RESUMO
High scalability of a novel bicyclic nucleoside building block, amido-bridged nucleic acid (AmNA), to diversify pharmacokinetic properties of therapeutic antisense oligonucleotides is described. N2'-functionalization of AmNA with a variety of hydrophobic groups is straightforward. Combinations of these modules display similar antisense knockdown effects and improve cellular uptake, relative to sequence-matched conventional 2',4'-bridged nucleic acid (2',4'-BNA) in vivo.
Assuntos
Aminas/química , Sistemas de Liberação de Medicamentos , Interações Hidrofóbicas e Hidrofílicas , Fígado/metabolismo , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/farmacocinética , Animais , Apolipoproteína C-III/genética , Apolipoproteína C-III/metabolismo , Ensaio de Imunoadsorção Enzimática , Camundongos , Oligonucleotídeos Antissenso/administração & dosagem , Compostos Organofosforados/síntese química , Compostos Organofosforados/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Chemically modified DNA aptamers specific to human α-thrombin were obtained from oligodeoxyribonucleotide (ODN) libraries by using a capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX) method. These libraries contained 2'-O,4'-C-methylene-bridged/linked bicyclic ribonucleotides (B/L nucleotides) in the primer region and/or C5-modified thymidine bearing N(6)-ethyladenine (t) in the nonprimer region. Modified DNA aptamers showed high binding affinities to the target, with dissociation constants (Kd) values in the range of subnanomolar to several ten nanomolar levels. The introduction of base modification significantly suppressed the frequency of G-quadruplex motifs, which are often seen in thrombin-binding DNA aptamers. The resulting alternatives contained the 10-mer consensus sequence t5Gt2G2, which is frequently found in modified DNA aptamers with subnanomolar protein binding affinities. Furthermore, some base- and sugar-modified DNA aptamers with the 12-mer consensus sequence t2G2tC(A/G)A2G2t displayed binding activities that were dependent on the presence of B/L nucleotides in the primer region. Such aptamers were interestingly not recovered from a natural DNA library or from DNA libraries modified with either B/L nucleotides or t's. This emerging characteristic binding property will enable the creation of a direct selection methodology for DNA-based molecular switches that are triggered by chemical conversion of B/L nucleotides introduced to constant sequence regions in ODN libraries.
Assuntos
Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Primers do DNA/metabolismo , Eletroforese Capilar/métodos , Oligonucleotídeos/metabolismo , Técnica de Seleção de Aptâmeros/métodos , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Primers do DNA/química , Primers do DNA/genética , Quadruplex G , Humanos , Dados de Sequência Molecular , Oligonucleotídeos/química , Oligonucleotídeos/genética , Análise de Sequência de DNA , Especificidade por SubstratoRESUMO
Artificial thymidine monomers possessing amide or N-methylamide bridges were designed, synthesized, and introduced into oligonucleotides. UV-melting experiments showed that these oligonucleotides preferred single-stranded RNA (ssRNA) to single-stranded DNA (ssDNA) in duplex formation. Both amide- and N-methylamide-modified oligonucleotides led to a significant increase in the binding affinity to ssRNA by up to +4.7 and +3.7°C of the Tm value per modification, respectively, compared with natural oligonucleotide. In addition, their oligonucleotides showed high stability against 3'-exonuclease.
Assuntos
Amidas/química , DNA/química , Desenho de Fármacos , RNA/química , Timidina/síntese química , Estrutura Molecular , Timidina/químicaAssuntos
Amidas/química , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/metabolismo , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Apolipoproteínas B/genética , Sequência de Bases , Linhagem Celular , Técnicas de Química Sintética , Humanos , Oligonucleotídeos/síntese química , Oligonucleotídeos/genética , Oligonucleotídeos Antissenso/síntese química , Oligonucleotídeos Antissenso/genética , Temperatura de TransiçãoRESUMO
A novel derivative of 2',4'-bridged nucleic acid, named hydroxamate-bridged nucleic acid (HxNA), containing a six-membered perhydro-1,2-oxazin-3-one ring, was designed and synthesized. The introduction of a carbonyl function along with an N-O linkage in the six-membered bridged structure is the unique structural feature of the novel 2',4'-bridged nucleic acid analogue. The design was carried out to restrict the flexibility of the sugar moiety through the trigonal planarity of carbonyl function, which would improve the properties of the modification. The synthesized monomer was incorporated into oligonucleotides, and their properties were examined. The HxNA-modified oligonucleotides exhibited selectively high affinity toward complementary ssRNA. Furthermore, the nuclease resistance of the HxNA-modified oligonucleotide was found to be higher than that of the corresponding natural and 2',4'-BNA/LNA-modified oligonucleotides. Interestingly, exposure of HxNA modified oligonucleotide to 3'-exonuclease resulted in gradual opening of the bridge, which stopped further digestion. Moreover, ring-opening of only one modification at the 3'-end of the oligonucleotides was observed, even if two or three HxNA modifications were present in the sequence. The results demonstrate the strong potential of the HxNA modification as a switch for the generation of highly nuclease-resistant RNA selective oligonucleotide in situ, which could have potential applications in antisense technology.
Assuntos
Hidrocarbonetos Aromáticos com Pontes/síntese química , Oligonucleotídeos Antissenso/síntese química , Sequência de Bases , Cromatografia de Fase Reversa , DNA/química , Exonucleases/química , Humanos , Ácidos Hidroxâmicos/química , Anotação de Sequência Molecular , Conformação de Ácido Nucleico , Oxazinas/química , RNA/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Especificidade por SubstratoRESUMO
A novel bridged nucleic acid bearing cyclic urea structure was successfully synthesized and introduced into oligonucleotide, displaying attractive characteristics of highly RNA selective hybridization ability and excellent resistance towards nuclease degradation.
Assuntos
Crotalus/metabolismo , Ácidos Nucleicos/química , Oligonucleotídeos/síntese química , Diester Fosfórico Hidrolases/metabolismo , RNA/química , Ureia/química , Animais , Ciclização , Estrutura Molecular , Hibridização de Ácido NucleicoRESUMO
Three kinds of novel bridged nucleic acid (BNA) monomers bearing carbamate or urea bridged structures, were designed and synthesized. One of these monomers, a urea-type BNA monomer was successfully incorporated into oligodeoxynucleotides (ODNs), and properties of the ODNs were evaluated. The ODNs containing the urea-type BNA monomers showed RNA selective hybridizing profiles and significant enzymatic stability.