RESUMO
High-quality oligonucleotides are obtained by selective modification of sequences containing aldehyde apurinic sites with a new chromatographic tag followed by RP-HPLC separation. Hydroxylamine derivative 1 of a water soluble nonionic surfactant modifies oligonucleotides selectively at abasic sites leading to significantly increased retention.
Assuntos
Aldeídos/química , Oligonucleotídeos/química , Oligonucleotídeos/isolamento & purificação , Purinas/química , Tensoativos/química , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Hidroxilamina/química , Deleção de Sequência/fisiologia , Espectrometria de Massas por Ionização por Electrospray , Água/químicaRESUMO
It is demonstrated that a solution of dichloroacetic acid in toluene removes dimethoxytrityl groups from the 5'-terminus of an antisense phosphorothioate oligodeoxyribonucleotide (ISIS 5132/CGP69846A) during synthesis on solid support cleanly and efficiently. It is therefore suggested to replace health hazardous dichloromethane which is typically used in oligonucleotide synthesis as solvent for DMTr-removal by toluene.
Assuntos
Oligonucleotídeos Antissenso/síntese química , Proteínas Proto-Oncogênicas c-raf/genética , Sequência de Bases , Cloro/química , Cromatografia Líquida de Alta Pressão , Oligonucleotídeos Antissenso/isolamento & purificação , Solventes/químicaRESUMO
'Retro-inverso' peptide nucleic acid (PNA) monomers of thymine (T*: N-(amidomethyl)-N-(N1-thyminyl-acetyl)-beta-alanyl) (and adenine) have been prepared and introduced in PNA oligomers. A homo 'retro-inverso' T*8 PNA was found not to hybridize to a complementary DNA or RNA oligonucleotide, whereas introduction of one retro-inverso thymine unit into the middle of a normal PNA 15-mer resulted in a c.a. 8 degrees C destabilization of the complex of this oligomer with a complementary DNA or RNA oligomer. In an effort to compensate for the structural nucleobase 'phase-shift' caused by the T* monomer by also introducing a beta-alanine monomer it is concluded that the effect of the T* backbone is -7 degrees C when hybridizing to DNA and -4.5 degrees C when hybridizing to RNA. Nonetheless, the T* unit shows good sequence discrimination comparable to that of normal PNA. Molecular dynamics simulations indicate an unfavourable conformation of the backbone amide carbonyl group resulting in reduced interaction with the aqueous medium and an 'electrostatic clash' with the carbonyl of the nucleobase linker. These results show that a simple inversion of an amide bond in the PNA backbone has a dramatic, and hardly predictable, effect on the DNA mimicking properties of the oligomer.
