Phosphoryl Guanidine Modified Synthetic Oligonucleotides

Phosphoryl guanidine oligonucleotides (PGOs) are synthetic nucleic acid analogs designed to overcome common limitations of natural DNA and RNA in medical and research applications. PGOs are structurally similar to natural DNA or RNA but contain a phosphorus-nitrogen (PN) bond, also known as a phosphoramidate, which is less toxic than a phosphorothioate.

Oligonucleotides containing one or more tetramethyl phosphoryl guanidine (TMG) groups can bind their complementary DNA and RNA sequences with an affinity that is only slightly different from that of natural oligodeoxyribonucleotides, despite the steric bulk of the TMG group. Stereopure phosphoryl guanidine-backbone linkages are also often reported as PN linkages.

DNA duplex with a phosphoryl guanidine moiety, Rp-diastereomer [7B72]

In natural DNA or RNA, the backbone consists of phosphate groups that carry a negative charge. In PGOs, a phosphoryl guanidine (PN) group replaces one oxygen atom in the phosphate group. The PN group is often a 1,3-dimethylimidazol-2-imine or a tetramethylphosphorylguanidine moiety. This substitution creates an electrically neutral backbone with a chiral center at the phosphorus atom, resulting in a mixture of diastereomers during synthesis.

This chemical alteration confers several advantages for PGOs over natural oligonucleotides with phosphodiester linkages and other modifications, such as phosphorothioates. PGOs are highly resistant to degradation by nucleases, making them more stable in biological environments. However, despite the bulky modification, they retain the ability to hybridize specifically to complementary DNA or RNA targets.

Because PGOs lack the negative charge of natural DNA, which repels against the negatively charged cell membrane, PGOs can often penetrate cells more easily. Unlike phosphorothioate modifications, PGOs appear to have a lower non-specific binding to proteins, potentially reducing toxic side effects.

PGOs are also useful as primers in PCR and RT-PCR. Their neutral charge allows them to bind to targets with high specificity, often reducing "noise" or non-specific amplification by-products compared to standard DNA primers. PGOs are particularly useful in detecting highly structured RNA molecules where standard primers might fail.

Kuryushkin et al. (2014) introduced a new nucleic acid analog called “phosphoryl guanidine.” The researchers showed that oxidation of 3’,5’-dithymidine-β-cyanoethyl phosphite by iodine in pyridine in the presence of 1,1,3,3-tetramethyl guanidine (TMG) yields a dinucleotide with an internucleotide tetramethyl phosphoryl guanidine group (Tmg) as the main product, useful for the design of new biologically active modified oligonucleotides. This analog contains an internucleoside tetramethyl phosphoryl guanidine (Tmg) group. Oligonucleotides with a Tmg group are stable under conditions of solid-phase DNA synthesis and subsequent cleavage and deprotection with ammonia. Oligonucleotides with one or more Tmg groups bind their complementary DNA or RNA with affinities like those of natural oligodeoxyribonucleotides.

Fokine et al. (2018) showed that charge-neutral phosphoryl guanidine oligonucleotides (PGOs) can be analyzed using vertical slab denaturing electrophoresis at pH 11, or in the presence of SDS as a micelle-forming agent. The PGOs containing the band can be visualized by UV shadowing or by staining with Coomassie Brilliant Blue.  

Skvortsova et al. (2019) reported that antisense phosphoryl guanidine oligo-2′-O-methylribonucleotide penetrated intracellular mycobacteria and suppressed targeted gene expression. The study showed that 2′-OMe PGOs reduced mycobacterial growth in culture and inhibited translation of the target mRNA more effectively than their PS counterparts.

Golyshev et al. (2021) performed structural and hybridization analyses of octa-, deca-, and dodecamers with all phosphate residues modified by 1,3-dimethylimidazolidine-2-imine moieties. The study detected a decrease in the proportion of C2′-endo and an increased proportion of C1′-exo sugar conformations of the modified chain In PGO duplexes. However, in contrast to DNA, the PGO duplex formation resulted in a release of several cations with the water shell significantly more disordered near PGOs and their complexes.

Kanarskaya et al. (2021) investigated the structure and hybridization properties of PGOs in the presence of various cosolvents. The study found that cosolvents did not perturb the secondary structure of PGO/DNA duplexes and that several water molecules released upon the formation of DNA or PGO duplexes are positioned very close to the duplex. However, crowding conditions affected DNA/DNA and DNA/PGO thermodynamics similarly.

Kuryushkin et al. (2021) designed several gapmers with alternating linkages, either on a deoxyribose or a 2′-O-methylribose backbone. The addition of PG modifications increased nuclease resistance in serum-containing medium for more than 21 days. The utilized gapmers efficiently silenced MDR1 mRNA and restored tumor cell sensitivity to chemotherapeutics, thereby successfully mediating multidrug resistance. 

Dyudeeva et al. (2022) studied the use of uncharged phosphoryl guanidine oligodeoxyribonucleotides (PGOs) as primers for mouse leukemia virus reverse transcriptase (MMLV H-) to investigate features of the elongation of wholly and partially uncharged PGO primers useful for the analysis of highly structured RNA. The study showed that PGOs can be efficiently elongated in the presence of a fragment of human ribosomal RNA with a complex spatial structure, allowing characterization of the elongation of partially and completely uncharged PGOs in the reverse-transcription reaction carried out by MMLV H-. 

Kandasamy et al. (2022) reported the synthesis and impact of stereopure phosphoryl-guanidine backbone linkages on oligonucleotides acting via a gapmer-based RNase H-mediated mechanism, using Malat1 and C9orf72 as benchmarks.

The study found that incorporating various types of PN linkages into a stereopure oligonucleotide backbone can increase silencing potency in cultured neurons under free-uptake conditions by 10-fold compared to similarly modified stereopure phosphorothioate (PS) and phosphodiester (PO) molecules. PN linkages positioned in the wings of the gapmer increased the potency of MALAT1 oligonucleotides in neurons.

Chubarov et al. (2023) tested primers modified with phosphoryl guanidines to detect E542K and E545K mutations in the PIK3CA gene. This study revealed that it is possible to detect ~50 copies of mutant DNA at a proportion as low as 0.5% of the total DNA, with reasonable specificity, in the plasmid model system. The PG modification placed at the third internucleotide phosphate mimics an additional mismatch, which may be sufficient to increase primer specificity. PG modification may allow the design of a universal PCR primer to increase primer specificity. 

Prokhorova et al. (2024) demonstrate that crRNAs containing a combination of deoxyribonucleosides and single or multiple phosphoryl guanidine groups facilitate the modulation of CRISPR-Cas9 system activity while improving its specificity in vitro.

Reference

Chubarov A.S., Oscorbin I.P., Novikova L.M., Filipenko M.L., Lomzov A.A., Pyshnyi D.V. Allele-Specific PCR for PIK3CA Mutation Detection Using Phosphoryl Guanidine Modified Primers. Diagnostics. 2023;13:250. doi: 10.3390/diagnostics13020250.  [PMC] [PubMed]

Dyudeeva ES, Pyshnaya IA. Phosphoryl guanidine oligonucleotides as primers for RNA-dependent DNA synthesis using murine leukemia virus reverse transcriptase. Vavilovskii Zhurnal Genet Selektsii. 2022 Feb;26(1):5-13.  [PMC]

Golyshev VM, Pyshnyi DV, Lomzov AA. Effects of Phosphoryl Guanidine Modification of Phosphate Residues on the Structure and Hybridization of Oligodeoxyribonucleotides. J Phys Chem B. 2021 Mar 25;125(11):2841-2855.  [PubMed]

Fokina A, Wang M, Ilyina A, Klabenkova K, Burakova E, Chelobanov B, Stetsenko D. Analysis of new charge-neutral DNA/RNA analogues phosphoryl guanidine oligonucleotides (PGO) by gel electrophoresis. Anal Biochem. 2018 Aug 15;555:9-11. [PubMed]

Kanarskaya MA, Golyshev VM, Pyshnyi DV, Lomzov AA. Structure and hybridization properties of phosphoryl guanidine oligonucleotides under crowding conditions. Biochem Biophys Res Commun. 2021 Nov 5;577:110-115. [PubMed]

Kupryushkin MS, Pyshnyi DV, Stetsenko DA. Phosphoryl guanidines: a new type of nucleic Acid analogues. Acta Naturae. 2014 Oct;6(4):116-8. PMID: 25558402; PMCID: PMC4273099. [PMC]

Kandasamy P, Liu Y, Aduda V, Akare S, Alam R, Andreucci A, Boulay D, Bowman K, Byrne M, Cannon M, Chivatakarn O, Shelke JD, Iwamoto N, Kawamoto T, Kumarasamy J, Lamore S, Lemaitre M, Lin X, Longo K, Looby R, Marappan S, Metterville J, Mohapatra S, Newman B, Paik IH, Patil S, Purcell-Estabrook E, Shimizu M, Shum P, Standley S, Taborn K, Tripathi S, Yang H, Yin Y, Zhao X, Dale E, Vargeese C. Impact of guanidine-containing backbone linkages on stereopure antisense oligonucleotides in the CNS. Nucleic Acids Res. 2022 Jun 10;50(10):5401-5423. [PMC]

Kupryushkin MS, Filatov AV, Mironova NL, Patutina OA, Chernikov IV, Chernolovskaya EL, Zenkova MA, Pyshnyi DV, Stetsenko DA, Altman S, Vlassov VV. Antisense oligonucleotide gapmers containing phosphoryl guanidine groups reverse MDR1-mediated multiple drug resistance of tumor cells. Mol Ther Nucleic Acids. 2021 Nov 29;27:211-226. [PMC]

Prokhorova DV, Kupryushkin MS, Zhukov SA, Zharkov TD, Dovydenko IS, Yakovleva KI, Pereverzev IM, Matveeva AM, Pyshnyi DV, Stepanov GA. Effect of the Phosphoryl Guanidine Modification in Chimeric DNA-RNA crRNAs on the Activity of the CRISPR-Cas9 System In Vitro. ACS Chem Biol. 2024 Jun 21;19(6):1311-1319. [Lifescience]

Skvortsova YV, Salina EG, Burakova EA, Bychenko OS, Stetsenko DA, Azhikina TL. A New Antisense Phosphoryl Guanidine Oligo-2'-O-Methylribonucleotide Penetrates Into Intracellular Mycobacteria and Suppresses Target Gene Expression. Front Pharmacol. 2019 Sep 19;10:1049. [ PMC]

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