Development of antiparallel-type triplex-forming oligonucleotides containing quinoline derivatives capable of recognizing a T-A base pair in a DNA duplex.

A triplex-forming oligonucleotide (TFO) can bind to genomic DNA and inhibit the expression of genes with specific sequences. However, to date, there have been a few reports of modified bases in antiparallel TFOs that can recognize and strongly bind to T-A base pairs. In this study, we introduced several quinoline derivatives into antiparallel TFOs to develop modified bases that can recognize the T-A base pair and evaluated their ability to form triplexes and to discriminate between base pairs. The introduction of 2-acetamido-6-aminoquinoline6DAQac) into an antiparallel TFO allowed the selective recognition of a T-A base pair at both low and high salt concentrations.

Chemical synthesis and biochemical characterization of cyclic oligonucleotides containing acyl groups at both 5'- and 3'-terminal positions.

Modified oligonucleotides, whose ON-OFF switch of hybridization can be controlled by an external stimulus, are important to understanding life phenomena and efficient treatment of diseases. The ON-OFF switch can be completely controlled by chemical modification of the oligonucleotide such as cyclization. However, their chemical modifications of the previous cyclic oligonucleotides remain after the addition of an external stimulus. To overcome this problem, we carried out the first synthesis of cyclic oligonucleotides containing acyl groups at both 5'- and 3'-terminal positions, which can be hydrolyzed by intracellular esterase. The cyclic oligonucleotides were successfully synthesized via disulfide bond formation and the phosphoramidite method without base protection on polymer supports containing a silyl linker. Subsequently, we were able to introduce a functional group into the cyclic oligonucleotide using the corresponding isothiocyanate reagent. Additionally, a cyclic oligonucleotide with acyl groups was found to have a much lower binding ability than the corresponding linear oligonucleotide. Moreover, we demonstrated its structural conversion to the corresponding linear oligonucleotide with two thiol groups under reducing conditions using dithiothreitol. It was also confirmed that the two terminal acyl groups of the linear oligonucleotide were hydrolyzed by pig liver esterase. These results indicate that hybridization of cyclic acylated nucleic acid drugs with high nuclease resistance is regulated by intracellular esterase under the reducing conditions in the cell cytoplasm.

Chemical synthesis and properties of modified oligonucleotides containing 5'-amino-5'-deoxy-5'-hydroxymethylthymidine residues.

In this study, we designed 5'-amino-5'-deoxy-5'-hydroxymethylthymidine as a new oligonucleotide modification with an amino group directly attached to the 5'-carbon atom. We successfully synthesized two isomers of 5'-amino-5'-deoxy-5'-hydroxymethylthymidine via dihydroxylation of the 5'-vinyl group incorporated into 5'-deoxy-5'-C-methenylthymidine derivative. Moreover, it was found that the nuclease resistance, binding selectivity to single-stranded RNA, and triplex-forming ability of an oligonucleotide containing RT residues of the new compound were higher than those of the unmodified oligonucleotide.

The ability of a triplex-forming oligonucleotide to recognize T-A and C-G base pairs in a DNA duplex is enhanced by incorporating N-acetyl-2,7-diaminoquinoline.

A triplex-forming oligonucleotide (TFO) can recognize the homopurine-homopyrimidine sequence in DNA duplexes and inhibit the transcription of targeted mRNAs. Recently, we reported that N-acetyl-2,7-diamino-1,8-naphthyridine (DANac), incorporated into a TFO, has high binding ability and base recognition selectivity for the pyrimidine bases in the purine-rich chain of the DNA duplex at pH 7.4. However, it was found in this study that the difference in the Tm values between the pyrimidine bases and purine bases decreased by more than 4 °C at pH 6.0-7.0. To improve the low base recognition selectivity of the TFO, we designed a new artificial base, DAQac, with a quinoline skeleton. The Tm values of the triplexes containing DAQac:T-A or DAQac:C-G were more than 13 °C higher than those of the triplexes containing DAQac:A-T or DAQac:G-C at pH 7.4. We also observed that under more acidic conditions (pH 6.0-7.0), the base recognition selectivity of DAQac in a triplex was higher than that of DANac, although the binding ability of DAQac in a triplex was similar to that of DANac. Additionally, we found that DAQac, incorporated into the TFO, could accurately recognize the MeC-G base pair in the hairpin DNA, similar to the C-G base pair.

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.

pH-Dependent Switching of Base Pairs Using Artificial Nucleobases with Carboxyl Groups.

In this study, we report the synthesis of modified oligonucleotides consisting of benzoic acid or isophthalic acid residues as new nucleobases. As evaluated by UV thermal denaturation analysis at different pH conditions (5.0, 6.0, 7.0, and 8.0), these modified oligonucleotides exhibited pH-dependent recognition of natural nucleobases and one is first found to be capable of base pair switching in response to a pH change. The isophthalic acid residue incorporated into the oligonucleotide on a d-threoninol backbone could preferentially bind with adenine but with guanine in response to a change in the pH conditions from pH 5 to pH 7 (or 8) without significant difference in duplex stability. These findings would be valuable for further developing pH-responsive DNA-based molecular devices.