RESUMO
The grafting of cationic groups to synthetic oligonucleotides (ONs) in order to reduce the charge repulsion between the negatively charged strands of a duplex or triplex, and consequently to increase a complex's stability, has been extensively studied. Guanidinium groups, which are highly basic and positively charged over a wide pH range, could be an efficient ON modification to enhance their affinity for nucleic acid targets and to improve cellular uptake. A straightforward post-synthesis method to convert amino functions attached to ONs (on sugar, nucleobase or backbone) into guanidinium tethers has been perfected. In comparison to amino groups, such cationic groups anchored to alpha-oligonucleotide phosphoramidate backbones play important roles in duplex stability, particularly with RNA targets. This high affinity could be explained by dual recognition resulting from Watson-Crick or Hoogsteen base pairing combined with cationic/anionic backbone recognition between strands involving H-bond formation and salt bridging. Molecular-dynamics simulations corroborate interactions between the cationic backbones of the alpha-ONs and the anionic backbones of the nucleic acid targets. Moreover, ONs with guanidinium modification increased cellular uptake relative to negatively charged ONs. The cellular localization of these new cationic phosphoramidate ONs is mainly cytoplasmic. The uptake of these ON analogues might occur through endocytosis.
Assuntos
Guanidina/química , Oligonucleotídeos/química , Oligonucleotídeos/farmacocinética , Cátions/síntese química , Cátions/química , Cátions/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , DNA/química , DNA/efeitos dos fármacos , Células HeLa , Humanos , Modelos Moleculares , Estrutura Molecular , Oligonucleotídeos/síntese química , Conformação Proteica , RNA/química , RNA/efeitos dos fármacos , Relação Estrutura-Atividade , TemperaturaRESUMO
The ability of cationic phosphoramidate pyrimidine alpha-oligonucleotides (ONs) to form triplexes with DNA duplexes was investigated by UV melting experiments, circular dichroism spectroscopy and gel mobility shift experiments. Replacement of the phosphodiester linkages in alpha-ONs with positively charged phosphoramidate linkages results in more efficient triplex formation, the triplex stability increasing with the number of positive charges. At a neutral pH and in the absence of magnesium ions, it was found that a fully cationic phosphoramidate alpha-TFO (triplex-forming oligonucleotide) forms a highly stable triplex that melts at a higher temperature than the duplex target. No hysteresis between the annealing and melting curves was noticed; this indicates fast association. Moreover, the recognition of a DNA duplex with a cationic alpha-TFO through Hoogsteen base pairing is highly sequence-specific. To the best of our knowledge, this is the first report of stable triplexes in the pyrimidine motif formed by cationic alpha-oligonucleotides and duplex targets.
Assuntos
Amidas/química , DNA/química , Oligonucleotídeos/química , Ácidos Fosfóricos/química , Pirimidinas/química , Sequência de Bases , Dicroísmo Circular , Concentração de Íons de Hidrogênio , Modelos Químicos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Temperatura , Raios UltravioletaRESUMO
A potential means to improve the efficacy of steric-blocking antisense oligonucleotides (ON) is to increase their affinity for a target RNA. The grafting of cationic amino groups to the backbone of the ON is one way to achieve this, as it reduces the electrostatic repulsion between the ON and its target. We have examined the duplex stabilising effects of introducing cationic phosphoramidate internucleoside linkages into ON with a non-natural alpha-anomeric configuration. Cationic alpha-ON bound with high affinity to single-stranded DNA and RNA targets. Duplex stabilisation was proportional to the number of cationic modifications, with fully cationic ON having particularly high thermal stability. The average stabilisation was greatly increased at low ionic strength. The duplex formed between cationic alpha-ON and their RNA targets were not substrates for RNase H. The penalty in T(m) inflicted by a single mismatch, however, was high; suggesting that they are well suited as sequence-specific, steric-blocking, antisense agents. Using a well-described target sequence in the internal ribosome entry site of the human hepatitis C virus, we have confirmed this potential in a cell-free translation assay as well as in a whole cell assay. Interestingly, no vectorisation was necessary for the cationic alpha-ON in cell culture.
Assuntos
DNA de Cadeia Simples/genética , Hepacivirus/genética , Oligonucleotídeos Antissenso/química , Biossíntese de Proteínas , RNA/genética , Amidas/química , Sítios de Ligação/genética , Cátions/química , Linhagem Celular Tumoral , Reagentes de Ligações Cruzadas/química , Humanos , Luciferases/efeitos dos fármacos , Luciferases/genética , Luciferases/metabolismo , Desnaturação de Ácido Nucleico , Ácidos Nucleicos Heteroduplexes/química , Ácidos Nucleicos Heteroduplexes/genética , Ácidos Nucleicos Heteroduplexes/metabolismo , Hibridização de Ácido Nucleico , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacologia , Ácidos Fosfóricos/química , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ribonuclease H/metabolismo , Ribossomos/metabolismo , TemperaturaRESUMO
A new efficient synthesis of 2-amino-alpha-2'-deoxyadenosine and its incorporation into methoxyethylphosphoramidate alpha-oligodeoxynucleotides (ODNs) via H-phosphonate chemistry were reported. Thermal denaturation experiments demonstrated a significant stabilization of the complexes formed between these analogues and their RNA target (+2 degrees C/NH2A) relative to adenosine-containing phosphoramidate alpha-oligonucleotides. Concerning the binding specificity of these modified ODNs, unlike natural ODNs, discrimination against G pairing is higher and against C pairing is lower.
