Albany 2019: 20th Conversation - Abstracts

category image Albany 2019
Conversation 20
June 11-15 2019
Adenine Press (2019)

Comparative physico chemical and biological studies of phosphorylguanidine oligonucleotide diasteriomers

Synthetic oligonucleotide analogs are widely used in various fields of molecular biology, nanobiotechnology, and medicine. Recently, we developed a new type of phosphate-modified nucleic acids: phosphorylguanidine (PG) oligonucleotides (PGOs). Guanidine residue can be introduced into desired positions of an oligonucleotide by the standard automatic phosphitamide protocol of DNA synthesis (Stetsenko et al., 2014; Kuprushkin et al., 2014).

In the present work, studied the absolute configuration of phosphate atom in PGO. First, we analyzed monomodified oligomer d(TpCp*A) (* - shows N,N,N’,N’-tetramethylguanidine moiety). Reverse-phase HPLC analysis allowed to isolate two individual diastereomers of monomodified oligomer (fast and slow). Both diastereomers of d(TpCp*A) were not digested with a snake venom phosphodiesterase after 7 days, whereas native analog was fully degraded after 30 min. Ultraviolet and circular dichroism spectroscopy methods, as well as 1D and 2D NMR spectroscopy showed high similarity of the diastereomers. Only detailed computational analysis of restraint penalty energies via restrained molecular dynamics simulations allowed us to conclude that most likely, the fast isomer is the Sp-, and the slow one is the Rp-diastereomer.

To study PGO properties more deeply we designed DNA duplex (d(5′-CAGCGGCG*TG-3′)/ d(CACGCCGCTG)) with single N,N’-dimethyl-N,N’-ethylguanidine modification. Using RP-HPLC we isolated individual diastereomers of PGO. 1D (1H, 32P) and 2D (1H-1H NOESY, 1H-1H COSY, 1H-1H TOCSY, 1H-31P COSY, 1H-13C HSQC, 1H-13C HMBC) NMR spectra were recorded. After assignment of all non-exchangeable protons in NOESY spectra, using the restrained molecular dynamics method, we determined that the fast diastereomer is Rp and the slow- Sp. The structural analysis showed that the introduction of the PG group into the oligomer does not disturb the B-form of the double DNA helix.

Computer analysis of the Taq DNA polymerase complexes with PGO duplexes and experimental 10-mer PGO primer elongation were carried out. The significant difference in the processing of the Rp and Sp isomers was observed. The detailed comparative analysis of the data obtained allowed us to determine the origin of the different processing of the diastereomers by the enzyme.

The data obtained shows that the phosphorylguanidine oligonucleotides are promising for a number of applications such as PCR analysis, biosensors (Dmitrienko et al., 2016) and therapeutics.

This research has been supported by the Russian Science Foundation [grant No. 18-14-00357].


    Stetsenko, D. A.; Kupryushkin, M. S.; Pyshnyi, D. V. International patent application WO2016/028187 A1, August 22, 2014.

    Kupryushkin, M. S.; Pyshnyi, D. V.; Stetsenko, D. A. (2014). Phosphoryl guanidines: a new type of nucleic Acid analogues. Acta Naturae 6 (4), 116-118.

    Dmitrienko, E.; Naumova, O.; Fomin, B.; Kupryushkin, M.; Volkova, A.; Amirkhanov, N.; Semenov, D.; Pyshnaya, I.; Pyshnyi, D. (2016). Surface modification of SOI-FET sensors for label-free and specific detection of short RNA analyte. Nanomedicine, 11(16), 2073-2082.

Alexander A. Lomzov,
Valeria S. Apukhtina,
Andrey V. Shernyukov,
Maxim S. Kupryushkin,
Georgy Yu. Shevelev, and
Dmitrii V. Pyshnyi

Institute of Chemical Biology and Fundamental Medicine SB RAS
Novosibirsk State University
Novosibirsk 630090, Russia.

Ph: +7-383-363-5134
Fx: +7-383-363-5153
Email: lomzov@niboch.nsc.ru