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 1.5 to 6 pg/ml, intra-/interday accuracies in the range of 86.8-119.1% and 88.1-113.1%, respectively, and precision of ≤17.2%. Furthermore, crossreactivity of 3'n-1, a metabolite with a single base difference, was <1%. Our approach provides an auspicious method for distinguishing metabolites and detecting ASOs with high sensitivity and specificity.

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. In this study, we aimed to confirm the feasibility of CPP-conjugation approach for DOCK2-inhibitory peptides, and select preferable sequences as CPP moiety. First, we evaluated cell permeability of thirteen known CPPs and partial sequences of influenza A viral protein PB1-F2 using an internalization assay system based on luciferin-luciferase reaction, and then selected four CPPs with efficient cellular uptake. Among four conjugates of these CPPs and a DOCK2-inhibitory peptide, the inhibitory activity of a novel CPP, PB1-F2 fragment 5 (PF5), conjugate was comparable to oligoarginine conjugate and higher than that of the non-conjugated peptide. Finally, internalization assay revealed that oligoarginine and PF5 increased the cellular uptake of inhibitory peptides to the same extent. Hence, we demonstrated that CPP-conjugation approach is applicable to the development of novel anti-inflammatory drugs based on DOCK2 inhibition by investigating both cellular uptake and bioactivity.

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. The mirrored pre-miR-21 binding peptide sequences were successfully obtained, and were chemically synthesized using D-amino acids. D-peptide is expected to have favorable properties as a drug candidate such as protease resistance and low immunogenicity. As a result of binding evaluation of the D-peptide to pre-miR-21, the EC50 value was 440nM. In addition, the D-peptide possessed inhibition activity to miR-21 processing.

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

Fibroblast growth factor receptor-1c (FGFR1c)/ßKlotho (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. The peptide, F91-8A07 (LPGRTCREYPDLWWVRCY), was discovered from random peptide T7 phage display and selectively bound to the FGFR1c/KLB complex, but not to FGFR1c and KLB individually. After subsequent peptide dimerization using a short polyethyleneglycol (PEG) linker, the dimeric F91-8A07 peptide showed higher potent agonist activity than that of FGF21 in cultured primary human adipocytes. Moreover, the dimeric peptide led to an expression of the early growth response protein-1 (Egr-1) mRNA in vivo, which is a target gene of FGFR1c. To the best of our knowledge, this is the first report of a FGFR1c/KLB complex-selective artificial peptide agonist.

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.

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. Caged fluorescent nucleotides with other emission wavelengths (622 and 724 nm) were also synthesized in this study; they were uncaged by 360 nm irradiation, and emitted fluorescence in the presence of the complementary RNA. Such probes were applied to area-specific RNA imaging in a cell. Only probes in the defined irradiation area were activated by uncaging irradiation, and subnuclear mRNA diffusion in a living cell was monitored.

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. UV and 1H NMR spectrometric studies revealed the structural properties of the taurine-containing base moieties and the sugar conformations of these modified ribonucleosides.

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.

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., to detect the existence of only one methyl group in a long DNA strand, is not easy. A rapid and selective chemical reaction capable of distinguishing between M and C would become a useful method for efficiently analyzing the status of cytosine methylation at a specific site in a gene. We herein report M-selective oxidation. M was oxidized efficiently by exposure to a reaction mixture containing an osmium complex, making possible a clear distinction from very weak oxidation of C. We readily obtained information on the methylation status at a specific site by means of M-selective oxidation using functional bipyridine ligands.

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.

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. The mechanism that underlies this excellent optical discrimination of singly mismatched targets is clarified by comparison of the thermal-denaturation profiles and fluorescence properties of the dual probe and a covalently linked analogue. Optical discrimination of singly mismatched targets arises from a decrease in excimer emission intensity due to a failure to form a ternary complex (a decrease in thermal stability) and/or local mismatch-induced changes in the helix geometry, depending on the position of the mismatched base pair. The devised dual-probe assay constitutes a simple and sensitive system for the detection of single-nucleotide polymorphism and highlights that conformational restriction combined with the use of short probes conveys favorable properties to dual-probe constructs.

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. We have designed a bipyridine-attached adenine derivative for sequence-specific osmium complexation. Sequence-specific osmium complexation was achieved by hybridization of a short DNA molecule containing this functional nucleotide to a target DNA sequence and resulted in the formation of a cross-linked structure. The interstrand cross-link clearly distinguished methylated cytosines from unmethylated cytosines and was used to quantify the degree of methylation at a specific cytosine in the genome.

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. Nucleotide analogs containing a C2',C3'-oxymethylene linker (E-type furanose conformation) or a C2',C4'-propylene linker (N-type furanose conformation) had no significant effect on triplex stability proving that conformational restriction per se is insufficient to stabilise 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.