Kohji Seio

Solid-phase synthesis of a 5′-terminal TMG-capped trinucleotide block of U1 snRNA

Abstract A 5′-terminal 2,2,7-trimethylguanosine-capped trinucleotide block (m 3 2,2,7 G 5′ pppAmpUmpA) was successfully synthesized on a highly cross-linked polystyrene resin by a new method for introduction of the first nucleoside onto the resin and a newly developed pyrophosphorylating agent having a benzotriazolyloxy substitutent as the leaving group.

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 (dUBF) or 5‐(3‐methylbenzofuran‐2‐yl)deoxyuridine (dUMBF), for the detection of single‐stranded DNA or RNA by utilizing DNA triplex formation. Fluorescence measurements revealed that the probe containing dUMBF achieved superior fluorescence enhancement than that containing dUBF. 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 dUMBF.

Fluorescence Properties of Pyrimidopyrimidoindole Nucleoside dCPPI Incorporated into Oligodeoxynucleotides

A series of oligodeoxynucleotides labeled by a pyrimidopyrimidoindole deoxynucleoside (1a: dC(PPI)) and its derivatives 2a and 3a substituted with electron-donating and -withdrawing groups, respectively, were synthesized according to the phosphoramidite approach. The photophysical properties and quenching efficiencies of oligonucleotides incorporating dC(PPI) derivatives were studied in detail. The thermal denaturation experiments and molecular dynamics simulation of DNA duplexes incorporating dC(PPI) suggested that a modified base of dC(PPI) could form base pairs with guanine and adenine in canonical Watson-Crick and reverse-wobble geometries, respectively. The fluorescence of oligonucleotides incorporating dC(PPI) derivatives increased upon binding to the counter strands, except when dC(PPI) and guanine formed a base pair. It was revealed that dGMP quenched the fluorescence of the cyano derivative 3a most effectively, whereas it affected that of the methoxy derivative 2a least effectively. The involvement of the electron transfer from guanine to the dC(PPI) derivatives in the fluorescence quenching was supported by energy considerations.

New nucleotide pairs for stable DNA triplexes stabilized by stacking interaction.

New nucleotide pairs applicable to formation of DNA triplexes were developed. We designed oligonucleotides incorporating 5-aryl deoxycytidine derivatives (dC5Ars) and cyclic deoxycytidine derivatives, dCPPP and dCPPI, having an expanded aromatic area, as the second strand. As pairing partners, two types of abasic residues (C3: propylene linker, phi: abasic base) were chosen. It was concluded that, when the 5-aryl-modified cytosine bases paired with the abasic sites in TFOs in a space-fitting manner, the stability of the resulting triplexes significantly increased. The recognition of C3 toward dC5Ars was selective because of the stacking interactions between their aromatic part and the nucleobases flanking the abasic site. These results indicate the potential utility of new nucleotide triplets for DNA triplex formation, which might expand the variety of structures and sequences and might be useful for biorelated fields such as DNA nanotechnologies.

Linear Relationship between Deformability and Thermal Stability of 2'-O-Modified RNA Hetero Duplexes

We describe the relationship between the experimentally determined melting temperatures of 2′-O-modified-RNA/RNA duplexes and their deformability estimated from molecular dynamics simulations. To clarify this relationship, we synthesized several fully modified oligoribonucleotides such as 2′-O-cyanoethyl RNAs and 2′-O-methoxyethyl RNAs and compared the actual melting temperatures of the duplexes with their calculated deformabilities. An increase of the melting temperatures by 2′-O-modifications was found to correlate strongly with an increase of the helical elastic constants in U14/A14, (CU)7/(AG)7, and (GACU)3/(AGUC)3 sequences. Linear regression analyses could be used to estimate the melting temperature with an accuracy of ±2.0 °C in our model case. Although the strong correlation was observed in the same base sequence, the linear regression functions were different from each base sequence. Our results indicated the possibility of predicting the thermal stability of 2′-O-modified duplexes at the computer-aided molecular design stage.

Nano-Scale Alignment of Proteins on a Flexible DNA Backbone

Nano-scale alignment of several proteins with freedom of motion is equivalent to an enormous increase in effective local concentration of proteins and will enable otherwise impossible weak and/or cooperative associations between them or with their ligands. For this purpose, a DNA backbone made of six oligodeoxynucleotide (ODN) chains is designed in which five double-stranded segments are connected by four single-stranded flexible linkers. A desired protein with an introduced cysteine is connected covalently to the 5′-end of azido-ODN by catalyst-free click chemistry. Then, six protein-ODN conjugates are assembled with their complementary nucleotide sequences into a single multi-protein-DNA complex, and six proteins are aligned along the DNA backbone. Flexible alignment of proteins is directly observed by high-speed AFM imaging, and association of proteins with weak interaction is demonstrated by fluorescence resonance energy transfer between aligned proteins.

Synthesis and Triplex Formation of Oligonucleotides Containing 8-Thioxodeoxyadenosine

For more effective DNA triplex formation under neutral conditions, we synthesized triplex-forming oligonucleotides containing 8-thioxodeoxyadenosine (s(8)dA) residues in place of the protonated deoxycytidines required for the third base pairing with DNA duplexes. Consequently, it was found that s(8)dA exhibited much stronger hybridization ability than dC under neutral conditions when four s(8)dA bases were arranged in a consecutive sequence.

The pathogenic A4269G mutation in human mitochondrial tRNA Ile alters the T-stem structure and decreases the binding affinity for elongation factor Tu

The A4269G mutation in the human mitochondrial (mt) tRNAIle gene is associated with fatal cardiomyopathy. This mutation completely inhibits protein synthesis in mitochondria, thereby significantly reducing their respiratory activity. The steady‐state amount of tRNAIle in cells bearing the A4269G mutation is almost half that of control cells. We previously reported that this mutation causes tRNAIle to be unstable both in vivo and in vitro. To investigate whether the instability of the mutant tRNAIle is due to structural alterations, a nuclease‐probing experiment was performed with a mitochondrial enzymatic extract, which showed that the A4269G mutation destabilizes the T‐stem of the mutant tRNAIle. In addition, measurements of the binding affinity of the aminoacylated mutant tRNAIle for mt elongation factor Tu (EF‐Tu) showed that the mutant tRNAIle binds mt EF‐Tu less efficiently than the wild‐type does. This observation provides insight into the molecular pathology associated with tRNA dysfunction caused by this pathogenic point mutation.

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 (dgC) 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’-OMegC), 2’-OMe-4N-(2-guanidoethyl)-5-methyl-2-thiocytidine (2’-OMegCs), dgC and 4S-(2-guanidoethyl)-4-thiothymidine (gsT). Further, we observed that N-acetyl-2,7-diamino-1,8-naphtyridine (DANac) 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 DANac, dgC, 2’-OMe-2-thiothymidine (2’-OMesT) and 2’-OMe-8-thioxoadenosine (2’-OMesA) 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 pentathymidylate using a 4-monomethoxytritylthio (MMTrS) group as a 5′-hydroxyl protecting group: toward oligonucleotide synthesis without acid treatment

Abstract A phosphoramidite unit having 4-monomethoxytritylthio as a new 5′-hydroxyl protecting group was prepared and employed in oligonucleotide synthesis. The new phosphoramidite enabled the synthesis of oligonucleotides without the use of acids such as TFA or DCA.