RESUMEN
A cyclic nucleoside has been designed and synthesized to serve as a conformationally fixed building block for the development of functional oligonucleotides. The bridge was introduced between the nucleobase and the 5'-position to fix the rotation around the C4'-C5' bond, the base orientation, and the sugar puckering all at once. The 13-membered cyclic structure was introduced using a sulfonamide linkage, which retains an N-H group that can be used to attach an additional nucleoside moiety. The sulfonamide linkage was formed through the end-to-end cyclization of an intermediate that contained both a sulfonyltriazole and amino groups. Both 1H NMR and computational studies revealed that the sugar conformation, base orientation, and γ torsion angle were S-type, anti, and trans, respectively. As such, cyclic nucleosides show promise for introducing these specific distorted conformations into functional nucleic acids.
Asunto(s)
Ácidos Nucleicos , Nucleósidos , Nucleósidos/química , Conformación Molecular , Oligonucleótidos/química , AzúcaresRESUMEN
Antisense oligonucleotides (ASOs) are therapeutic modalities that are successfully used as pharmaceuticals. However, there remains a concern that treatment with ASOs may cleave mismatched RNAs other than the target gene, leading to numerous alterations in gene expression. Therefore, improving the selectivity of ASOs is of paramount importance. Our group has focused on the fact that guanine forms stable mismatched base pairs and has developed guanine derivatives with modifications at the 2-amino group, which potentially change the mismatch recognition ability of guanine and the interaction between ASO and RNase H. In this study, we evaluated the properties of ASOs containing two guanine derivatives, 2-N-carbamoyl-guanine and 2-N-(2-pyridyl)guanine. We conducted ultraviolet (UV) melting experiments, RNase H cleavage assays, in vitro knockdown assays, and off-target transcriptome analyses using DNA microarrays. Our results indicate that the target cleavage pattern of RNase H was altered by the modification with guanine. Furthermore, global transcript alteration was suppressed in ASO incorporating 2-N-(2-pyridyl)guanine, despite a decrease in the thermal mismatch discrimination ability. These findings suggest that chemical modifications of the guanine 2-amino group have the potential to suppress hybridization-dependent off-target effects and improve ASO selectivity.
Asunto(s)
Oligonucleótidos Antisentido , ARN , Oligonucleótidos Antisentido/química , ARN/metabolismo , Emparejamiento Base , Hibridación de Ácido Nucleico , Ribonucleasa H/metabolismoRESUMEN
RNase H acts as a key effector in gene knockdown by antisense oligonucleotides (ASOs). Although various chemical modifications have been developed to regulate RNase H-mediated cleavage, precise control is yet to be achieved. In this study, we tried to address the question of whether the interaction of phosphate groups or deoxyriboses is more important in the recognition of DNA/RNA duplex by RNase H. To answer this question, we investigated the effect of methylene group insertion at the 5'-upstream or 3'-downstream phosphorothioate groups on RNase H-mediated cleavage. By inserting a methylene group at the 5'-upside or 3'-downside, the distance between phosphates or deoxyriboses could be changed in a different pattern. Maximum suppression of the cleavage reaction was observed when a methylene group was inserted at the 5'-phosphate group of the nucleoside which is known to distinguish ribose and deoxyribose via stacking of the W221 residue in RNase H. This effect was observed in a different sequence as well as mismatched duplexes, suggesting the interaction of deoxyribose rings with RNase H is more important than that of phosphate groups. Our results will contribute to the designing of further molecular modifications that improve the selectivity of RNase H-mediated cleavage reactions which allows for the development of allele-specific ASOs.
Asunto(s)
Oligonucleótidos Antisentido , Ribonucleasa H , Ribonucleasa H/química , Oligonucleótidos Antisentido/química , Desoxirribosa , Oligonucleótidos/química , FosfatosRESUMEN
A synthetic scheme was developed to derive a modified ribothymidine bearing a 3-(N-methylsulfamoyl)propyl group on 2'-oxygen (TMSP). For synthesis initiation, a nucleophilic attack of 1,2-ethanediol on 5'-protected 2,2'-anhydro-ribothymidine was performed to selectively modify the 2'-position. After protection of the 3'-hydroxy group, the hydroxyethyl group was oxidized to the aldehyde, which was coupled with isobutyl (diethoxyphosphinyl)methanesulfonate through the Horner-Wadsworth-Emmons reaction to yield the sulfonate intermediate. The intermediate was further converted to the desired TMSP. Using the phosphoramidite units derived from nucleosides, we synthesized oligonucleotides incorporating TMSP. Oligonucleotides modified with TMSP were found to have duplex stability, resistance toward 3'-exonuclease digestion, and antisense activity comparable to that of the oligonucleotide modified with a previously reported 2'-O-methylcarbamoylethyl group. Based on these results and the generality of the synthetic scheme, 2'-O-sulfamoylalkyl modification is expected to be used for the modulation of the properties of oligonucleotides by changing the substituents on the nitrogen, enabling the oligonucleotides to possess suitable properties for antisense oligonucleotides.
Asunto(s)
Nucleósidos , Oligonucleótidos Antisentido , Aldehídos , Glicol de Etileno , Mesilatos , Nitrógeno , Oligonucleótidos , Oxígeno , Uridina/análogos & derivadosRESUMEN
Substitutions, insertions, and deletions derived from synthetic oligonucleotides are the hurdles for the synthesis of long DNA such as genomes. We quantified these synthetic errors by next-generation sequencing and revealed that the quality of the enzymatically amplified final combined product depends on the conditions of the preceding solid phase chemical synthesis, which generates the initial pre-amplified fragments. Among all possible substitutions, the G-to-A substitution was the most prominently observed substitution followed by G-to-T, C-to-T, T-to-C, and A-to-G substitutions. The observed error rate for G-to-A substitution was influenced by capping conditions, suggesting that the capping step played a major role in the generation of G-to-A substitution. Because substitutions observed in long DNA were derived from the generation of non-canonical nucleosides during chemical synthesis, non-canonical nucleosides resistant to side reactions could be used as error-proof nucleosides. As an example of such error-proof nucleosides, we evaluated 7-deaza-2´-deoxyguanosine and 8-aza-7-deaza-2´-deoxyguanosine and showed 50-fold decrease in the error rate of G-to-A substitution when phenoxyacetic anhydride was used as capping reagents. This result is the first example that improves the quality of synthesized sequences by using non-canonical nucleosides as error-proof nucleosides. Our results would contribute to the development of highly accurate template DNA synthesis technologies.
Asunto(s)
ADN , Nucleósidos , Oligonucleótidos/genética , Técnicas de Síntesis en Fase SólidaRESUMEN
Unnatural base pairs (UBPs) which exhibit a selectivity against pairing with canonical nucleobases provide a powerful tool for the development of nucleic acid-based technologies. As an alternative strategy to the conventional UBP designs, which involve utility of different recognition modes at the Watson-Crick interface, we now report that the exclusive base pairing can be achieved through the spatial separation of recognition units. The design concept was demonstrated with the alkynylated purine (NPu, OPu) and pyridazine (NPz, OPz) nucleosides endowed with nucleobase-like 2-aminopyrimidine or 2-pyridone ('pseudo-nucleobases') on their major groove side. These alkynylated purines and pyridazines exhibited exclusive and stable pairing properties by the formation of complementary hydrogen bonds between the pseudo-nucleobases in the DNA major groove as revealed by comprehensive Tm measurements, 2D-NMR analyses, and MD simulations. Moreover, the alkynylated purine-pyridazine pairs enabled dramatic stabilization of the DNA duplex upon consecutive incorporation while maintaining a high sequence-specificity. The present study showcases the separation of the recognition interface as a promising strategy for developing new types of UBPs.
Asunto(s)
Ácidos Nucleicos , Nucleósidos , Emparejamiento Base , ADN/química , Enlace de Hidrógeno , Conformación de Ácido Nucleico , Ácidos Nucleicos/química , Nucleósidos/químicaRESUMEN
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.
Asunto(s)
Oligodesoxirribonucleótidos , Péptidos , Oligonucleótidos/química , Péptidos/químicaRESUMEN
To expand the variety of 2'-O-modified oligonucleotides, we synthesized 2'-O-carbamoylethyl-modified oligonucleotides bearing ethyl, n-propyl, n-butyl, n-pentyl, and n-octyl groups on their nitrogen atoms. The corresponding nucleosides were synthesized using 2'-O-benzyloxycarbonylethylthymidine, which was easily converted into the carboxylic acid through hydrogeneration; subsequent condensation with the appropriate amine gave the desired nucleoside. We evaluated the effect of the 2'-O-alkylcarbamoylethyl modifications on duplex stability by analyzing melting temperature, which revealed the formation of isostable duplexes. In addition, we also revealed that these modifications, especially octylcarbamoylethyl, endowed these oligonucleotides with resistance toward a 3'-exonuclease. These results highlight the usefulness of the 2'-O-alkylcarbamoylethyl modification for various biological applications.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Exonucleasas/antagonistas & inhibidores , Oligonucleótidos/farmacología , ARN Complementario/antagonistas & inhibidores , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Exonucleasas/metabolismo , Conformación de Ácido Nucleico , Oligonucleótidos/síntesis química , Oligonucleótidos/química , ARN Complementario/metabolismo , Temperatura de TransiciónRESUMEN
Duchenne muscular dystrophy (DMD) is a fatal X-linked disorder caused by nonsense or frameshift mutations in the DMD gene. Among various treatments available for DMD, antisense oligonucleotides (ASOs) mediated exon skipping is a promising therapeutic approach. For successful treatments, however, it is requisite to rigorously optimise oligonucleotide chemistries as well as chemical modifications of ASOs. To achieve this, here, we aim to develop a novel enhanced green fluorescence protein (EGFP)-based reporter assay system that allows us to perform efficient and high-throughput screenings for ASOs. We design a new expression vector with a CAG promoter to detect the EGFP fluorescence only when skipping of mdx-type exon 23 is induced by ASOs. Then, an accurate screening was successfully conducted in C57BL/6 primary myotubes using phosphorodiamidate morpholino oligomer or locked nucleic acids (LNA)/2'-OMe mixmers with different extent of LNA inclusion. We accordingly generated a novel transgenic mouse model with this EGFP expression vector (EGFP-mdx23 Tg). Finally, we confirmed that the EGFP-mdx23 Tg provided a highly sensitive platform to check the effectiveness as well as the biodistribution of ASOs for exon skipping therapy. Thus, the assay system provides a simple yet highly sensitive platform to optimise oligonucleotide chemistries as well as chemical modifications of ASOs.
Asunto(s)
Exones/genética , Terapia Genética/métodos , Empalme del ARN/fisiología , Animales , Modelos Animales de Enfermedad , Distrofina/genética , Exones/fisiología , Femenino , Genes Reporteros/genética , Proteínas Fluorescentes Verdes , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Morfolinos/genética , Fibras Musculares Esqueléticas/metabolismo , Distrofia Muscular de Duchenne/genética , Oligonucleótidos/genética , Oligonucleótidos Antisentido/metabolismo , Cultivo Primario de Células , Empalme del ARN/genéticaRESUMEN
We developed new DNA triplexes that contain four base triads T-A·T, A-ψ·CBr, G-PIC·YO, and C-G·Py+, where CBr, YO, Py, ψ, and PIC are 5-bromocytosine, 5-methyl-4-pyrimidone, 2-aminopyridine, the aglycons of deoxypseudouridine, and deoxypseudoisocytidine, respectively. DNA duplex incorporating T-A, A-ψ, G-PIC, and C-G, and triplex forming oligonucleotide incorporating T, CBr, YO, and Py formed the triplex as evaluated by Tm measurements. The triplex formation was successfully applied to the inhibition of transcription of the DNA duplex incorporating T7-promoter sequence modified by the above modified bases.
Asunto(s)
ADN/química , Oligonucleótidos/química , Aminopiridinas/química , Secuencia de Bases , Citosina/análogos & derivados , Citosina/química , Enlace de Hidrógeno , Modelos Moleculares , Conformación de Ácido Nucleico , Transición de Fase , Regiones Promotoras Genéticas , Temperatura de TransiciónRESUMEN
Enzymatic incorporation of deoxynucleoside 5'-triphosphate bearing the photocleavable protecting group is a useful method for the preparation of photocaged oligodeoxynucleotides. Here, we describe the synthesis of new photocaged deoxynucleoside triphosphates N1-(2-nitrobenzyl)-deoxypseudouridine triphosphate (dNBΨTP) and N1-(6-nitropiperonyloxymethyl)-deoxypseudouridine triphosphate (dNPOMΨTP). We successfully synthesized dNBΨTP and dNPOMΨTP and applied them to enzymatic synthesis of photocaged oligonucleotides. In addition, we also synthesized phosphoramidites of N1-(2-nitrobenzyl)- and N1-(6-nitropiperonyloxymethyl)-deoxypseudouridine to enable chemical synthesis of photocaged oligonucleotides incorporating them. The photocleavable 2-nitrobenzyl and 6-nitropiperonyloxymethyl in oligonucleotides were cleaved by irradiation at 365 nm for 30 and 10 s, respectively. We also studied the enzymatic incorporation of dNBΨTP and dNPOMΨTP using the Klenow fragment exo-. As a result, it was clarified that dNPOMΨTP could be incorporated to oligonucleotide 193 times more efficiently than dNBΨTP, as judged by Vmax/Km. We also performed the incorporation of at least eight dNPOMΨ residues in a 35-mer oligodeoxynucleotide. It has also been revealed that the oligodeoxynucleotides incorporating photocaged deoxypseudouridine were useful for photocontrol of DNA triplex formation.
Asunto(s)
ADN , Oligonucleótidos , Replicación del ADN , PolifosfatosRESUMEN
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.
Asunto(s)
Oligonucleótidos/química , Ribonucleasas/química , Animales , Ratones , Conformación de Ácido Nucleico , Oligonucleótidos/síntesis química , Ribonucleasas/metabolismoRESUMEN
Fluorescence turn-on sensors for adenosine were developed using DNA triplexes modified with a fluorescent molecular rotor 5-(3-methylbenzofuran-2-yl)deoxyuridine (dUMBF) and abasic sites. Binding of adenosine to the abasic site next to the dUMBF changed the microenvironment and conformation (from the twisted to planar state) of dUMBF and enhanced the fluorescence. Adenosine could be selectively detected over other nucleosides and adenosine phosphates. The binding of adenosine was confirmed by UV-thermal melting experiments. Further, the conformational changes of dUMBF from the twisted to coplanar state upon binding of adenosine was supported by MD simulations.
Asunto(s)
Adenosina/análisis , Benzofuranos/química , ADN/química , Desoxiuridina/análogos & derivados , Colorantes Fluorescentes/química , Secuencia de Bases , Técnicas Biosensibles/métodos , Metilación , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Espectrometría de Fluorescencia/métodosRESUMEN
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.
RESUMEN
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.
Asunto(s)
Oligonucleótidos/química , Tiouridina/química , Estructura Molecular , Tiouridina/agonistas , Tiouridina/síntesis químicaRESUMEN
Herein, we determined the crystal structure of a DNA duplex containing consecutive 6-thioguanine-6-thioguanine disulfides. The disulfide bonds were reversibly formed and cleaved in the presence of Cu(ii) ions and glutathione. To our knowledge, this is the first reaction in which metal ions efficiently accelerated disulfide bond formation between thio-bases in duplexes.
RESUMEN
An RNase H-dependent antisense oligonucleotide (ASO), having the 2'-O-(2-N-methylcarbamoylethyl) (MCE) modification, was evaluated in vitro and in vivo. The antisense activities of an ASO having the MCE modification were comparable with those of an ASO having the 2'-O-methoxyethyl (MOE) modification in both in vitro and in vivo experiments. In contrast, the hepatotoxic potential of the ASO having the MCE modification was lower than that of the ASO having the MOE modification. Thus, these findings suggested that the MCE modification could be used as an alternative to the MOE modification.
Asunto(s)
Oligodesoxirribonucleótidos/administración & dosificación , Oligonucleótidos Antisentido/administración & dosificación , ARN Mensajero/administración & dosificación , Ribonucleasa H/genética , Animales , Humanos , Hígado/efectos de los fármacos , Ratones , Nucleótidos/efectos adversos , Nucleótidos/química , Nucleótidos/genética , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/genética , Oligonucleótidos Antisentido/efectos adversos , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/genética , ARN Mensajero/química , ARN Mensajero/genética , Ribonucleasa H/administración & dosificación , Ribonucleasa H/químicaRESUMEN
Deoxynucleoside 5'-triphosphate was synthesized with 3-oxo-2 H-pyridazin-6-yl (PzO)-a uracil analogue lacking a 2-keto group-as the nucleobase. Theoretical analyses and hybridization experiments indicated that PzO recognizes adenine (A) for formation of a Watson-Crick base pair. Primer extension reactions using nucleoside 5'-triphosphate and the Klenow fragment revealed that the synthetic nucleoside 5'-triphosphate was incorporated into the 3' end of the primer through recognition of A in the template strand. Moreover, the 3'-nucleotide residue harboring PzO as the base was resistant to the 3'-exonuclease activity of Klenow fragment exo+. The primer bearing the PzO base at the 3' end could function in subsequent chain elongation. These properties of PzO were attributed to the presence of an endocyclic nitrogen atom at the position ortho to the glycosidic bond, which was presumed to form an H-bond with the amino acid residue of DNA polymerase for effective recognition of the 3' end of the primer for primer extension. These results provide a basis for designing new nucleobases by combining a nitrogen atom at the position ortho to the glycosidic bond and base-pairing sites for Watson-Crick hydrogen bonding.
Asunto(s)
Cartilla de ADN/genética , Piridazinas/química , Nucleótidos de Timina/química , Emparejamiento Base , Cartilla de ADN/metabolismo , Electrones , Enlace de Hidrógeno , Modelos Moleculares , Electricidad Estática , Nucleótidos de Timina/metabolismoRESUMEN
This study aimed to synthesize triplex-forming oligonucleotides (TFOs) containing 2'-deoxy-6-thioxanthosine (s6X) and 2'-deoxy-6-thioguanosine (s6Gs) residues and examined their triplex-forming ability. Consecutive arrangement of s6X and s6Gs residues increased the triplex-forming ability of the oligonucleotides more than 50 times, compared with the unmodified TFOs. Moreover, the stability of triplex containing a mismatched pair was much lower than that of the full-matched triplex, though s6X could form a s6X-GC mismatched pair via tautomerization of s6X. The present results reveal excellent properties of modified TFOs containing s6Xs and s6Gs residues, which may be harnessed in gene therapy and DNA nanotechnology.
Asunto(s)
ADN/síntesis química , Oligonucleótidos/química , Ribonucleósidos/química , Emparejamiento Base , Secuencia de Bases , ADN/química , Desoxiguanosina/análogos & derivados , Desoxiguanosina/química , Oligonucleótidos/síntesis química , Tionucleósidos/química , XantinasRESUMEN
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 60s by UV irradiation at 365nm. Experiments using NBGTP or NBUTP in T7-RNA transcription reactions showed that NBGTP could be useful for the photocontrol of transcription by UV irradiation.