Oligodeoxynucleotides Modified with 2'-O-(Cysteinylaminobutyl)carbamoylethylribothymidine Residues for Native Chemical Ligation with Peptide at Internal Positions.

We used native chemical ligation (NCL) to synthesize a 2'-O-{N-[N-(S-tert-butylthiocysteinyl)aminobutyl]carbamoylethyl} (CysBCE) ribothymidine-derived oligonucleotide to expand the variety of peptide conjugation sites, allowing the incorporation of peptides at the 2'-hydroxy group when the oligonucleotide forms a duplex with the complementary strand. The NCL reaction with a peptide thioester and the modified oligonucleotide proceeded smoothly even when the CysBCE modification was in the middle of the oligonucleotide sequence. In addition, we incorporated two CysBCEs into an oligonucleotide to conjugate two peptides to one oligonucleotide. The results indicated that the tandem NCL reactions proceeded efficiently when the oligonucleotide hybridized to the complementary strand to avoid intramolecular disulfide formation between the two CysBCE groups. This method could be useful for peptide conjugation on the 2'-position.

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.

Tolerance of N2-heteroaryl modifications on guanine bases in a DNA G-quadruplex.

To systematically determine the effect of N2-heteroaryl modification on the stability of G-quadruplex structures, six types of N2-heteroarylated deoxyguanosines were incorporated into oligonucleotides with intramolecular quadruplex-forming sequences obtained from the human telomere sequence. A UV melting experiment, electrophoretic mobility shift assay, and circular dichroism were performed to evaluate the influence of N2-heteroaryl modification. Among the N2-heteroaryl modifications used in this study, N2-(pyrimidin-2-yl) modification markedly destabilized the G-quadruplex structure. Interestingly, N2-heteroaryl modification in the middle guanine base of the fourth strand was well-tolerated and formed a G-quadruplex, suggesting that the fourth strand is the least important strand in sustaining the G-quadruplex structure.

Modification of oligonucleotides with weak basic residues via the 2'-O-carbamoylethyl linker for improving nuclease resistance without loss of duplex stability and antisense activity.

For the improvement of nuclease resistance, four kinds of new modifications through a carbamoylethyl linker were designed. Among them, the 2'-O-[2-N-{2-(benzimidazol-1-yl)ethyl}carbamoylethyl] modification showed 20-fold longer half-life when treated with a 3' to 5' exonuclease compared to the 2'-O-methoxyethyl (MOE) modification, which is used in approved drugs. In addition, this large modification did not disturb the binding affinity or RNase H-dependent antisense activity. From these findings, it could be concluded that an adequate linker, such as carbamoylethyl in this study, could extend the utility of 2'-O-modification without loss of the properties of nucleic acids. This strategy would be useful for the development of nucleic acid therapeutics.

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.

A triad molecular conductor: simultaneous control of charge and molecular arrangements.

Molecular and charge arrangements in the solid state were controlled by a new building block: a triad molecule. Owing to the appropriate flexibilities in both molecular structure and electron distribution of the triad, the apparently simple salt exhibits an unstable metallic phase, which is promising for superconducting transitions.