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.

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.

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 60 s by UV irradiation at 365 nm. Experiments using NBGTP or NBUTP in T7-RNA transcription reactions showed that NBGTP could be useful for the photocontrol of transcription by UV irradiation.Download high-res image (71KB)Download full-size image

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

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

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.

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 (PzO) and phthalazin-1-one (PhO) as thymine analogs. The PNAs having an aPz or a PzO 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.

To discriminate among miRNA length variants, we synthesized conformationally restricted or unrestricted oligonucleotides containing a cyclohexyl phosphate residue. These oligonucleotides formed duplexes with length-matched complementary miRNAs more tightly than with length variants. The use of one of these modified oligodeoxynucleotides as a reverse transcription primer enabled a novel RT-PCR that can discriminate among miRNA length variants.

Single- and double-stranded oligodeoxynucleotides (ODNs) incorporating both 2-aminopurine (2AP) and an indole-fused cytosine analog (PPI) were prepared and studied for their fluorescence properties. PPI and 2AP can be excited simultaneously by irradiation at 300 nm, with emission observed at 500 nm for PPI and 370 nm for 2AP. We demonstrated the utility of these properties in the dual fluorescence labeling of ODNs giving well-separated emission peaks. In addition, both of the fluorescence signals of a doubly modified ODN changed independently, reflecting the local duplex formation at the regions containing 2AP or PPI. Potential applications of this strategy for the dual fluorescence labeling of oligonucleotides with 2AP and PPI include monitoring local structure alterations of functional nucleic acids and the multiplex detection of biologically important nucleic acids.

We have previously reported DNA triplexes containing the unnatural base triad G-PPI·C3, in which PPI is an indole-fused cytosine derivative incorporated into DNA duplexes and C3 is an abasic site in triplex-forming oligonucleotides (TFOs) introduced by a propylene linker. In this study, we developed a new unnatural base triad A-ψ·CR1 where ψ and CR1 are base moieties 2′-deoxypseudouridine and 5-substituted deoxycytidine, respectively. We examined several electron-withdrawing substituents for R1 and found that 5-bromocytosine (CBr) could selectively recognize ψ. In addition, we developed a new PPI derivative, PPIMe, having a methyl group on the indole ring in order to achieve selective triplex formation between DNA duplexes incorporating various Watson–Crick base pairs, such as T-A, C-G, A-ψ, and G-PPIMe, and TFOs containing T, C, CBr, and C3. We studied the selective triplex formation between these duplexes and TFOs using UV-melting and gel mobility shift assays.

We have developed new artificial oligonucleotides which distinguish short RNA targets from long ones. The modification of the 5′ termini of oligonucleotides by using adenosine derivatives that possess a bulky cyclohexyl phosphate moiety at their base moiety and a phosphate group at the position of their 5′-hydroxyl group maximized their short RNA selectivity. The 2′-O-methyl-RNA (5′-XCmAmAmCmCmUmAmCmUm) having these modifications exhibits ca. 10 °C higher Tm in the duplexes with the complementary short RNA (3′-GUUGGAUGA-5′) than with the long RNA (3′-AUUAUAUUGGUUA-5′). The oligodeoxynucleotides having the same modification exhibited similar selectivity. Such short-RNA selective binding of terminally modified oligonucleotides can be employed to distinguish between mature microRNAs and pre-microRNAs.

2′-O-[N-(4-Aminobutylcarbamoyl)]uridine (Uabcm) was synthesized and incorporated into oligonucleotides. The oligonucleotides incorporating Uabcm formed more stable duplexes with their complementary and mismatched RNAs than those containing 2′-O-carbamoyluridine (Ucm). The stability of duplex with a Uabcm-rG base pair showed higher thermostability than the duplex having unmodified U-rG base pair. The Uabcm residue showed enhanced resistance to snake venome phosphodiesterase.

We examined the synthesis of oligodeoxynucleotides containing all four nucleobases using the 4-methoxytritylthio (MMTrS) group for protection of the 5′-hydroxyl group. The MMTrS group could be introduced into 3′-O-TBDMS-deoxycytidine, -deoxyadenosine and -deoxyguanosine with the appropriate base protecting groups using strong bases such as n-butyl lithium and lithium hexamethyldisilazide. Because the MMTrS group could be removed by oxidation with an aqueous I2 solution, the oxidation of internucleotidic phosphite intermediates could be performed simultaneously. Thus, the nucleotide chain could be extended in a three-step protocol that comprised coupling, capping and oxidation/deprotection. Oligodeoxynucleotides with 10 and 20 mixed-base sequences could be synthesized using this protocol.

We have developed new artificial oligonucleotide probes that show selective recognition for short RNA targets over long RNA targets. Our results suggested that modification of the termini of the oligonucleotide probes by bulky substituents such as cyclohexyl and 4-(3,6,9-trioxaundecylenedioxy)phenyl (Bzcr) groups significantly improved the selectivity of the probes toward the short RNA targets. The selectivity was further improved by the addition of a phosphate group on the cyclohexane ring. Although much improved selectivity toward short RNA targets is desirable in a general sense, it is particularly applicable to the selective detection of matured-miRNA over pre-miRNAs.

Previously, we reported 2-N-carbamoylguanine (cmG) as a guanine analog. We further studied the synthetic protocol and hybridization properties of oligodeoxynucleotides (ODNs) incorporating cmG. These ODNs were synthesized using the phosphoramidite of cmG without protection of the 6-O position. However, the isolated products were contaminated with deacylated products having guanine in place of cmG. The detailed analysis of the synthetic process suggested that the deacylation resulted from the reaction of the carbamoyl moiety with capping reagents. Protection of the 6-O position suppressed the side reaction. The thermal stability of the DNA duplexes incorporating cmG was analyzed. An analysis of Tm values revealed that the base discrimination ability of cmG was comparable to or higher than that of the canonical guanine depending on the flanking bases.

We have developed new artificial oligonucleotides which distinguish short RNA targets from long ones. The modification of the 5′ termini of oligonucleotides by using adenosine derivatives that possess a bulky cyclohexyl phosphate moiety at their base moiety and a phosphate group at the position of their 5′-hydroxyl group maximized their short RNA selectivity. The 2′-O-methyl-RNA (5′-XCmAmAmCmCmUmAmCmUm) having these modifications exhibits ca. 10 °C higher Tm in the duplexes with the complementary short RNA (3′-GUUGGAUGA-5′) than with the long RNA (3′-AUUAUAUUGGUUA-5′). The oligodeoxynucleotides having the same modification exhibited similar selectivity. Such short-RNA selective binding of terminally modified oligonucleotides can be employed to distinguish between mature microRNAs and pre-microRNAs.

We have developed new artificial oligonucleotide probes that show selective recognition for short RNA targets over long RNA targets. Our results suggested that modification of the termini of the oligonucleotide probes by bulky substituents such as cyclohexyl and 4-(3,6,9-trioxaundecylenedioxy)phenyl (Bzcr) groups significantly improved the selectivity of the probes toward the short RNA targets. The selectivity was further improved by the addition of a phosphate group on the cyclohexane ring. Although much improved selectivity toward short RNA targets is desirable in a general sense, it is particularly applicable to the selective detection of matured-miRNA over pre-miRNAs.

We have previously reported DNA triplexes containing the unnatural base triad G-PPI·C3, in which PPI is an indole-fused cytosine derivative incorporated into DNA duplexes and C3 is an abasic site in triplex-forming oligonucleotides (TFOs) introduced by a propylene linker. In this study, we developed a new unnatural base triad A-ψ·CR1 where ψ and CR1 are base moieties 2′-deoxypseudouridine and 5-substituted deoxycytidine, respectively. We examined several electron-withdrawing substituents for R1 and found that 5-bromocytosine (CBr) could selectively recognize ψ. In addition, we developed a new PPI derivative, PPIMe, having a methyl group on the indole ring in order to achieve selective triplex formation between DNA duplexes incorporating various Watson–Crick base pairs, such as T-A, C-G, A-ψ, and G-PPIMe, and TFOs containing T, C, CBr, and C3. We studied the selective triplex formation between these duplexes and TFOs using UV-melting and gel mobility shift assays.

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.

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

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.

To discriminate among miRNA length variants, we synthesized conformationally restricted or unrestricted oligonucleotides containing a cyclohexyl phosphate residue. These oligonucleotides formed duplexes with length-matched complementary miRNAs more tightly than with length variants. The use of one of these modified oligodeoxynucleotides as a reverse transcription primer enabled a novel RT-PCR that can discriminate among miRNA length variants.