DNA triplex-based fluorescence turn-on sensors for adenosine using a fluorescent molecular rotor 5-(3-methylbenzofuran-2-yl) deoxyuridine.

Fluorescence turn-on sensors for adenosine were developed using DNA triplexes modified with a fluorescent molecular rotor 5-(3-methylbenzofuran-2-yl)deoxyuridine (dUMBF) and abasic sites. Binding of adenosine to the abasic site next to the dUMBF changed the microenvironment and conformation (from the twisted to planar state) of dUMBF and enhanced the fluorescence. Adenosine could be selectively detected over other nucleosides and adenosine phosphates. The binding of adenosine was confirmed by UV-thermal melting experiments. Further, the conformational changes of dUMBF from the twisted to coplanar state upon binding of adenosine was supported by MD simulations.

Synthesis of 2'-O-(N-methylcarbamoylethyl) 5-methyl-2-thiouridine and its application to splice-switching oligonucleotides.

The effect of 2'-O-(N-methylcarbamoyl)ethyl (MCE) modification on splice-switching oligonucleotides (SSO) was systematically evaluated. The incorporation of five MCE nucleotides at the 5'-termini of SSOs effectively improved the splice switching effect. In addition, the incorporation of 2'-O-(N-methylcarbamoylethyl)-5-methyl-2-thiouridine (s2TMCE), a duplex-stabilizing nucleotide with an MCE modification, into SSOs further improved splice switching. These SSOs may be useful for the treatment of genetic diseases associated with splicing errors.

Application of 2'-O-(2-N-Methylcarbamoylethyl) Nucleotides in RNase H-Dependent Antisense Oligonucleotides.

An RNase H-dependent antisense oligonucleotide (ASO), having the 2'-O-(2-N-methylcarbamoylethyl) (MCE) modification, was evaluated in vitro and in vivo. The antisense activities of an ASO having the MCE modification were comparable with those of an ASO having the 2'-O-methoxyethyl (MOE) modification in both in vitro and in vivo experiments. In contrast, the hepatotoxic potential of the ASO having the MCE modification was lower than that of the ASO having the MOE modification. Thus, these findings suggested that the MCE modification could be used as an alternative to the MOE modification.

Synthesis of and triplex formation in oligonucleotides containing 2'-deoxy-6-thioxanthosine.

This study aimed to synthesize triplex-forming oligonucleotides (TFOs) containing 2'-deoxy-6-thioxanthosine (s6X) and 2'-deoxy-6-thioguanosine (s6Gs) residues and examined their triplex-forming ability. Consecutive arrangement of s6X and s6Gs residues increased the triplex-forming ability of the oligonucleotides more than 50 times, compared with the unmodified TFOs. Moreover, the stability of triplex containing a mismatched pair was much lower than that of the full-matched triplex, though s6X could form a s6X-GC mismatched pair via tautomerization of s6X. The present results reveal excellent properties of modified TFOs containing s6Xs and s6Gs residues, which may be harnessed in gene therapy and DNA nanotechnology.

Deoxynucleoside Triphosphate Containing Pyridazin-3-one Aglycon as a Thymidine Triphosphate Substitute for Primer Extension and Chain Elongation by Klenow Fragments.

Deoxynucleoside 5'-triphosphate was synthesized with 3-oxo-2 H-pyridazin-6-yl (PzO)-a uracil analogue lacking a 2-keto group-as the nucleobase. Theoretical analyses and hybridization experiments indicated that PzO recognizes adenine (A) for formation of a Watson-Crick base pair. Primer extension reactions using nucleoside 5'-triphosphate and the Klenow fragment revealed that the synthetic nucleoside 5'-triphosphate was incorporated into the 3' end of the primer through recognition of A in the template strand. Moreover, the 3'-nucleotide residue harboring PzO as the base was resistant to the 3'-exonuclease activity of Klenow fragment exo+. The primer bearing the PzO base at the 3' end could function in subsequent chain elongation. These properties of PzO were attributed to the presence of an endocyclic nitrogen atom at the position ortho to the glycosidic bond, which was presumed to form an H-bond with the amino acid residue of DNA polymerase for effective recognition of the 3' end of the primer for primer extension. These results provide a basis for designing new nucleobases by combining a nitrogen atom at the position ortho to the glycosidic bond and base-pairing sites for Watson-Crick hydrogen bonding.

Synthesis of photocaged 6-O-(2-nitrobenzyl)guanosine and 4-O-(2-nitrobenzyl) uridine triphosphates for photocontrol of the RNA transcription reaction.

6-O-(2-Nitrobenzyl)guanosine and 4-O-(2-nitrobenzyl)uridine triphosphates (NBGTP, NBUTP) were synthesized, and their biochemical and photophysical properties were evaluated. We synthesized NBUTP using the canonical triphosphate synthesis method and NBGTP from 2',3'-O-TBDMS guanosine via a triphosphate synthesis method by utilizing mild acidic desilylation conditions. Deprotection of the nitrobenzyl group in NBGTP and NBUTP proceeded within 60s by UV irradiation at 365nm. Experiments using NBGTP or NBUTP in T7-RNA transcription reactions showed that NBGTP could be useful for the photocontrol of transcription by UV irradiation.

Synthesis of oligonucleotides containing 2-N-heteroarylguanine residues and their effect on duplex/triplex stability.

To systematically understand the effect of 2-N-heteroarylguanine (GHA) modification on the stability of higher-order DNA structures, nucleoside derivatives and oligodeoxyribonucleotides containing guanine residues modified with four kinds of hereroaryl groups on the 2-amino group were synthesized. The stabilities of the DNA duplex and the parallel-oriented DNA triplex containing these GHAs were studied by measuring their melting temperatures (Tm). Tm experiments and DFT calculations of the modified guanine nucleobases suggested that the base pair formation energy and stability of the two conformations, i.e., the open- and closed-type conformations, are key to determining the stability of the DNA duplex. Finally, the DNA triplex was destabilized when modified guanine residues were introduced into triplex-forming oligonucleotides.

Deformability Calculation for Estimation of the Relative Stability of Chemically Modified RNA Duplexes.

Chemical modification of RNA duplexes alters their stability. We have attempted to develop a computational approach to estimate the thermal stability of chemically modified duplexes. These studies revealed that the deformability of chemically modified RNA duplexes, calculated from molecular dynamics simulations, could be used as a good indicator for estimating the effect of chemical modification on duplex thermal stability. This unit describes how deformability calculation can be applied to estimate the relative stability of chemically modified RNA duplexes. © 2017 by John Wiley & Sons, Inc.

Fluorescence enhancement of oligodeoxynucleotides modified with green fluorescent protein chromophore mimics upon triplex formation.

Green fluorescent protein (GFP)-based molecular-rotor chromophores were attached to the 5-positions of deoxyuridines, and subsequently, incorporated into the middle positions of oligodeoxynucleotides. These oligonucleotides were designed to form triplex DNA in order to encapsulate the GFP chromophores, mimicking GFP structures. Upon triplex formation, the embedded GFP chromophores exhibited fluorescence enhancement, suggesting the potential application of these fluorescent probes for the detection of nucleic acids.

Enzymatic synthesis and reverse transcription of RNAs incorporating 2'-O-carbamoyl uridine triphosphate.

Enzymatic synthesis and the reverse transcription of RNAs containing 2'-O-carbamoyl uridine were evaluated. A mild acidic deprotection procedure allowed the synthesis of 2'-O-carbamoyl uridine triphosphate (UcmTP). UcmTP was incorporated correctly into long RNAs, and its fidelity during reverse transcription using SuperScript III was sufficient for RNA aptamer selection.

Photo-controlled binding of MutS to photo-caged DNA duplexes incorporating 4-O-(2-nitrobenzyl) or 4-O-[2-(2-nitrophenyl)propyl]thymidine.

Mismatch binding protein MutS binding to bulge structure in DNA duplexes was controlled by UV irradiation. 4-O-(2-Nitrobenzyl)thymidine or 4-O-[2-(2-nitrophenyl)propyl]thymidine was incorporated into DNA duplexes a bulged position. The MutS did not bind to the caged DNA duplexes but bound after removing the 2-nitrobenzyl or 2-(2-nitrophenyl)propyl group by photo-irradiation. By using photo-caged DNA duplex, we revealed that binding of MutS to the uncaged DNA downstream of the T7 RNA promoter weakly inhibited transcription by T7 RNA polymerase.

Effective Strategy for Conformer-Selective Detection of Short-Lived Excited State Species: Application to the IR Spectroscopy of the N1H Keto Tautomer of Guanine.

The ultrafast deactivation processes in the excited state of biomolecules, such as the most stable tautomers of guanine, forbid any state-of-the-art gas phase spectroscopic studies on these species with nanosecond lasers. This drawback can be overcome by grafting a chromophore having a long-lived excited state to the molecule of interest, allowing thus a mass-selective detection by nanosecond R2PI and therefore double resonance IR/UV conformer-selective spectroscopic studies. The principle is presently demonstrated on the keto form of a modified 9-methylguanine, for which the IR/UV double resonance spectrum in the C═O stretch region, reported for the first time, provides evidence for extensive vibrational couplings within the guanine moiety. Such a successful strategy opens up a route to mass-selective IR/UV spectroscopic investigations on molecules exhibiting natural chromophores having ultrashort-lived excited states, such as DNA bases, their complexes as well as peptides containing short-lived aromatic residues.

Identification of the Three Genes Involved in Controlling Production of a Phytotoxin Tropolone in Burkholderia plantarii.

UNLABELLED: Tropolone, a phytotoxin produced by Burkholderia plantarii, causes rice seedling blight. To identify genes involved in tropolone synthesis, we systematically constructed mutations in the genes encoding 55 histidine kinases and 72 response regulators. From the resulting defective strains, we isolated three mutants, KE1, KE2, and KE3, in which tropolone production was repressed. The deleted genes of these mutants were named troR1, troK, and troR2, respectively. The mutant strains did not cause rice seedling blight, and complementation experiments indicated that TroR1, TroK, and TroR2 were involved in the synthesis of tropolone in B. plantarii However, tropolone synthesis was repressed in the TroR1 D52A, TroK H253A, and TroR2 D46A site-directed mutants. These results suggest that the putative sensor kinase (TroK) and two response regulators (TroR1 and TroR2) control the production of tropolone in B. plantarii IMPORTANCE: A two-component system is normally composed of a sensor histidine kinase (HK) and a cognate response regulator (RR) pair. In this study, HK (TroK) and two RRs (TroR1 and TroR2) were found to be involved in controlling tropolone production in B. plantarii These three genes may be part of a bacterial signal transduction network. Such networks are thought to exist in other bacteria to regulate phytotoxin production, as well as environmental adaptation and signal transduction.

7-(Benzofuran-2-yl)-7-deazadeoxyguanosine as a fluorescence turn-ON probe for single-strand DNA binding protein.

7-(Benzofuran-2-yl)-7-deazadeoxyguanosine ((BF)dG) was synthesized and incorporated into an oligodeoxynucleotide (ODN). The single-stranded ODN containing (BF)dG shows 91-fold fluorescence enhancement upon binding of single-strand DNA binding protein.

Synthesis of 5-[3-(2-aminopyrimidin-4-yl)aminopropyn-1-yl]uracil derivative that recognizes Ade-Thy base pairs in double-stranded DNA.

5-[3-(2-Aminopyrimidin-4-yl)aminopropyn-1-yl]uracil (Ura(Pyr)) was designed as a new nucleobase to recognize Ade-Thy base pair in double-stranded DNA. We successfully synthesized the dexoynucleoside phosphoramidite having Ura(Pyr) and incorporated it into triplex forming oligonucleotides (TFOs). Melting temperature analysis revealed that introduction of Ura(Pyr) into TFOs could effectively stabilize their triplex structures without loss of base recognition capabilities.

Design, Synthesis, and Properties of Phosphoramidate 2',5'-Linked Branched RNA: Toward the Rational Design of Inhibitors of the RNA Lariat Debranching Enzyme.

Two RNA fragments linked by means of a 2',5' phosphodiester bridge (2' hydroxyl of one fragment connected to the 5' hydroxyl of the other) constitute a class of nucleic acids known as 2'-5' branched RNAs (bRNAs). In this report we show that bRNA analogues containing 2'-5' phosphoramidate linkages (bN-RNAs) inhibit the lariat debranching enzyme, a 2',5'-phosphodiesterase that has recently been implicated in neurodegenerative diseases associated with aging. bN-RNAs were efficiently generated using automated solid-phase synthesis and suitably protected branchpoint building blocks. Two orthogonally removable groups, namely the 4-monomethoxytrityl (MMTr) group and the fluorenylmethyl-oxycarbonyl (Fmoc) groups, were evaluated as protecting groups of the 2' amino functionality. The 2'-N-Fmoc methodology was found to successfully produce bN-RNAs on solid-phase oligonucleotide synthesis. The synthesized bN-RNAs resisted hydrolysis by the lariat debranching enzyme (Dbr1) and, in addition, were shown to attenuate the Dbr1-mediated hydrolysis of native bRNA.

Complete genome sequence of Lactobacillus acetotolerans RIB 9124 (NBRC 13120) isolated from putrefied (hiochi) Japanese sake.

Lactobacillus acetotolerans RIB 9124 (NBRC 13120) was isolated from putrefied (hiochi) Japanese sake. Here we report the complete genome sequence of this organism. This paper is the first report demonstrating the fully sequenced and completely annotated genome of a L. acetotolerans strain.

Synthesis and triplex-forming properties of oligonucleotides capable of recognizing corresponding DNA duplexes containing four base pairs.

A triplex-forming oligonucleotide (TFO) could be a useful molecular tool for gene therapy and specific gene modification. However, unmodified TFOs have two serious drawbacks: low binding affinities and high sequence-dependencies. In this paper, we propose a new strategy that uses a new set of modified nucleobases for four-base recognition of TFOs, and thereby overcome these two drawbacks. TFOs containing a 2'-deoxy-4N-(2-guanidoethyl)-5-methylcytidine (d(g)C) residue for a C-G base pair have higher binding and base recognition abilities than those containing 2'-OMe-4N-(2-guanidoethyl)-5-methylcytidine (2'-OMe (g)C), 2'-OMe-4N-(2-guanidoethyl)-5-methyl-2-thiocytidine (2'-OMe (g)Cs), d(g)C and 4S-(2-guanidoethyl)-4-thiothymidine ((gs)T). Further, we observed that N-acetyl-2,7-diamino-1,8-naphtyridine ((DA)Nac) has a higher binding and base recognition abilities for a T-A base pair compared with that of dG and the other DNA derivatives. On the basis of this knowledge, we successfully synthesized a fully modified TFO containing (DA)Nac, d(g)C, 2'-OMe-2-thiothymidine (2'-OMe (s)T) and 2'-OMe-8-thioxoadenosine (2'-OMe (s)A) with high binding and base recognition abilities. To the best of our knowledge, this is the first report in which a fully modified TFO accurately recognizes a complementary DNA duplex having a mixed sequence under neutral conditions.

Synthesis of peptide nucleic acids containing pyridazine derivatives as cytosine and thymine analogs, and their duplexes with complementary oligodeoxynucleotides.

Synthesis of peptide nucleic acids (PNAs) is reported with new pyridazine-type nucleobases: 3-aminopyridazine (aPz) and 1-aminophthalazine (aPh) as cytosine analogs, and pyridazin-3-one (Pz(O)) and phthalazin-1-one (Ph(O)) as thymine analogs. The PNAs having an aPz or a Pz(O) formed duplexes with each complementary oligodeoxynucleotide forming a base pair with G or A, respectively, as evaluated by using UV melting analyses and circular dichroism (CD) spectra.

Controlling the fluorescence of benzofuran-modified uracil residues in oligonucleotides by triple-helix formation.

We developed fluorescent turn-on probes containing a fluorescent nucleoside, 5-(benzofuran-2-yl)deoxyuridine (dU(BF)) or 5-(3-methylbenzofuran-2-yl)deoxyuridine (dU(MBF)), for the detection of single-stranded DNA or RNA by utilizing DNA triplex formation. Fluorescence measurements revealed that the probe containing dU(MBF) achieved superior fluorescence enhancement than that containing dU(BF). NMR and fluorescence analyses indicated that the fluorescence intensity increased upon triplex formation partly as a consequence of a conformational change at the bond between the 3-methylbenzofuran and uracil rings. In addition, it is suggested that the microenvironment around the 3-methylbenzofuran ring contributed to the fluorescence enhancement. Further, we developed a method for detecting RNA by rolling circular amplification in combination with triplex-induced fluorescence enhancement of the oligonucleotide probe containing dU(MBF).