How RNase HI (Escherichia coli) promoted site-selective hydrolysis works on RNA in duplex with carba-LNA and LNA substituted antisense strands in an antisense strategy context?

2017 ◽  
Vol 13 (5) ◽  
pp. 921-938
Author(s):  
Oleksandr Plashkevych ◽  
Qing Li ◽  
Jyoti Chattopadhyaya
Keyword(s):  
Escherichia Coli ◽  
Nucleic Acid ◽  
Kinetic Study ◽  
Rnase H ◽  
Locked Nucleic Acid ◽  
Cleavage Sites ◽  
Selective Hydrolysis ◽  
Rnase Hi ◽  
Site Selective

Kinetic study of 36 AON–RNA heteroduplexes single modified by locked nucleic acid (LNA) or by carba-LNA show site-dependent modulation of RNase H promoted cleavage of RNA strand by 2 to 5 fold with preferential 5′-GpN-3′ cleavage sites, giving up to 70% of the products.

scholarly journals An Investigation into the Potential of Targeting Escherichia coli rne mRNA with Locked Nucleic Acid (LNA) Gapmers as an Antibacterial Strategy

Molecules ◽  
2021 ◽  
Vol 26 (11) ◽  
pp. 3414
Author(s):  
Layla R. Goddard ◽  
Charlotte E. Mardle ◽  
Hassan Gneid ◽  
Ciara G. Ball ◽  
Darren M. Gowers ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Nucleic Acid ◽  
Translation Initiation ◽  
Antisense Oligonucleotide ◽  
Antisense Oligonucleotides ◽  
Mrna Translation ◽  
Rna Degradation ◽  
Rnase H ◽  
Locked Nucleic Acid ◽  
Translation Initiation Region

The increase in antibacterial resistance is a serious challenge for both the health and defence sectors and there is a need for both novel antibacterial targets and antibacterial strategies. RNA degradation and ribonucleases, such as the essential endoribonuclease RNase E, encoded by the rne gene, are emerging as potential antibacterial targets while antisense oligonucleotides may provide alternative antibacterial strategies. As rne mRNA has not been previously targeted using an antisense approach, we decided to explore using antisense oligonucleotides to target the translation initiation region of the Escherichia coli rne mRNA. Antisense oligonucleotides were rationally designed and were synthesised as locked nucleic acid (LNA) gapmers to enable inhibition of rne mRNA translation through two mechanisms. Either LNA gapmer binding could sterically block translation and/or LNA gapmer binding could facilitate RNase H-mediated cleavage of the rne mRNA. This may prove to be an advantage over the majority of previous antibacterial antisense oligonucleotide approaches which used oligonucleotide chemistries that restrict the mode-of-action of the antisense oligonucleotide to steric blocking of translation. Using an electrophoretic mobility shift assay, we demonstrate that the LNA gapmers bind to the translation initiation region of E. coli rne mRNA. We then use a cell-free transcription translation reporter assay to show that this binding is capable of inhibiting translation. Finally, in an in vitro RNase H cleavage assay, the LNA gapmers facilitate RNase H-mediated mRNA cleavage. Although the challenges of antisense oligonucleotide delivery remain to be addressed, overall, this work lays the foundations for the development of a novel antibacterial strategy targeting rne mRNA with antisense oligonucleotides.

scholarly journals Evaluation of Multiple-Turnover Capability of Locked Nucleic Acid Antisense Oligonucleotides in Cell-Free RNase H-Mediated Antisense Reaction and in Mice

2014 ◽  
Vol 24 (4) ◽  
pp. 283-290 ◽  
Author(s):  
Tsuyoshi Yamamoto ◽  
Naoko Fujii ◽  
Hidenori Yasuhara ◽  
Shunsuke Wada ◽  
Fumito Wada ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Antisense Oligonucleotides ◽  
Rnase H ◽  
Locked Nucleic Acid

scholarly journals Selection, Optimization, and Pharmacokinetic Properties of a Novel, Potent Antiviral Locked Nucleic Acid-Based Antisense Oligomer Targeting Hepatitis C Virus Internal Ribosome Entry Site

2011 ◽  
Vol 55 (7) ◽  
pp. 3105-3114 ◽  
Author(s):  
Carl Laxton ◽  
Kevin Brady ◽  
Sterghios Moschos ◽  
Paul Turnpenny ◽  
Jaiessh Rawal ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Nucleic Acid ◽  
Antiviral Activity ◽  
Internal Ribosome Entry Site ◽  
Rnase H ◽  
Locked Nucleic Acid ◽  
Response Curves ◽  
Entry Site ◽  
Internal Ribosome Entry

ABSTRACTWe have screened 47 locked nucleic acid (LNA) antisense oligonucleotides (ASOs) targeting conserved (>95% homology) sequences in the hepatitis C virus (HCV) genome using the subgenomic HCV replicon assay and generated both antiviral (50% effective concentration [EC50]) and cytotoxic (50% cytotoxic concentration [CC50]) dose-response curves to allow measurement of the selectivity index (SI). This comprehensive approach has identified an LNA ASO with potent antiviral activity (EC50= 4 nM) and low cytotoxicity (CC50>880 nM) targeting the 25- to 40-nucleotide region (nt) of the HCV internal ribosome entry site (IRES) containing the distal and proximal miR-122 binding sites. LNA ASOs targeting previously known accessible regions of the IRES, namely, loop III and the initiation codon in loop IV, had poor SI values. We optimized the LNA ASO sequence by performing a 1-nucleotide walk through the 25- to 40-nt region and show that the boundaries for antiviral efficacy are extremely precise. Furthermore, we have optimized the format for the LNA ASO using different gapmer and mixomer patterns and show that RNase H is required for antiviral activity. We demonstrate that RNase H-refractory ASOs targeting the 25- to 40-nt region have no antiviral effect, revealing important regulatory features of the 25- to 40-nt region and suggesting that RNase H-refractory LNA ASOs can act as potential surrogates for proviral functions of miR-122. We confirm the antisense mechanism of action using mismatched LNA ASOs. Finally, we have performed pharmacokinetic experiments to demonstrate that the LNA ASOs have a very long half-life (>5 days) and attain hepatic maximum concentrations >100 times the concentration required forin vitroantiviral activity.

Infection of Escherichia coli with bacteriophages

1969 ◽  
Vol 27 ◽  
pp. 370-371
Author(s):  
Manfred E. Bayer
Keyword(s):  
Escherichia Coli ◽  
Nucleic Acid ◽  
Cell Surface ◽  
Virus Type ◽  
Infection Process ◽  
Adsorption Site ◽  
The Other ◽  
Specific Receptors ◽  
Bacterial Receptors

The first step in the infection of a bacterium by a virus consists of a collision between cell and bacteriophage. The presence of virus-specific receptors on the cell surface will trigger a number of events leading eventually to release of the phage nucleic acid. The execution of the various "steps" in the infection process varies from one virus-type to the other, depending on the anatomy of the virus. Small viruses like ØX 174 and MS2 adsorb directly with their capsid to the bacterial receptors, while other phages possess attachment organelles of varying complexity. In bacteriophages T3 (Fig. 1) and T7 the small conical processes of their heads point toward the adsorption site; a welldefined baseplate is attached to the head of P22; heads without baseplates are not infective.

Trinucleotide Cap Analogue Bearing a Locked Nucleic Acid Moiety: Synthesis, mRNA Modification, and Translation for Therapeutic Applications

2021 ◽  
Author(s):  
Annamalai Senthilvelan ◽  
Tyson Vonderfecht ◽  
Muthian Shanmugasundaram ◽  
Indra Pal ◽  
Jason Potter ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Locked Nucleic Acid ◽  
Acid Moiety ◽  
Therapeutic Applications
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