TY - JOUR

T1 - A new antisense phosphoryl guanidine oligo-2′-O-methylribonucleotide penetrates into intracellular mycobacteria and suppresses target gene expression

AU - Skvortsova, Yulia V.

AU - Salina, Elena G.

AU - Burakova, Ekaterina A.

AU - Bychenko, Oksana S.

AU - Stetsenko, Dmitry A.

AU - Azhikina, Tatyana L.

N1 - Copyright © 2019 Skvortsova, Salina, Burakova, Bychenko, Stetsenko and Azhikina.

PY - 2019/9/19

Y1 - 2019/9/19

N2 - The worldwide spread of multidrug-resistant Mycobacterium tuberculosis strains prompted the development of new strategies to combat tuberculosis, one of which is antisense therapy based on targeting bacterial mRNA by oligonucleotide derivatives. However, the main limitation of antisense antibacterials is poor cellular uptake because of electrostatic charge. Phosphoryl guanidine oligo-2′-O-methylribonucleotides (2′-OMe PGOs) are a novel type of uncharged RNA analogues with high RNA affinity, which penetrate through the bacterial cell wall more efficiently. In this study, we investigated the uptake and biological effects of 2′-OMe PGO in mycobacteria. The results indicated that 2′-OMe PGO specific for the alanine dehydrogenase-encoding ald gene inhibited the growth of Mycobacterium smegmatis and downregulated ald expression at both the transcriptional and translational levels through an RNase H-independent mechanism, showing higher biological activity than its phosphorothioate oligonucleotide counterpart. Confocal microscopy revealed that the anti-ald 2′-OMe PGO was taken up by intracellular mycobacteria residing in RAW 264.7 macrophages without exerting toxic effects on eukaryotic cells, indicating that 2′-OMe PGO was able to efficiently cross two cellular membranes. In addition, 2′-OMe PGO inhibited the transcription of the target ald gene in M. smegmatis-infected macrophages. Thus, we demonstrated, for the first time, a possibility of targeting gene expression and inhibiting growth of intracellular mycobacteria by antisense oligonucleotide derivatives. Strong antisense activity and efficient uptake of the new RNA analogue, 2′-OMe PGO, by intracellular microorganisms revealed here may promote the development of novel therapeutic strategies to treat TB and prevent the emergence of drug-resistant mycobacterial strains.

AB - The worldwide spread of multidrug-resistant Mycobacterium tuberculosis strains prompted the development of new strategies to combat tuberculosis, one of which is antisense therapy based on targeting bacterial mRNA by oligonucleotide derivatives. However, the main limitation of antisense antibacterials is poor cellular uptake because of electrostatic charge. Phosphoryl guanidine oligo-2′-O-methylribonucleotides (2′-OMe PGOs) are a novel type of uncharged RNA analogues with high RNA affinity, which penetrate through the bacterial cell wall more efficiently. In this study, we investigated the uptake and biological effects of 2′-OMe PGO in mycobacteria. The results indicated that 2′-OMe PGO specific for the alanine dehydrogenase-encoding ald gene inhibited the growth of Mycobacterium smegmatis and downregulated ald expression at both the transcriptional and translational levels through an RNase H-independent mechanism, showing higher biological activity than its phosphorothioate oligonucleotide counterpart. Confocal microscopy revealed that the anti-ald 2′-OMe PGO was taken up by intracellular mycobacteria residing in RAW 264.7 macrophages without exerting toxic effects on eukaryotic cells, indicating that 2′-OMe PGO was able to efficiently cross two cellular membranes. In addition, 2′-OMe PGO inhibited the transcription of the target ald gene in M. smegmatis-infected macrophages. Thus, we demonstrated, for the first time, a possibility of targeting gene expression and inhibiting growth of intracellular mycobacteria by antisense oligonucleotide derivatives. Strong antisense activity and efficient uptake of the new RNA analogue, 2′-OMe PGO, by intracellular microorganisms revealed here may promote the development of novel therapeutic strategies to treat TB and prevent the emergence of drug-resistant mycobacterial strains.

KW - Antibacterial agents

KW - Antisense oligonucleotides

KW - Cellular uptake

KW - Macrophages

KW - Multidrug resistance

KW - RNase H

KW - Tuberculosis

KW - multidrug resistance

KW - TUBERCULOSIS

KW - tuberculosis

KW - ALANINE DEHYDROGENASE

KW - cellular uptake

KW - OLIGONUCLEOTIDES

KW - DELIVERY

KW - macrophages

KW - SMEGMATIS

KW - INHIBITION

KW - antibacterial agents

KW - antisense oligonucleotides

KW - GROWTH

KW - DRUGS

UR - http://www.scopus.com/inward/record.url?scp=85073008679&partnerID=8YFLogxK

U2 - 10.3389/fphar.2019.01049

DO - 10.3389/fphar.2019.01049

M3 - Article

C2 - 31632266

AN - SCOPUS:85073008679

VL - 10

JO - Frontiers in Pharmacology

JF - Frontiers in Pharmacology

SN - 1663-9812

IS - SEP

M1 - 1049

ER -