scholarly journals Synthesis and Evaluation of Artificial Nucleic Acid Bearing an Oxanorbornane Scaffold

Molecules ◽  
2020 ◽  
Vol 25 (7) ◽  
pp. 1732
Author(s):  
Hibiki Komine ◽  
Shohei Mori ◽  
Kunihiko Morihiro ◽  
Kenta Ishida ◽  
Takumi Okuda ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Structural Flexibility ◽  
Biophysical Properties ◽  
Conformational Restriction ◽  
Conformationally Restricted ◽  
Stability Enhancement ◽  
Duplex Formation ◽  
The Given ◽  
Modified Oligonucleotide ◽  
Single Bonds

Natural oligonucleotides have many rotatable single bonds, and thus their structures are inherently flexible. Structural flexibility leads to an entropic loss when unwound oligonucleotides form a duplex with single-stranded DNA or RNA. An effective approach to reduce such entropic loss in the duplex-formation is the conformational restriction of the flexible phosphodiester linkage and/or sugar moiety. We here report the synthesis and biophysical properties of a novel artificial nucleic acid bearing an oxanorbornane scaffold (OxNorNA), where the adamant oxanorbornane was expected to rigidify the structures of both the linkage and sugar parts of nucleic acid. OxNorNA phosphoramidite with a uracil (U) nucleobase was successfully synthesized over 15 steps from a known sugar-derived cyclopentene. Thereafter, the given phosphoramidite was incorporated into the designed oligonucleotides. Thermal denaturation experiments revealed that oligonucleotides modified with the conformationally restricted OxNorNA-U properly form a duplex with the complementally DNA or RNA strands, although the Tm values of OxNorNA-U-modified oligonucleotides were lower than those of the corresponding natural oligonucleotides. As we had designed, entropic loss during the duplex-formation was reduced by the OxNorNA modification. Moreover, the OxNorNA-U-modified oligonucleotide was confirmed to have extremely high stability against 3′-exonuclease activity, and its stability was even higher than those of the phosphorothioate-modified counterparts (Sp and Rp). With the overall biophysical properties of OxNorNA-U, we expect that OxNorNA could be used for specialized applications, such as conformational fixation and/or bio-stability enhancement of therapeutic oligonucleotides (e.g., aptamers).

Stopped-flow kinetics of locked nucleic acid (LNA)–oligonucleotide duplex formation: studies of LNA–DNA and DNA–DNA interactions

2001 ◽  
Vol 354 (3) ◽  
pp. 481-484 ◽  
Author(s):  
Ulla CHRISTENSEN ◽  
Nana JACOBSEN ◽  
Vivek K. RAJWANSHI ◽  
Jesper WENGEL ◽  
Troels KOCH
Keyword(s):  
Nucleic Acid ◽  
Dissociation Constants ◽  
Locked Nucleic Acid ◽  
Stopped Flow ◽  
Complementary Dna ◽  
Conformationally Restricted ◽  
Dna And Rna ◽  
Melting Curves ◽  
Oligonucleotide Duplex ◽  
Duplex Formation

The locked nucleic acid (LNA) monomer is a conformationally restricted nucleotide analogue with an extra 2′-O,4′-C-methylene bridge added to the ribose ring. Oligonucleotides that contain LNA monomers have shown greatly enhanced thermal stability when hybridized to complementary DNA and RNA and are considered most promising candidates for efficient recognition of a given mixed sequence in a nucleic acid duplex and as an antisense molecule. Here the kinetics and thermodynamics of a series of oligonucleotide duplex formations of DNA–DNA and DNA–LNA octamers were studied using stopped-flow absorption measurements at 25°C and melting curves. The reactions of the DNA octamer 5′-CAGGAGCA-3′ with its complementary DNA octamer 5′-TGCTCCTG-3′, and with the LNA octamers 5′-TLGCTCCTG-3′ (LNA-1), 5′-TLGCTLCCTG-3′ (LNA-2) and 5′-TLGCTLCCTLG-3′(LNA-3), containing respectively one, two or three thymidine 2′-O,4′-C-methylene-(D-ribofuranosyl) nucleotide monomers, designated TL, were studied. In all cases were seen fast second-order association reactions with kobs = 2×107M-1˙s-1. At 25°C the dissociation constants of the duplexes obtained from melting curves were: DNA–DNA, 10nM; DNA–LNA-1, 20nM; DNA–LNA-2, 2nM; and DNA–LNA-3, 0.3nM; thus the greatly enhanced duplex stability induced by LNA is confirmed. Since the association rates were all equal this increase in stability is due to slower rates of dissociation of the complexes.

scholarly journals Synthesis and properties of oligonucleotides modified with an N-methylguanidine-bridged nucleic acid (GuNA[Me]) bearing adenine, guanine, or 5-methylcytosine nucleobases

2021 ◽  
Vol 17 ◽  
pp. 622-629
Author(s):  
Naohiro Horie ◽  
Takao Yamaguchi ◽  
Shinji Kumagai ◽  
Satoshi Obika
Keyword(s):  
Nucleic Acid ◽  
Binding Affinity ◽  
Antisense Oligonucleotides ◽  
Modified Oligonucleotides ◽  
Biophysical Properties ◽  
Chemical Modifications ◽  
Antisense Technology ◽  
Strong Affinity ◽  
The Stability

Chemical modifications have been extensively used for therapeutic oligonucleotides because they strongly enhance the stability against nucleases, binding affinity to the targets, and efficacy. We previously reported that oligonucleotides modified with an N-methylguanidine-bridged nucleic acid (GuNA[Me]) bearing the thymine (T) nucleobase show excellent biophysical properties for applications in antisense technology. In this paper, we describe the synthesis of GuNA[Me] phosphoramidites bearing other typical nucleobases including adenine (A), guanine (G), and 5-methylcytosine (mC). The phosphoramidites were successfully incorporated into oligonucleotides following the method previously developed for the GuNA[Me]-T-modified oligonucleotides. The binding affinity of the oligonucleotides modified with GuNA[Me]-A, -G, or -mC toward the complementary single-stranded DNAs or RNAs was systematically evaluated. All of the GuNA[Me]-modified oligonucleotides were found to have a strong affinity for RNAs. These data indicate that GuNA[Me] could be a useful modification for therapeutic antisense oligonucleotides.

scholarly journals Demonstration of Hepatitis C Virus RNA withIn SituHybridization Employing a Locked Nucleic Acid Probe in Humanized Liver of Infected Chimeric Mice and in Needle-Biopsied Human Liver

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Kazuya Shiogama ◽  
Ken-ichi Inada ◽  
Michinori Kohara ◽  
Hidemi Teramoto ◽  
Yasuyoshi Mizutani ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Nucleic Acid ◽  
Oligonucleotide Probe ◽  
High Sensitivity ◽  
Locked Nucleic Acid ◽  
Hcv Rna ◽  
Chimeric Mice ◽  
Rt Pcr ◽  
Modified Oligonucleotide

Background.In situhybridization (ISH) with high sensitivity has been requested to demonstrate hepatitis C virus (HCV) RNA in formalin-fixed, paraffin-embedded (FFPE) sections of the liver.Methods. ISH employing a locked-nucleic-acid- (LNA-)modified oligonucleotide probe and biotin-free catalyzed signal amplification system (CSAII) was applied to HCV-RNA detection in the liver tissue. Nested reverse-transcription polymerase chain reaction (RT-PCR) was performed for HCV genotyping using total RNA extracted from FFPE sections. The target tissues included FFPE tissue sections of humanized livers in HCV-infected chimeric mice (HCV genotypes 1a, 1b, and 2a and noninfected) and of needle-biopsied livers from HCV-infected patients.Results. HCV-RNA was demonstrated with the ISH technique in HCV-infected liver tissues from both chimeric mice and 9 (82%) of 11 patients with HCV infection. The HCV signals were sensitive to RNase. Nested RT-PCR confirmed the genotype in 8 (73%) of 11 livers (type 1b: 6 lesions and type 2a: 2 lesions). HCV-RNA was not identified in chronic hepatitis B lesions, fatty liver, autoimmune hepatitis, and hepatocellular carcinoma.Conclusion. ISH using the LNA-modified oligonucleotide probe and CSAII was applicable to detecting HCV-RNA in routinely prepared FFPE liver specimens.

The conformational restriction of synthetic peptides, including a malaria peptide, for use as immunogens

1989 ◽  
Vol 323 (1217) ◽  
pp. 565-572 ◽  
Keyword(s):  
Hydrogen Bond ◽  
Plasmodium Falciparum ◽  
Amino Acid ◽  
Hydrogen Bonds ◽  
Synthetic Peptides ◽  
Synthetic Methods ◽  
Amide Hydrogen ◽  
Conformational Restriction ◽  
Conformationally Restricted ◽  
New Strategy

A new strategy is advanced for the conformational restriction of peptidyl immunogens. Our approach is to replace putative amide-amide hydrogen bonds with covalent hydrogen-bond mimics. Because on average every other amino acid in a protein engages in this bond, the syntheses of diversely shaped peptides can be contemplated. Synthetic methods for introducing a potential hydrogen-bond mimic into a peptide with α-helical potential is reported and the structural consequences are discussed. The replacement of the hydrogen bond with a chemical link will modify as well as shape the peptide. To explore the consequences of these changes, a potential synthetic vaccine for malaria, the repeating tetrapeptide Asn-Pro-Asn-Ala, was conformationally restricted. Antibodies to the shaped malarial peptide showed a strong cross reaction with Plasmodium falciparum sporozoites.

scholarly journals Synthesis and biophysical properties of carbamate-locked nucleic acid (LNA) oligonucleotides with potential antisense applications

2019 ◽  
Vol 17 (21) ◽  
pp. 5341-5348 ◽  
Author(s):  
Cameron Thorpe ◽  
Sven Epple ◽  
Benjamin Woods ◽  
Afaf H. El-Sagheer ◽  
Tom Brown
Keyword(s):  
Nucleic Acid ◽  
Locked Nucleic Acid ◽  
Biophysical Properties

Carbamate-LNA oligonucleotides have improved biophysical properties for theraputic applications.

Advances in the synthesis and antisense technology applications of bridged nucleic acid monomers

2021 ◽  
Vol 25 ◽  
Author(s):  
Priyanka Mangla ◽  
Balaji Olety ◽  
Vivek K. Sharma
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Antisense Oligonucleotides ◽  
Cost Effective ◽  
Biophysical Properties ◽  
Technology Applications ◽  
Effective Synthesis ◽  
Target Rna ◽  
Ribose Sugar ◽  
Tremendous Impact

: Bridged nucleic acids (BNA) or locked nucleic acids (LNA) are a class of nucleic acids modification, which is obtained by connecting the 2'-O and 4'-C of ribose sugar using a methylene bridge. This ‘bridging or locking’ (hence the name) of ribose sugar has a tremendous impact both on the biological and biophysical properties of therapeutic nucleic acids. They have enhanced stability against nucleases and also have higher binding affinity for the target RNA. Owing to these advantages, BNA is one of the most preferred nucleic acid modifications of antisense oligonucleotides (ASOs). However, the synthesis of BNA monomers which are lengthy and low-yielding, requires extensive protection and deprotection of the sugar functionalities. In this article, we aim to review challenges associated with their synthesis, and discuss recent chemical, chemo-enzymatic, and transglycosylation strategies employed for efficient and cost-effective synthesis of BNA monomers and selected BNA analogues.

Modeling Stopped-Flow Data for Nucleic Acid Duplex Formation Reactions: The Importance of Off-Path Intermediates

2013 ◽  
Vol 117 (30) ◽  
pp. 8966-8976 ◽  
Author(s):  
Jacqueline R. Sikora ◽  
Brittany Rauzan ◽  
Rachel Stegemann ◽  
Alice Deckert
Keyword(s):  
Nucleic Acid ◽  
Stopped Flow ◽  
Flow Data ◽  
Duplex Formation

Thermodynamic and Kinetic Characterization of Duplex Formation between 2′-O, 4′-C-Methylene-modified Oligoribonucleotides, DNA and RNA

2007 ◽  
Vol 27 (6) ◽  
pp. 327-333 ◽  
Author(s):  
Ulla Christensen
Keyword(s):  
Nucleic Acid ◽  
Full Length ◽  
Locked Nucleic Acid ◽  
Complementary Dna ◽  
High Affinity ◽  
Thermodynamics And Kinetics ◽  
Dna And Rna ◽  
Reaction Thermodynamics ◽  
Duplex Formation

2′-O,4′-C-methylene-linked ribonucleotide derivatives, named LNA (locked nucleic acid) and BNA (bridged nucleic acid) are nucleic acid analogoues that have shown high-affinity recognition of DNA and RNA, and the employment of LNA oligomers for antisense activity, gene regulation and nucleic acid diagnostics seems promising. Here we show kinetic and thermodynamic results on the interaction of a series of 10 bases long LNA–DNA mixmers, gabmers as well as full length LNA's with the complementary DNA, RNA and LNA oligonucleotides in the presence and absence of 10 mM Mg2+- ions. Our results show no significant differences in the reaction thermodynamics and kinetics between the LNA species, only a tendency to stronger duplex formation with the gabmer and mixmer. Introduction of a few LNA's thus may be a better strategy, than using full length LNA's to obtain an oligonucleotide that markedly increases the strength of duplexes formed with the complementary DNA and RNA.

Sign in / Sign up

Export Citation Format

Share Document