborane moiety to the N7 position of dGTP results in a nucleotide that is accepted in lieu of normal dGTP by a number of DNA polymerases.

Synthesis of 5-substituted 2'-deoxycytidine 5'-(alpha-P-borano)triphosphates, their incorporationinto DNA and effects on exonuclease.

Direct PCR sequencing with boronated nucleotides provides an alternative to current PCR sequencing methods. The positions of boranophosphate-modified nucleotides incorporated randomly into DNA during PCR can be revealed directly by exonuclease digestion to give sequencing ladders. Cytosine nucleotides, however, are especially sensitive to exonuclease digestion and provide suboptimal sequencing ladders. Therefore, a series of 5-substituted analogs of 2'-deoxycytidine 5'-(alpha-P-borano)triphosphates (dCTPalphaB) were synthesized with the hope of increasing the nuclease resistance of deoxycytosine residues and thereby enhancing the deoxycytosine band intensities. These dCTP analogs contain a boranophosphate modification at the alpha-phosphate group in 2'-deoxycytidine 5'-triphosphate (dCTP) as well as a 5-methyl, 5-ethyl, 5-bromo or 5-iodo substitution for the 5-hydrogen of cytosine. The two diastereomers of each new dCTP derivative were separated by reverse phase HPLC. The first eluted diastereomer (putatively Rp) of each dCTP analog was a substrate for T7 DNA polymerase (Sequenase) and had an incorporation efficiency similar to normal dCTP and dCTPalphaB, with the 5-iodo-dCTPalphaB analog being the least efficient. Substitution at the C-5 position of cytosine by alkyl groups (ethyl and methyl) markedly enhanced the dCTPalphaB resistance towards exonuclease III (5-Et-dCTPalphaB >5-Me-dCTPalphaB >dCTPalphaB approximately 5-Br-dCTPalphaB >5-I-dCTPalphaB), thereby generating DNA sequences that better define the deoxycytosine positions. The introduction of modified dCTPalphaB should increase the utility of direct DNA sequencing with boronated nucleoside 5'-triphosphates.