RESUMO
Phosphorothioate oligodeoxynucleotides containing the C-5 propyne analogs of uridine and cytidine bind RNA with high affinity and are potent antisense inhibitors of gene expression. In a cellular assay, gene-specific antisense inhibition occurred at nanomolar concentrations of oligonucleotide, was dose-dependent and exquisitely sensitive to sequence mismatches, and was correlated with the melting temperature and length of oligonucleotide. Activity was independent of RNA target site and cell type but was detectable only when the oligonucleotides were microinjected or delivered with cell-permeabilizing agents. These oligonucleotides may have important applications in therapy and in studies of gene function.
Assuntos
Oligonucleotídeos Antissenso/farmacologia , Nucleotídeos de Pirimidina/farmacologia , RNA/efeitos dos fármacos , Tionucleotídeos/farmacologia , Alcinos/farmacologia , Animais , Sequência de Bases , Linhagem Celular , Chlorocebus aethiops , Humanos , Dados de Sequência Molecular , Oligonucleotídeos Antissenso/farmacocinética , Nucleotídeos de Pirimidina/farmacocinética , Ratos , Tionucleotídeos/farmacocinéticaRESUMO
Factors that govern the specificity of an antisense oligonucleotide (ON) for its target RNA include accessibility of the targeted RNA to ON binding, stability of ON/RNA complexes in cells, and susceptibility of the ON/RNA complex to RNase H cleavage. ON specificity is generally proposed to be dependent on its length. To date, virtually all previous antisense experiments have used 12-25 nt-long ONs. We explored the antisense activity and specificity of short (7 and 8 nt) ONs modified with C-5 propyne pyrimidines and phosphorothioate internucleotide linkages. Gene-selective, mismatch sensitive, and RNase H-dependent inhibition was observed for a heptanucleotide ON. We demonstrated that the flanking sequences of the target RNA are a major determinant of specificity. The use of shorter ONs as antisense agents has the distinct advantage of simplified synthesis. These results may lead to a general, cost-effective solution to the development of antisense ONs as therapeutic agents.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Oligonucleotídeos Antissenso/farmacologia , RNA/efeitos dos fármacos , Animais , Sequência de Bases , Linhagem Celular , Chlorocebus aethiops , Análise Custo-Benefício , Células HeLa , Humanos , Conformação de Ácido Nucleico , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/metabolismo , RNA/química , RNA/metabolismo , Ribonuclease H/metabolismoRESUMO
Sterically hindered N6-dialkylformamidine protected deoxyadenosine is more stable to acidic depurination than N6-benzoyldeoxyadenosine and is potentially a valuable protecting group in the synthesis of deoxyoligonucleotides.
Assuntos
Oligodesoxirribonucleotídeos/síntese química , Oligonucleotídeos/síntese química , Adenosina/análogos & derivados , Amidinas , Desoxiadenosinas , Formamidas , Indicadores e Reagentes , Cinética , Relação Estrutura-AtividadeRESUMO
Deoxynucleoside H-phosphonates are used in the chemical synthesis of deoxyoligonucleotides up to 107 bases in length. The biological activity of the synthetic DNA is assessed by cloning into M13 and sequencing. An improved synthesis of protected deoxynucleoside H-phosphonates is also described.
Assuntos
DNA/síntese química , Fenômenos Químicos , Química , Desoxirribonucleosídeos , Peso Molecular , OrganofosfonatosRESUMO
The binding of Rev to the Rev-response element (RRE) of the human immunodeficiency virus (HIV) is essential for RNA transport and expression of structural proteins such as gp160 encoded by env. To determine if env expression could be disrupted by complementary oligodeoxynucleotides (ODNs), band-shift studies were used to identify RRE sites that are essential for the formation of Rev-RRE complexes [Chin, D. J. (1992) J. Virol. 66, 600-607] or the stability of preformed complexes. In this report, we describe complete disruption of preformed Rev-RRE complexes by a subset of 15 ODNs complementary to stem-loop V. The most potent ODN complementary to bases CUGGGGCAUCAAGC disrupted 50% of preformed complexes at 1.2 microM, a 400-fold molar excess over the RNA. Expression of env in COS7 cells was blocked by nuclear microinjection of ODNs with C-5 propyne-modified pyrimidines and phosphorothioate linkages. Inhibition was highly dependent upon RNA target position, internucleotide chemistry, ODN sequence, and concentration. Unmodified phosphodiester or phosphorothioate ODNs were inactive. For the most potent ODN, 50% of the injected cells' env expression (I50) was blocked with 0.1 microM. A translational block is unlikely since these ODNs blocked expression of a luciferase vector in which the RRE was placed downstream of the termination codon. Consistent with their in vitro effects upon Rev-RRE complexes, stem-loop V ODNs were 9-fold more active than stem-loop II ODNs in blocking env expression while having a reduced (I50 = 0.27 microM) but equivalent potency against luciferase-RRE. These results suggest that disruption of Rev-RRE complexes may assist in blocking env expression.
Assuntos
Alcinos/farmacologia , Expressão Gênica/efeitos dos fármacos , Genes env , Genes rev , HIV-1/genética , Oligodesoxirribonucleotídeos/farmacologia , Sequência de Bases , Sítios de Ligação , Linhagem Celular , DNA Complementar/farmacologia , DNA Viral/química , DNA Viral/metabolismo , Produtos do Gene env/genética , Produtos do Gene env/metabolismo , Técnicas de Transferência de Genes , Proteína gp160 do Envelope de HIV , Microinjeções , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Ribonuclease H/metabolismo , beta-Galactosidase/genéticaRESUMO
Triple-helix formation by oligodeoxynucleotides in a sequence-specific manner is limited to polypurine tracts of duplex DNA. To increase the number of biologically relevant targets for triple-helix formation, we have utilized oligodeoxynucleotides containing a 3'-3' internucleotide junction to allow for binding to opposite strands of duplex DNA. Molecular modeling was used to aid in the design of the xylose dinucleoside linker 1 that is rigid and minimizes the number of conformers to minimize the entropy of binding. Thermal denaturation studies show that a 3'-3'-linked oligodeoxynucleotide, bearing nine nucleotides on each side of the linker, has a higher Tm (47.6 degrees C) than that of a 21-mer binding to a single polypurine tract (45.3 degrees C). Binding domain minimization studies and sequence-specific alkylation of a target duplex demonstrate a high degree of cooperativity between the two triple-helix binding domains, thus allowing for an increase in the number of biologically relevant targets for triple-helix formation.
Assuntos
Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Composição de Bases , Sequência de Bases , Sítios de Ligação , Temperatura Alta , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Desnaturação de Ácido Nucleico , Oligodesoxirribonucleotídeos/metabolismo , Purinas/metabolismo , Xilose/químicaRESUMO
The synthesis, hybridization properties and antisense activities of oligodeoxynucleotides (ODNs) containing 7-(1-propynyl)-7-deaza-2'-deoxyguanosine (pdG) and 7-(1-propynyl)-7-deaza-2'-deoxyadenosine (pdA) are described. The suitably protected nucleosides were synthesized and incorporated into ODNs. Thermal denaturation (Tm) of these ODNs hybridized to RNA demonstrates an increased stability relative to 7-unsubstituted deazapurine and unmodified ODN controls. Antisense inhibition by these ODNs was determined in a controlled microinjection assay and the results demonstrate that an ODN containing pdG is approximately 6 times more active than the unmodified ODN. 7-Propyne-7-deaza-2'-deoxyguanosine is a promising lead analog for the development of antisense ODNs with increased potency.
Assuntos
DNA Antissenso/química , Desoxiadenosinas/química , Desoxiguanosina/análogos & derivados , Conformação de Ácido Nucleico , Ácidos Nucleicos Heteroduplexes/química , Oligodesoxirribonucleotídeos/química , Sequência de Bases , DNA Antissenso/genética , Desoxiguanosina/química , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/genéticaRESUMO
Triple helix formation with pyrimidine deoxyoligonucleotides for the sequence-specific recognition of DNA duplex targets suffers from a decrease in affinity as the pH of the medium increases to that of physiological fluids. A solution to this problem has been identified and entails the substitution of N6-methyl-8-oxo-2'-deoxyadenosine (M) for the 5-methyl-deoxycytosine base residues. The triple helix forming ability of an oligonucleotide consisting of thymidine and M residues is pH independent in the physiological range. Furthermore, M has been found to be superior to the previously used 5-methyldeoxycytidine and deoxyguanosine in conferring increased affinity for duplex DNA under physiological salt conditions.
Assuntos
Oligodesoxirribonucleotídeos/química , Sequência de Bases , Desoxicitidina/análogos & derivados , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Hibridização de Ácido NucleicoRESUMO
Antisense oligodeoxynucleotides (ODNs) are capable of inhibiting gene expression via a RNase H mechanism in which the complementary RNA is degraded by RNase H. C-5 propyne dU phosphorothioate ODNs bind selectively and with high affinity to RNA within cells leading to potent antisense inhibition of RNA translation. The effect that increasing steric bulk of C-5-substituted deoxyuridine analogs has on affinity for RNA and ability to inhibit gene expression is discussed. The relative binding affinity was measured by thermal denaturation (Tm) analysis, and antisense activity was determined by inhibition of SV40 T-antigen (TAg) expression in CV1 cells. The results show that antisense activity is not directly correlated to Tm measurements. In vitro analysis (RNase H cleavage, on-rates, and off-rates) and pre-formed ODN/RNA experiments indicate that RNase H activity and intracellular dissociation appear to be major determinants of the antisense potency of the various substituted ODNs. The results of our analysis point to the unique ability of C-5 propyne dU ODNs to selectively bind to RNA within cells and activate cleavage of RNA by RNase H leading to potent inhibition of gene expression.
Assuntos
Expressão Gênica/efeitos dos fármacos , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/farmacologia , Animais , Antígenos Transformantes de Poliomavirus/genética , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cinética , Microinjeções , Estrutura Molecular , Oligonucleotídeos Antissenso/síntese química , RNA Complementar/genética , RNA Complementar/metabolismo , Ribonuclease H/metabolismoRESUMO
Antisense gene inhibition occurs when an oligonucleotide (ON) has sufficient binding affinity such that it hybridizes its reverse complementary target RNA and prevents translation either by causing inactivation of the RNA (possibly by RNase H) or by interfering with a cellular process such as stalling a ribosome. The mechanisms underlying these processes were explored. Cellular antisense inhibition was evaluated in a microinjection assay using ON modifications which precluded or allowed in vitro RNase H cleavage of ON/RNA hybrids. RNase H-independent inhibition of protein synthesis could be achieved by targeting either the 5'-untranslated region or the 5'-splice junction of SV40 large T antigen using 2'-O-allyl phosphodiester ONs which contained C-5 propynylpyrimidines (C-5 propyne). Inhibition at both sites was 20-fold less active than inhibition using RNase H-competent C-5 propyne 2'-deoxy phosphorothioate ONs. In vitro analysis of association and dissociation of the two classes of ONs with complementary RNA showed that the C-5 propyne 2'-O-allyl phosphodiester ON bound to RNA as well as the C-5 propyne 2'-deoxy phosphorothioate ON. In vitro translation assays suggested that the two classes of ONs should yield equivalent antisense effects in the absence of RNase H. Next, ON/T antigen RNA hybrids were injected into the nuclei and cytoplasm of cells. Injection of C-5 propyne 2'-O-allyl phosphodiester ON/RNA hybrids resulted in expression of T antigen, implying that the ONs dissociated from the RNA in cells which likely accounted for their low potency. In contrast, when C-5 propyne 2'-deoxy phosphorothioate ON/T antigen RNA complexes were injected into the nucleus, the duplexes were stable enough to completely block T antigen translation, presumably by RNA inactivation. Thus, a dramatic finding is that C-5 propyne 2'-deoxy phosphorothioate ONs, once hybridized to RNA, are completely effective at preventing mRNA translation. The implication is that further increases in complex stability coupled with effective RNase H cleavage will not result in enhanced potency. We predict that the development of more effective ONs will only come from modifications which increase the rate of ON/RNA complex formation within the nucleus.