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.

Synthesis and properties of oligonucleotides modified with 2'-O-(2-carboxyethyl)nucleotides and their carbamoyl derivatives.

2'-O-Methyl oligoribonucleotides with four kinds of 2'-O-modified uridine derivatives were synthesised. Their duplex stability, hydration behavior and exonuclease resistance were studied by spectroscopic analyses and molecular dynamics simulations. Consequently, 2'-O-modification of the uridine residue with 2-carbamoylethyl or 2-(N-methylcarbamoyl)ethyl groups resulted in a significant improvement of the exonuclease resistance without the loss of duplex stability.

A new modified cytosine base capable of base pairing with guanine using four hydrogen bonds.

Oligonucleotides, containing 4-N-(1H-pyrrol-2-ylcarbonyl)deoxycytidine (dC(Pyc)) and related derivatives, were synthesized via deprotection using 1.5 M NaOMe/MeOH. Among them, oligodeoxynucleotides containing dC(Pyc) exhibited a higher hybridization affinity for DNA and RNA than the unmodified oligodeoxynucleotides. Comparative analysis between dC(Pyc) and its derivatives by molecular dynamic simulation indicated that the C(Pyc) residue could form four hydrogen bonds with the opposite G nucleobase keeping a more planar structure than the C(Inc) residue where the Pyc group was replaced with a 1H-indol-2-ylcarbonyl group.

Properties of 5- and/or 2-modified 2'-O-cyanoethyl uridine residue: 2'-O-cyanoethyl-5-propynyl-2-thiouridine as an efficient duplex stabilizing component.

We systematically synthesized eight types of 5- and/or 2-modified uridine derivatives and evaluated their effect on duplex stability. The incorporation of 2'-O-cyanoethyl-2-thio-5-propynyluridine (p(5)s(2)UOCE) into RNA was significantly effective for stabilization of RNA/RNA (+8.5 °C) and DNA/RNA (+10.4 °C) duplexes. These striking effects were maintained in oligonucleotides with different sequences or multiple incorporations. In addition, p(5)s(2)UOCE increased selectivity toward the correct AU Watson-Crick base pair over the most stable mismatched base pair in both RNA/RNA and DNA/RNA duplexes. Hence, p(5)s(2)UOCE could be useful for various applications of modified oligonucleotides that need high duplex stability and base pairing selectivity.

Assembly of pyrene-modified DNA/RNA duplexes incorporating a G-rich single strand region.

The structural properties of a DNA/RNA duplex having a pyrene residue at the 5' end of DNA and a G-rich single strand region at the 3' end of RNA were studied in detail. Fluorescence and ultracentrifugation analyses indicated the formation of a complex containing four DNA/RNA duplexes, which required a pyrene residue, G-rich sequence, RNA-type backbone, and high salt concentration.

Fluorescent properties of oligonucleotides doubly modified with an indole-fused cytosine analog and 2-aminopurine.

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.

Prediction of the stability of modified RNA duplexes based on deformability analysis: oligoribonucleotide derivatives modified with 2'-O-cyanoethyl-5-propynyl-2-thiouridine as a promising component.

We describe a method to predict the stability of a modified RNA duplex. Ten unique modified RNA duplexes showed a linear relationship between the calculated and experimentally determined duplex stabilities.

Synthesis and properties of cationic 2'-O-[N-(4-aminobutyl)carbamoyl] modified oligonucleotides.

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

Development of an efficient method for phosphorodiamidate bond formation by using inorganic salts.

Phosphorodiamidate morpholino oligonucleotides (PMOs) have been extensively applied in antisense strategies for gene regulation because of their high stability in serum and low toxicity. However, chain elongation of PMOs requires long reaction time because few efficient methods have been developed for the formation of phosphorodiamidate bonds. In this Letter, we examined the effect of various additives to improve the reaction efficiency for formation of the phosphorodiamidate bond in the synthesis of PMOs. The addition of certain inorganic salts to the reaction media was found to be more effective. Particularly, lithium bromide was the most effective reagent and led to considerable acceleration (ca. 10-fold improvement).

Formation of new base pairs between inosine and 5-methyl-2-thiocytidine derivatives.

In this paper, we report DNA and 2'-OMe-RNA probes containing 5-methyl-2-thiocytidine (m(5)s(2)C) residues that can bind selectively and strongly to the corresponding RNA targets containing inosine residues by the significant stacking effect and steric hindrance of the 2-thiocarbonyl group.

Short-RNA selective binding of oligonucleotides modified using adenosine and guanosine derivatives that possess cyclohexyl phosphates as substituents.

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'-XC(m)A(m)A(m)C(m)C(m)U(m)A(m)C(m)U(m)) having these modifications exhibits ca. 10 °C higher T(m) in the duplexes with the complementary short RNA (3'-GUUGGAUGA-5') than with the long RNA (3'-AUUAUAUGUUGGAUGAUGGUUA-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.

DNA duplexes and triplex-forming oligodeoxynucleotides incorporating modified nucleosides forming stable and selective triplexes.

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·C(R1) where ψ and C(R1) are base moieties 2'-deoxypseudouridine and 5-substituted deoxycytidine, respectively. We examined several electron-withdrawing substituents for R1 and found that 5-bromocytosine (C(Br)) could selectively recognize ψ. In addition, we developed a new PPI derivative, PPI(Me), 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-PPI(Me), and TFOs containing T, C, C(Br), and C3. We studied the selective triplex formation between these duplexes and TFOs using UV-melting and gel mobility shift assays.

Synthesis of 5'-terminal capped oligonucleotides using O-N phosphoryl migration of phosphoramidite derivatives.

Trivalent phosphoramidite derivatives could be readily converted by reacting with 1-hydroxy-7-azabenzotriazole to phosphotriester intermediates; these intermediates reacted smoothly with phosphorylated compounds to give pyrophosphate derivatives. This new phosphorylation approach enabled a facile and rapid synthesis of 5'-adenylated DNA oligomers (A(5')ppDNA) on resins using a silyl-type linker. Our new approach could be applied to the synthesis of a 2'-OMe-RNA oligomer containing the 5'-terminal 2,2,7-trimethylguanosine cap structure.

Stable triplex formation using the strong stacking effect of consecutive thionucleoside moieties.

In this study, it was found that the arrangement of consecutive thiocarbonyl groups of s(2)T and m(5)s(2)C remarkably stabilized the pre-protonated form of the triplex, and that the stabilization of the pre-protonated form increased the pKa value of a cytosine derivative in the triplex.

Biochemical behavior of N-oxidized cytosine and adenine bases in DNA polymerase-mediated primer extension reactions.

To clarify the biochemical behavior of 2'-deoxyribonucleoside 5'-triphosphates and oligodeoxyribonucleotides (ODNs) containing cytosine N-oxide (C(o)) and adenine N-oxide (A(o)), we examined their base recognition ability in DNA duplex formation using melting temperature (T(m)) experiments and their substrate specificity in DNA polymerase-mediated replication. As the result, it was found that the T(m) values of modified DNA-DNA duplexes incorporating 2'-deoxyribonucleoside N-oxide derivatives significantly decreased compared with those of the unmodified duplexes. However, single insertion reactions by DNA polymerases of Klenow fragment (KF) (exo(-)) and Vent (exo(-)) suggested that C(o) and A(o) selectively recognized G and T, respectively. Meanwhile, the kinetic study showed that the incorporation efficiencies of the modified bases were lower than those of natural bases. Ab initio calculations suggest that these modified bases can form the stable base pairs with the original complementary bases. These results indicate that the modified bases usually recognize the original bases as partners for base pairing, except for misrecognition of dATP by the action of KF (exo(-)) toward A(o) on the template, and the primers could be extended on the template DNA. When they misrecognized wrong bases, the chain could not be elongated so that the modified base served as the chain terminator.

Synthesis of oligodeoxynucleotides using fully protected deoxynucleoside 3'-phosphoramidite building blocks and base recognition of oligodeoxynucleotides incorporating N3-cyano-ethylthymine.

Oligodeoxynucleotide (ODN) synthesis, which avoids the formation of side products, is of great importance to biochemistry-based technology development. One side reaction of ODN synthesis is the cyanoethylation of the nucleobases. We suppressed this reaction by synthesizing ODNs using fully protected deoxynucleoside 3'-phosphoramidite building blocks, where the remaining reactive nucleobase residues were completely protected with acyl-, diacyl-, and acyl-oxyethylene-type groups. The detailed analysis of cyanoethylation at the nucleobase site showed that N3-protection of the thymine base efficiently suppressed the Michael addition of acrylonitrile. An ODN incorporating N3-cyanoethylthymine was synthesized using the phosphoramidite method, and primer extension reactions involving this ODN template were examined. As a result, the modified thymine produced has been proven to serve as a chain terminator.

Oligonucleotide synthesis involving deprotection of amidine-type protecting groups for nucleobases under acidic conditions.

Amidine-type protecting groups, i.e., N,N-dimethylformamidine (dmf) and N,N-dibutylformamidine (dbf) groups, introduced into nucleobases were rapidly removed under mild acidic conditions using imidazolium triflate (IMT) or 1-hydroxybenztriazole (HOBt). This new deprotection strategy allowed a 2'-O-methyl-RNA derivative bearing a base-labile group to be efficiently synthesized using a silyl-type linker. It was also found that our new method could be applied to the synthesis of an unmodified RNA oligomer.

Synthesis and properties of terminally modified oligonucleotides capable of short-RNA selective hybridization.

We have developed 2'-O-methyl-RNAs having phosphorylated cyclohexane at the 5' and/or 3'-terminal adenine. These 2'-O-methyl-RNAs formed less stable duplex with the longer target RNA than that with the short target RNA. We tried to improve the short-RNA selective hybridization property by the synthesis of 2'-O-methyl-RNAs having terminal guanine modifications.

Synthesis and triplex formation of oligonucleotides containing 8-thioxodeoxyadenosine and 5-methyl-2-thiodeoxycytosine.

For more effective DNA triplex formation under neutral conditions, we synthesized triplex-forming oligonucleotides (TFO) containing 8-thioxodeoxyadenine (s(8)A) residues in place of the protonated cytosines (Cs) required for the third base pairing with DNA duplexes. Consequently, it was found that s(8)A exhibited much stronger hybridization ability than C under neutral conditions when four s(8)A bases were arranged in a consecutive sequence. Moreover, we also synthesized TFOs containing 5-methyl-2-thiocytosines and examined their ability of triplex formation.

Synthesis and hybridization properties of oligonucleotides having 4-N-(pyrrol-2-ylcarbonyl)deoxycytidine.

A new 4-N-acylated deoxycytidine derivative, 4-N-(1H-pyrrol-2-ylcarbonyl)deoxycytidine, was synthesized and found to be stable under rather basic conditions. Oligodeoxynucleotides (ODNs) incorporating this modified deoxynucleoside at various positions were successfully synthesized by using a pivaloyloxymethyl (POM) group for protection of the pyrrole residue. The POM group was removed by treatment with 1.5 M NaOMe/MeOH. ODNs containing modified deoxycytidines exhibited hybridization properties superior to those of the unmodified ODNs. We found the acylation of the cytosine base with an aromatic acyl-type substituent led to significant increase of the thermo stability of DNA duplexes. This is the first noteworthy observation in this kind of modification. The synthesis and hybridization properties of 4-N-(1H-pyrrol-2-ylcarbonyl)deoxycytidine derivatives will be also reported.