Phosphoramidate Azole Oligonucleotides for Single Nucleotide Polymorphism Detection by PCR.

Detection of the Kirsten rat sarcoma gene (KRAS) mutational status is an important factor for the treatment of various malignancies. The most common KRAS-activating mutations are caused by single-nucleotide mutations, which are usually determined by using PCR, using allele-specific DNA primers. Oligonucleotide primers with uncharged or partially charged internucleotide phosphate modification have proved their ability to increase the sensitivity and specificity of various single nucleotide mutation detection. To enhance the specificity of single nucleotide mutation detection, the novel oligonucleotides with four types of uncharged and partially charged internucleotide phosphates modification, phosphoramide benzoazole (PABA) oligonucleotides (PABAO), was used to prove the concept on the KRAS mutation model. The molecular effects of different types of site-specific PABA modification in a primer or a template on a synthesis of full-length elongation product and PCR efficiency were evaluated. The allele-specific PCR (AS-PCR) on plasmid templates showed a significant increase in analysis specificity without changes in Cq values compared with unmodified primer. PABA modification is a universal mismatch-like disturbance, which can be used for single nucleotide polymorphism discrimination for various applications. The molecular insights of the PABA site-specific modification in a primer and a template affect PCR, structural features of four types of PABAO in connection with AS-PCR results, and improvements of AS-PCR specificity support the further design of novel PCR platforms for various biological targets testing.

Properties of phosphoramide benzoazole oligonucleotides (PABAOs). I. Structure and hybridization efficiency of N-benzimidazole derivatives.

In this work, we for the first time conducted a detailed study on the structure, dynamics, and hybridization properties of N-benzimidazole group-bearing phosphoramide benzoazole oligonucleotides (PABAOs) that we developed recently. By circular dichroism we established that the introduction of the modifications does not disrupt the B conformation of the DNA double helix. The formation of complexes is approximated by a two-state model. Complexes of PABAOs with native oligodeoxriboynucleotides form efficiently, and the introduction of such modifications reduces thermal stability of short duplexes (8-10 bp) by ∼5°Ð¡ per modification. Using UV-spectroscopy analysis, a neutral charge of the phosphate residue modified by the N-benzimidazole moiety in the pH range of 3-9.5 was found. The results confirm possible usefulness of PABAOs for both basic research and biomedical applications.

Synthesis of Oligonucleotides Carrying Inter-nucleotide N-(Benzoazole)-phosphoramide Moieties.

In this work, we present new oligonucleotide derivatives containing inter-nucleotide N-benzimidazole, N-benzoxazole, N-benzothiazole, and 1,3-dimethyl-N-benzimidazole (benzoazoles) phosphoramide groups. These modifications were introduced via the Staudinger reaction with appropriate azides during standard automated solid-phase oligonucleotide synthesis. The principal structural difference between the new azido modifiers and those already known is that they are bulk heterocyclic structures, similar to purine nucleoside bases. Modified oligonucleotides with one and two modifications at different positions and multiple modified heteronucleotide sequences were obtained with high yields. The possibility of multiple modifications in the process of automatic DNA synthesis is fundamental and critical for further application of our oligonucleotide derivatives. Initial studies on the properties of new oligonucleotides were carried out. The stability of the oligodeoxyribonucleotide duplex containing phosphoramide groups of N-benzoazoles with complementary DNA or RNA is slightly lower than that of native complexes.

Synthesis of the new nucleoside 5'-alpha-iminophosphates using Staudinger reaction.

Efficient protocols were developed for the synthesis of a new compounds - nucleoside 5'-α-iminophosphates using the Staudinger reaction. These substances are alpha-phosphate mimetic nucleotide in which an oxygen atom is replaced by a corresponding imino (=N-R)-group. Various 5'-iminomonophosphates of nucleosides were obtained. A chemical method for the synthesis of triphosphate derivatives based on the iminomonophosphates has been designed. Thymidine 5'-(1,3-dimethylimidazolidin-2-ylidene)-triphosphate (ppp(DMI)T) was synthesized, its hydrolytic stability and substrate properties in relation to some DNA polymerases was firstly studied. It was shown that ppp(DMI)T can serve as substrate for enzyme catalyzed template-independent DNA synthesis by human terminal deoxynucleotidyltransferase TdT.