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Can Vitamin B12 Assist the Internalization of Antisense LNA Oligonucleotides into Bacteria?

ABSTRACT: The emergence of bacterial resistance to traditional small-molecule antibiotics is fueling the search for innovative strategies to treat infections. Inhibiting the expression of essential bacterial genes using antisense oligonucleotides (ASOs), particularly composed of nucleic acid mimics (NAMs), has emerged as a promising strategy. However, their efficiency depends on their association with vectors that can translocate the bacterial envelope. Vitamin B₁₂ is among the largest molecules known to be taken up by bacteria and has very recently started to gain interest as a trojan-horse vector. Gapmers and steric blockers were evaluated as ASOs against Escherichia coli (E. coli). Both ASOs were successfully conjugated to B₁₂ by copper-free azide-alkyne click-chemistry. The biological effect of the two conjugates was evaluated together with their intracellular localization in E. coli. Although not only B₁₂ but also both B₁₂-ASO conjugates interacted strongly with E. coli, they were mostly colocalized with the outer membrane. Only 6-9% were detected in the cytosol, which showed to be insufficient for bacterial growth inhibition. These results suggest that the internalization of B₁₂-ASO conjugates is strongly affected by the low uptake rate of the B₁₂ in E. coli and that further studies are needed before considering this strategy against biofilms in vivo.

SUBMITTER: Pereira S

PROVIDER: S-EPMC8065541 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Highly Potent GalNAc-Conjugated Tiny LNA Anti-miRNA-122 Antisense Oligonucleotides.

Project description:The development of clinically relevant anti-microRNA antisense oligonucleotides (anti-miRNA ASOs) remains a major challenge. One promising configuration of anti-miRNA ASOs called "tiny LNA (tiny Locked Nucleic Acid)" is an unusually small (~8-mer), highly chemically modified anti-miRNA ASO with high activity and specificity. Within this platform, we achieved a great enhancement of the in vivo activity of miRNA-122-targeting tiny LNA by developing a series of N-acetylgalactosamine (GalNAc)-conjugated tiny LNAs. Specifically, the median effective dose (ED50) of the most potent construct, tL-5G3, was estimated to be ~12 nmol/kg, which is ~300-500 times more potent than the original unconjugated tiny LNA. Through in vivo/ex vivo imaging studies, we have confirmed that the major advantage of GalNAc over tiny LNAs can be ascribed to the improvement of their originally poor pharmacokinetics. We also showed that the GalNAc ligand should be introduced into its 5' terminus rather than its 3' end via a biolabile phosphodiester bond. This result suggests that tiny LNA can unexpectedly be recognized by endogenous nucleases and is required to be digested to liberate the parent tiny LNA at an appropriate time in the body. We believe that our strategy will pave the way for the clinical application of miRNA-targeting small ASO therapy.

| S-EPMC8228246 | biostudies-literature

Knockdown of circular RNAs using LNA-modified antisense oligonucleotides

Project description:Circular RNAs (circRNAs) constitute an abundant class of covalently closed non-coding RNA molecules that are formed by backsplicing from eukaryotic protein-coding genes. Recent studies have shown that circRNAs can act as microRNA or protein decoys as well as transcriptional regulators. However, the functions of most circRNAs are still poorly understood. Because circRNA sequences overlap with their linear parent transcripts, depleting specific circRNAs without affecting host gene expression remains a challenge. Here, we assessed the utility of LNA-modified antisense oligonucleotides (ASOs) to knock down circRNAs for loss-of-function studies. We identified 5807 circRNAs in total RNA sequencing data from 4 liver cancer cell lines and used the back splice junction (BSJ) sequences of 7 validated circRNAs as target sites for designing different LNA-modified ASOs for circRNA knockdown. We found that while most RNase H-dependent gapmer ASOs mediate effective knockdown of their target circRNAs, some gapmers reduce the levels of the linear parent transcript and may also cause degradation of unintended off-targets. The circRNA targeting specificity can be enhanced using design-optimized gapmer ASOs or LNA/DNA mixmer ASOs, which display potent and specific circRNA knockdown with a minimal effect on the host genes or predicted off-targets. In summary, our results demonstrate that LNA-modified ASOs complementary to BSJ sequences mediate robust knockdown of circRNAs in vitro and, thus, represent a useful tool to explore the biological roles of circRNAs in loss-of-function studies in cultured cells and animal models.

2022-11-01 | GSE145592 | GEO

PCSK9 LNA antisense oligonucleotides induce sustained reduction of LDL cholesterol in nonhuman primates.

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Project description:Usually, small interfering RNAs and most antisense molecules need mechanical or chemical delivery methods to down-modulate the targeted mRNA. However, these delivery approaches complicate the interpretations of biological consequences. We show that locked nucleic acid (LNA)-based antisense oligonucleotides (LNA-ONs) readily down-modulate genes of interest in multiple cell lines without any delivery means. The down-modulation of genes was quick, robust, long-lasting and specific followed by potent down-modulation of protein. The efficiency of the effect varied among the 30 tumor cell lines investigated. The most robust effects were found in those cells where nuclear localization of the LNA-ON was clearly observed. Importantly, without using any delivery agent, we demonstrated that HER3 mRNA and protein could be efficiently down-modulated in cells and a tumor xenograft model. These data provide a simple and efficient approach to identify potential drug targets and animal models. Further elucidation of the mechanism of cellular uptake and trafficking of LNA-ONs may enhance not only the therapeutic values of this platform but also antisense molecules in general.

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Project description:Antisense oligonucleotides (ASOs) are becoming important drugs for hard to treat diseases. Modifications to their DNA backbones are essential to inhibit degradation in vivo, but they can reduce binding affinity to RNA targets. To address this problem we have combined the enzymatic resistance of carbamate (CBM) DNA backbone analogues with the thermodynamic stability conferred by locked nucleic acid sugars (LNA). Using a dinucleotide phosphoramidite strategy and automated solid phase synthesis, we have synthesised a set of oligonucleotides modified with multiple LNA-CBM units. The LNA sugars restore binding affinity to RNA targets, and in this respect LNA position with respect to the CBM linkage is important. Oligonucleotides containing carbamate flanked on its 5'and 3'-sides by LNA form stable duplexes with RNA and unstable duplexes with DNA, which is desirable for antisense applications. Carbamate-LNA modified oligonucleotides also show increased stability in the presence of snake venom and foetal bovine serum compared to LNA or CBM backbones alone.

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Project description:We report the structure activity relationships of short 14-mer phosphorothioate gapmer antisense oligonucleotides (ASOs) modified with α-L-locked nucleic acid (LNA) and related modifications targeting phosphatase and tensin homologue (PTEN) messenger RNA in mice. α-L-LNA represents the α-anomer of enantio-LNA and modified oligonucleotides show LNA like binding affinity for complementary RNA. In contrast to sequence matched LNA gapmer ASOs which showed elevations in plasma alanine aminotransferase (ALT) levels indicative of hepatotoxicity, gapmer ASOs modified with α-L-LNA and related analogs in the flanks showed potent downregulation of PTEN messenger RNA in liver tissue without producing elevations in plasma ALT levels. However, the α-L-LNA ASO showed a moderate dose-dependent increase in liver and spleen weights suggesting a higher propensity for immune stimulation. Interestingly, replacing α-L-LNA nucleotides in the 3'- and 5'-flanks with R-5'-Me-α-L-LNA but not R-6'-Me- or 3'-Me-α-L-LNA nucleotides, reversed the drug induced increase in organ weights. Examination of structural models of dinucleotide units suggested that the 5'-Me group increases steric bulk in close proximity to the phosphorothioate backbone or produces subtle changes in the backbone conformation which could interfere with recognition of the ASO by putative immune receptors. Our data suggests that introducing steric bulk at the 5'-position of the sugar-phosphate backbone could be a general strategy to mitigate the immunostimulatory profile of oligonucleotide drugs. In a clinical setting, proinflammatory effects manifest themselves as injection site reactions and flu-like symptoms. Thus, a mitigation of these effects could increase patient comfort and compliance when treated with ASOs.Molecular Therapy - Nucleic Acids (2012) 1, e47; doi:10.1038/mtna.2012.34; published online 18 September 2012.

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Project description:The duplex stability with target mRNA and the gene silencing potential of a novel bridged nucleic acid analogue are described. The analogue, 2',4'-BNA(NC) antisense oligonucleotides (AONs) ranging from 10- to 20-nt-long, targeted apolipoprotein B. 2',4'-BNA(NC) was directly compared to its conventional bridged (or locked) nucleic acid (2',4'-BNA/LNA)-based counterparts. Melting temperatures of duplexes formed between 2',4'-BNA(NC)-based antisense oligonucleotides and the target mRNA surpassed those of 2',4'-BNA/LNA-based counterparts at all lengths. An in vitro transfection study revealed that when compared to the identical length 2',4'-BNA/LNA-based counterpart, the corresponding 2',4'-BNA(NC)-based antisense oligonucleotide showed significantly stronger inhibitory activity. This inhibitory activity was more pronounced in shorter (13-, 14-, and 16-mer) oligonucleotides. On the other hand, the 2',4'-BNA(NC)-based 20-mer AON exhibited the highest affinity but the worst IC(50) value, indicating that very high affinity may undermine antisense potency. These results suggest that the potency of AONs requires a balance between reward term and penalty term. Balance of these two parameters would depend on affinity, length, and the specific chemistry of the AON, and fine-tuning of this balance could lead to improved potency. We demonstrate that 2',4'-BNA(NC) may be a better alternative to conventional 2',4'-BNA/LNA, even for "short" antisense oligonucleotides, which are attractive in terms of drug-likeness and cost-effective bulk production.

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Project description:There is mounting interest in developing antisense and siRNA oligonucleotides into therapeutic entities; however, this potential has been limited by poor access of oligonucleotides to their pharmacological targets within cells. Transfection reagents, such as cationic lipids and polymers, are commonly utilized to improve functional delivery of nucleic acids including oligonucleotides. Cellular entry of large plasmid DNA molecules with the assistance of these polycationic carriers is mediated by some form of endocytosis; however, the mechanism for delivery of small oligonucleotide molecules has not been well established. In this study, splice-shifting oligonucleotides have been formulated into cationic lipoplexes and polyplexes, and their internalization mechanisms have been examined by using pharmacological and genetic inhibitors of endocytosis. The results showed that intercellular distribution of the oligonucleotides to the nucleus governs their pharmacological response. A mechanistic study revealed that oligonucleotides delivered by lipoplexes enter the cells partially by membrane fusion and this mechanism accounts for the functional induction of the target gene. In contrast, polyplexes are internalized by unconventional endocytosis pathways that do not require dynamin or caveolin. These studies may help rationally design novel delivery systems with superior transfection efficiency but lower toxicity.

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Electronic Structures of LNA Phosphorothioate Oligonucleotides.

Project description:Important oligonucleotides in anti-sense research have been investigated in silico and experimentally. This involves quantum mechanical (QM) calculations and chromatography experiments on locked nucleic acid (LNA) phosphorothioate (PS) oligonucleotides. iso-potential electrostatic surfaces are essential in this study and have been calculated from the wave functions derived from the QM calculations that provide binding information and other properties of these molecules. The QM calculations give details of the electronic structures in terms of e.g., energy and bonding, which make them distinguish or differentiate between the individual PS diastereoisomers determined by the position of sulfur atoms. Rules are derived from the electronic calculations of these molecules and include the effects of the phosphorothioate chirality and formation of electrostatic potential surfaces. Physical and electrochemical descriptors of the PS oligonucleotides are compared to the experiments in which chiral states on these molecules can be distinguished. The calculations demonstrate that electronic structure, electrostatic potential, and topology are highly sensitive to single PS configuration changes and can give a lead to understanding the activity of the molecules.

| S-EPMC5537454 | biostudies-literature

Specific RNP capture with antisense LNA/DNA mixmers.

Project description:RNA-binding proteins (RBPs) play essential roles in RNA biology, responding to cellular and environmental stimuli to regulate gene expression. Important advances have helped to determine the (near) complete repertoires of cellular RBPs. However, identification of RBPs associated with specific transcripts remains a challenge. Here, we describe "specific ribonucleoprotein (RNP) capture," a versatile method for the determination of the proteins bound to specific transcripts in vitro and in cellular systems. Specific RNP capture uses UV irradiation to covalently stabilize protein-RNA interactions taking place at "zero distance." Proteins bound to the target RNA are captured by hybridization with antisense locked nucleic acid (LNA)/DNA oligonucleotides covalently coupled to a magnetic resin. After stringent washing, interacting proteins are identified by quantitative mass spectrometry. Applied to in vitro extracts, specific RNP capture identifies the RBPs bound to a reporter mRNA containing the Sex-lethal (Sxl) binding motifs, revealing that the Sxl homolog sister of Sex lethal (Ssx) displays similar binding preferences. This method also revealed the repertoire of RBPs binding to 18S or 28S rRNAs in HeLa cells, including previously unknown rRNA-binding proteins.

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