RESUMO
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.
Assuntos
Citosina/química , Guanina/química , Pareamento de Bases , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Estrutura Molecular , Oligonucleotídeos/síntese química , Oligonucleotídeos/químicaRESUMO
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.
Assuntos
DNA/química , RNA/química , Tiouridina/análogos & derivados , Uridina/síntese química , Conformação de Ácido Nucleico , Tiouridina/química , Uridina/análogos & derivados , Uridina/químicaRESUMO
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.
Assuntos
Oligonucleotídeos/química , Oligonucleotídeos/síntese química , Uridina/química , Simulação de Dinâmica Molecular , Estrutura Molecular , Uridina/análogos & derivadosRESUMO
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.
Assuntos
DNA/química , Guanina/química , Pirenos/química , RNA/química , Sequência de Bases , DNA/metabolismo , Quadruplex G , Conformação de Ácido Nucleico , RNA/metabolismo , Espectrometria de Fluorescência , UltracentrifugaçãoRESUMO
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.
Assuntos
2-Aminopurina/análogos & derivados , 2-Aminopurina/química , Citosina/análogos & derivados , Citosina/química , Indóis/química , Oligonucleotídeos/química , Coloração e Rotulagem/métodos , Sequência de Bases , Corantes Fluorescentes , Imagem Molecular , Dados de Sequência Molecular , Espectrometria de FluorescênciaRESUMO
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.
Assuntos
Adenina/análogos & derivados , Óxidos N-Cíclicos/química , Citosina/análogos & derivados , DNA Polimerase Dirigida por DNA/metabolismo , DNA/química , Adenina/química , Adenina/metabolismo , Pareamento de Bases , Óxidos N-Cíclicos/metabolismo , Citosina/química , Citosina/metabolismo , DNA Polimerase I/metabolismo , Primers do DNA , Desoxirribonucleotídeos/síntese química , Desoxirribonucleotídeos/metabolismo , Desnaturação de Ácido Nucleico , Oligodesoxirribonucleotídeos/síntese química , Oligodesoxirribonucleotídeos/química , Especificidade por Substrato , Temperatura , Moldes GenéticosRESUMO
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).
Assuntos
Brometos/farmacologia , Compostos de Lítio/farmacologia , Morfolinos/química , Morfolinos/síntese química , Sais/química , Cromatografia Líquida de Alta Pressão , Estrutura MolecularRESUMO
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.
Assuntos
Oligonucleotídeos/síntese química , RNA/química , Uridina/análogos & derivados , Uridina/síntese química , Animais , Pareamento de Bases , Cátions , Cromatografia de Fase Reversa , Estabilidade de Medicamentos , Humanos , Conformação de Ácido Nucleico , Diester Fosfórico Hidrolases/metabolismo , Interferência de RNA , Venenos de SerpentesRESUMO
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.
Assuntos
DNA/química , Nucleosídeos/química , Oligodesoxirribonucleotídeos/química , Sequência de Bases , Modelos Moleculares , Conformação de Ácido Nucleico , Compostos Organofosforados/químicaRESUMO
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.
Assuntos
Adenosina/metabolismo , Guanosina/metabolismo , MicroRNAs/química , MicroRNAs/metabolismo , Oligonucleotídeos/metabolismo , Adenosina/química , Sequência de Bases , Sítios de Ligação , Guanosina/química , Oligonucleotídeos/química , Fosfatos/química , Fosfatos/metabolismoRESUMO
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.
Assuntos
Citidina/análogos & derivados , Sondas de DNA/química , Inosina/análogos & derivados , RNA/química , Pareamento de Bases , Sítios de Ligação , Citidina/química , Hibridização de Ácido NucleicoRESUMO
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.
Assuntos
Oligodesoxirribonucleotídeos/síntese química , Compostos Organofosforados/química , Cromatografia Líquida de Alta Pressão , Estrutura Molecular , Oligodesoxirribonucleotídeos/químicaRESUMO
New 3'-terminal deoxyribonucleoside-loading reagents having a silyl-type linker were developed. They were effectively introduced into polymer supports under the conditions of Huisgen [3 + 2] cycloaddition without base protection. Moreover, four unmodified DNA oligomers d[TACCTAAATCCAX] (X = T, A, C, and A) and a base-labile modified DNA 12mer d[A*C*T*C*C*GT*C*T*A*C*G] 16 (A* = 6-N-acetyl-8-aza-7-deaza-2'-deoxyadenosine, C* = 4-N-acetyl-2'-deoxyctydine, T* = 2-thio-T) were successfully synthesized by cleavage of the silyl-type linker using Bu(4)NF under neutral conditions in our N-unprotected phosphoramidite method. In this paper, we also report a new reaction of chlorination of cytosine base using 1,3-dichloro-5,5-dimethylhydantoin.
Assuntos
Nucleosídeos/química , Polímeros/química , Catálise , Compostos Clorados/química , Cromatografia Líquida de Alta Pressão , Cobre/química , DNA/química , Estrutura MolecularRESUMO
It was found that N-arylcarbamoyl and N-(phenylsulfonyl)carbamoyl (psc) groups could be effectively introduced onto the amino groups of deoxycytidine and deoxyadenosine derivatives and could be removed thermolytically. We succeeded in synthesizing DNA probes incorporating these thermo-removable protecting groups and developed a new system for molecular switching by changing the protection- and deprotection-modes using simple heating and re-carbamoylation with isocyanates. This reversible process enabled us to control the hybridization ability of the DNA probes.
Assuntos
Sondas de DNA/síntese química , Precursores de Ácido Nucleico/síntese química , Carbamatos/química , Desoxiadenosinas/química , Desoxicitidina/química , Temperatura AltaRESUMO
A 5'-terminal 2,2,7-trimethylguanosine (TMG) cap structure plays an important role in transport of TMG-capped snRNAs from the cytoplasm to the nucleus in living cells. This property can be expected to be used for carrying artificial nucleic acids into the nucleus in gene therapy. In this paper, a new TMG-capping reagent 1 was developed and used for the synthesis of oligodeoxynucleotides having a 5'-terminal TMG-capped pyrophosphate linkage (m(3)(2,2,7)G(5')pp-DNA) lacking one phosphate group using a silyl-type linker on polymer supports.
Assuntos
DNA/química , Guanosina/análogos & derivados , Oligodesoxirribonucleotídeos/química , Sequência de Bases , DNA/síntese química , Técnicas de Transferência de Genes , Guanosina/síntese química , Guanosina/química , Estrutura Molecular , Oligodesoxirribonucleotídeos/síntese químicaRESUMO
In RNA synthesis without base protection, a new method for O-selective condensation with more than 99% selectivity was developed by 6-nitro-HOBt-mediated cleavage of undesired P(III)-N bonds on nucleobase moieties. Moreover, we for the first time succeeded in synthesizing oligoRNAs without base protection.
Assuntos
Nitrogênio/química , Fósforo/química , RNA/síntese química , Cromatografia Líquida de Alta Pressão , Dimerização , Estrutura Molecular , Fosfitos/química , RNA/químicaRESUMO
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.
Assuntos
Aptâmeros de Nucleotídeos/síntese química , RNA Polimerases Dirigidas por DNA/metabolismo , Transcrição Reversa , Uridina Trifosfato/química , Proteínas Virais/metabolismo , HumanosRESUMO
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.
Assuntos
Oligorribonucleotídeos/química , RNA/química , Tiouridina/análogos & derivados , Conformação de Ácido Nucleico , Estabilidade de RNA , Tiouridina/química , Temperatura de TransiçãoRESUMO
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.
Assuntos
Oligonucleotídeos/síntese química , Compostos Organofosforados/química , Estrutura Molecular , FosforilaçãoRESUMO
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.