Sensitive and specific quantification of antisense oligonucleotides using probe alteration-linked self-assembly reaction technology.

Quantitative bioanalysis is essential when establishing pharmacokinetic properties during the drug development process. To overcome challenges of sensitivity, specificity and process complexity associated with the conventional analysis of antisense oligonucleotides (ASOs), a new approach to nonenzymatic hybridization assays using probe alteration-linked self-assembly reaction (PALSAR) technology as a signal amplifier was evaluated. PALSAR quantification of ASOs in mouse tissue and plasma was able to achieve a high sensitivity ranging from 1.

Investigation on cellular uptake and pharmacodynamics of DOCK2-inhibitory peptides conjugated with cell-penetrating peptides.

Protein-protein interaction between dedicator of cytokinesis 2 (DOCK2) and Ras-related C3 botulinum toxin substrate 1 (Rac1) is an attractive intracellular target for transplant rejection and inflammatory diseases. Recently, DOCK2-selective inhibitory peptides have been discovered, and conjugation with oligoarginine cell-penetrating peptide (CPP) improved inhibitory activity in a cell migration assay. Although a number of CPPs have been reported, oligoarginine was only one example introduced to the inhibitory peptides.

Discovery of peptidic miR-21 processing inhibitor by mirror image phage display: A novel method to generate RNA binding D-peptides.

A novel method to generate RNA binding D-peptide has been developed. To achieve the screening method, phage display was applied to "Mirrored" RNA (L-enantiomer of RNA). We have selected pre-miR21 as an initial screening target to demonstrate the method.

Discovery of an artificial peptide agonist to the fibroblast growth factor receptor 1c/βKlotho complex from random peptide T7 phage display.

Fibroblast growth factor receptor-1c (FGFR1c)/βKlotho (KLB) complex is a receptor of fibroblast growth factor 21 (FGF21). Pharmacologically, FGF21 shows anti-obesity and anti-diabetic effects upon peripheral administration. Here, we report the development of an artificial peptide agonist to the FGFR1c/KLB heterodimer complex.

Design and synthesis of caged fluorescent nucleotides and application to live-cell RNA imaging.

A binary photocontrolled nucleic acid probe that contains a nucleotide modified with one photolabile nitrobenzyl unit and two hybridization-sensitive thiazole orange units has been designed for area-specific fluorescence imaging of RNA in a cell. The synthesized probe emitted very weak fluorescence regardless of the presence of the complementary RNA, whereas it showed hybridization-sensitive fluorescence emission at 532 nm after photoirradiation at 360 or 405 nm for uncaging. Fluorescence suppression of the caged probe was attributed to a decrease in the duplex-formation ability.

On-chip DNA methylation analysis using osmium complexation.

The development of a reaction for detecting the presence/absence of one methyl group in a long DNA strand is a chemically and biologically challenging research subject. A newly designed chemical assay on a chip for the typing of DNA methylation has been developed. A methylation-detection probe fixed at the bottom of microwells was crosslinked with methylated DNA mediated by osmium complexation and contributes to selective amplification of methylated DNA.

Functionalization of 2'-amino-LNA with additional nucleobases.

Thymin-1-yl-acetic acid and adenin-9-yl-acetic acid have been coupled to the N2'-atom of a 2'-amino-LNA thymine nucleoside, and these "double-headed" LNA monomers have been incorporated into oligodeoxyribonucleotides via their corresponding phosphoramidite derivatives. Oligonucleotides containing these modified nucleotides show in most cases increased thermal stability when forming duplexes with complementary DNA, even allowing multiple incorporations. Incorporation of "double-headed" LNA monomers in both strands also led to stable duplexes, however, no indication of Watson-Crick base pairing between the extra nucleobases could be found.

Chemical synthesis and properties of 5-taurinomethyluridine and 5-taurinomethyl-2-thiouridine.

Unique taurine-containing uridine derivatives, 5-taurinomethyluridine (tau m5U) and 5-taurinomethyl-2-thiouridine (tau m5s2U), which were discovered in mammalian mitochondrial tRNAs, exist at the first position of the anticodon. In this paper, we report the first efficient synthesis of tau m5U and tau m5s2U and describe their physicochemical properties. These modified ribonucleosides were synthesized by the reaction of 5-substituted uridine derivatives with a tetrabutylammonium salt of taurine that is highly reactive and well-soluble in common organic solvents.

Synthesis and characterization of the 5-methyl-2'-deoxycytidine glycol-dioxoosmium-bipyridine ternary complex in DNA.

It has been established that approximately 80% of a 5-methyl-2'-deoxycytidine glycol-dioxoosmium(vi)-bipyridine ternary complex, which is known to be produced as one of various consequences of oxidative damage of DNA and is formed in a key step of a recently developed DNA methylation detection method, has the 5R,6S configuration.

Dual-probe system using pyrenylmethyl-modified amino-LNA for mismatch detection.

A dual-probe containing pyrenylmethyl amino-LNA has been developed for sensitive mismatch detection. While hybridization with complementary DNA/RNA results in very strong excimer signals, exposure to singly mismatched DNA/RNA targets results in significantly decreased excimer emission.

Chemistry of osmium-DNA complexes.

A precise chemistry of ternary complex formation between osmium reagents, 2,2'-bipyridine (Bpy) and DNA strand has been characterized. Enzymatically digested products of ternary complexes formed for a thymidine in an oligonucleotide has given two diastereoisomers corresponding the 5S,6R- and 5R,6S-isomers of T-Os-Bpy in 70:30 ratio.

DNA methylation analysis using metal complex formation.

Analysis of the cytosine methylation status of a gene is very important for understanding the expression mechanism of genetic information. However, to distinguish 5-methylcytosine (M) from C, i.e.

Sensitive SNP dual-probe assays based on pyrene-functionalized 2'-amino-LNA: lessons to be learned.

A homogenous fluorescence dual-probe assay containing 2'-N-(pyren-1-ylmethyl)-2'-amino-LNA (locked nucleic acid) building blocks has been developed for effective mismatch-sensitive nucleic acid detection. The pyrene units, which are connected to the rigid bicyclic furanose derivative of 2'-amino-LNA through a short linker, are positioned at the 3' and 5' ends of a dual-probe system. Whereas hybridization with complementary DNA/RNA results in very strong excimer signals, as the pyrene units are in close proximity to one another in the ternary complex, exposure to most singly mismatched DNA/RNA targets results in significantly lower excimer emission intensity.

An osmium-DNA interstrand complex: application to facile DNA methylation analysis.

Nucleic acids often acquire new functions by forming a variety of complexes with metal ions. Osmium, in an oxidized state, also reacts with C5-methylated pyrimidines. However, control of the sequence specificity of osmium complexation with DNA is still immature, and the value of the resulting complexes is unknown.

LNA (locked nucleic acid) and analogs as triplex-forming oligonucleotides.

The triplex-forming abilities of some conformationally restricted nucleotide analogs are disclosed and compared herein. 2'-Amino-LNA monomers proved to be less stabilising to triplexes than LNA monomers when incorporated into a triplex-forming third strand. N2'-functionalisation of 2'-amino-LNA monomers with a glycyl unit induced the formation of exceptionally stable triplexes.

Oligoribonucleotide synthesis by the use of 1-(2-cyanoethoxy)ethyl (CEE) as a 2'-hydroxy protecting group.

A novel method for the synthesis of oligoribonucleotides has been developed by the use of 1-(2-cyanoethoxy)ethyl (CEE) as a 2'-hydroxy protecting group. The CEE group was introduced to the 2'-position of an N-acyl-3',5'-O-silyl protected ribonucleoside under acidic conditions. A combination of the use of N-acyl and 2'-O-CEE protecting groups enables a reliable and complete two step deprotection, first with NH3aq-EtOH, then with TBAF in THF, without cleavage of internucleotidic linkages.