Synthesis of a Cyclic Dinucleotide Analogue with Ambiguous Bases, 5-Aminoimidazole-4-carboxamide.

Cyclic dinucleotides (CDNs) are second messengers composed of two purine nucleotides. In recent years, the structural diversity of CDNs and their functionality in biological processes are being intensely studied. Herein we report the chemical synthesis of cyclic di-5-aminoimidazole-4-carboxamide-1-β-d-ribofuranosyl monophosphate (c-di-ZMP) (), which consists of two 5-amino-4-imidazolecarboxamide ribonucleotides (Z-ribonucleotides) linked via two phosphodiester linkages.

Gene Expression of 4'-Thioguanine DNA via 4'-Thiocytosine RNA.

DNA and RNA nucleotides are ubiquitous molecules that store and transmit genetic information. The emergence of synthetic elements that fulfill the function of DNA and RNA provides an alternative gene expression system. Herein, we demonstrate the gene expression of 4'-thioguanine DNA (dG DNA) via 4'-thiocytosine RNA (dC RNA) to give green fluorescent protein (GFPuv) in a single test tube.

The novel functional nucleic acid iRed effectively regulates target genes following cytoplasmic delivery by faint electric treatment.

An intelligent shRNA expression device (iRed) contains the minimum essential components needed for shRNA production in cells, and could be a novel tool to regulate target genes. However, general delivery carriers consisting of cationic polymers/lipids could impede function of a newly generated shRNA via electrostatic interaction in the cytoplasm. Recently, we found that faint electric treatment (fET) of cells enhanced delivery of siRNA and functional nucleic acids into the cytoplasm in the absence of delivery carriers.

Gene Silencing Using 4'-thioDNA as an Artificial Template to Synthesize Short Hairpin RNA Without Inducing a Detectable Innate Immune Response.

The development of a versatile technique to induce RNA interference (RNAi) without immune stimulation in vivo is of interest as existing approaches to trigger RNAi, such as small interfering RNA (siRNA) and plasmid DNA (pDNA) expressing short hairpin RNA (shRNA), present drawbacks arising from innate immune stimulation. To overcome them, an intelligent shRNA expression device (iRed) designed to induce RNAi was developed. The minimum sequence of iRed encodes only the U6 promoter and shRNA.

Faithful PCR Amplification of an Unnatural Base-Pair Analogue with Four Hydrogen Bonds.

In vitro replication of an unnatural imidazopyridopyridine:naphthyridine base pair, (i.e., ImN(N):NaO(O)), having four hydrogen bonds was investigated.

Chemistry, properties, and in vitro and in vivo applications of 2'-O-methoxyethyl-4'-thioRNA, a novel hybrid type of chemically modified RNA.

We report the synthesis, properties, and in vitro and in vivo applications of 2'-O-methoxyethyl-4'-thioRNA (MOE-SRNA), a novel type of hybrid chemically modified RNA. In its hybridization with complementary RNA, MOE-SRNA showed a moderate improvement of Tm value (+3.4 °C relative to an RNA:RNA duplex).

First synthesis of fully modified 4'-selenoRNA and 2'-OMe-4'-selenoRNA based on the mechanistic considerations of an unexpected strand break.

This study investigated oligonucleotide (ON) synthesis containing 4'-selenoribonucleoside(s) under standard phosphoramidite conditions. Careful operation using a manual ON synthetic system revealed that an unexpected strand break occurred to afford a C2-symmetric homodimer as a byproduct. In addition, this side reaction occurred during I2 oxidation.

PCR amplification of 4'-thioDNA using 2'-deoxy-4'-thionucleoside 5'-triphosphates.

2'-Deoxy-4'-thioribonucleic acid (4'-thioDNA) having a sulfur atom instead of an oxygen atom in the furanose ring has a nuclease resistance and hybridization ability higher than that of natural DNA. Despite its great potential for various biological applications, a long 4'-thioDNA having all four kinds of 2'-deoxy-4'-thionucleosides has not been reported. In this study, we describe systematic analysis of the incorporation of 2'-deoxy-4'-thionucleoside 5'-triphosphates (dSNTPs) using various DNA polymerases.

Synthesis of a novel 1,2-dithianenucleoside via Pummerer-like reaction, followed by Vorbruggen glycosylation between a 1,2-dithiane derivative and uracil.

The novel 1,2-dithianenucleoside was designed as a hybrid type of modification between 4'-thioribonucleoside and altritol nucleoside. The desired compound, i.e.

Gene suppression via U1 small nuclear RNA interference (U1i) machinery using oligonucleotides containing 2'-modified-4'-thionucleosides.

Gene suppression via U1 small nuclear RNA interference (U1i) is considered to be one of the most attractive approaches, and takes the place of general antisense, RNA interference (RNAi), and anti-micro RNA machineries. Since the U1i can be induced by short oligonucleotides (ONs), namely U1 adaptors consisting of a 'target domain' and a 'U1 domain', we prepared adaptor ONs using 2'-modified-4'-thionucleosides developed by our group, and evaluated their U1i activity. As a result, the desired gene suppression via U1i was observed in ONs prepared as a combination of 2'-fluoro-4'-thionucleoside and 2'-fluoronucleoside units as well as only 2'-fluoronucleoside units, while those prepared as combination of 2'-OMe nucleoside/2'-OMe-4'-thionucleoside and 2'-fluoronucleoside units did not show significant activity.

A practical post-modification synthesis of oligodeoxynucleotides containing 4,7-diaminoimidazo[5',4':4,5]pyrido[2,3-d]pyrimidine nucleoside.

We describe herein the practical post-modification synthesis of oligodeoxynucleotides (ODNs) containing 4,7-diaminoimidazo[5',4':4,5]pyrido[2,3-d]pyrimidine nucleoside (ImN(N)). Since the ImN(N) nucleoside unit possessing tribenzoyl groups on its exocyclic amino groups as the protecting group was quite unstable under acidic conditions, cleavage of its glycosidic linkage in ODN has been suggested throughout the conditions of solid-phase synthesis. As an alternative approach, we investigated a post-modification synthesis of the desired ODNs containing the ImN(N) unit.

New imidazopyridopyrimidine:naphthyridine base-pairing motif, ImN(N):NaO(O), consisting of a DAAD:ADDA hydrogen bonding pattern, markedly stabilize DNA duplexes.

The new imidazopyridopyrimidine:naphthyridine base-pairing motifs, ImO(O):NaN(N) and ImN(N):NaO(O), were designed. Among the base pairs examined, DNA duplexes containing ImN(N):NaO(O) pair(s) consisting of a DAAD:ADDA hydrogen bonding pattern (D = donor, A = acceptor) were markedly stabilized thermally and thermodynamically.