Synthesis and properties of RNA analogs— oligoribonucleotide N3 ′→ P5 ′ phosphoramidates

Author： Matray Tracy J. Gryaznov Sergei M. Testa S.M. Liu Y. Cech T.R. Herschlag D. Burke H.K. Ekland E.H. Freier S.M. Pieken W.A. Paolella G. Boli M. Mei H.-Y. Mei H.-Y. Mei H.-Y. Gryaznov S. Chen J.-K. Gryaznov S. Zielinski W.S. Joyce G.F. Azhayev A.V. Ozols A.M. Schultz R.G. McCurdy S.N. Nelson J.S. Azhayev A.V. Vorbruggen H. Ozols A.M. Zou R. Schulhof J.C. Neilson T. Divacar K.J. Luyten I. Gryaznov S. Santalucia J. Caruthers M.H.

Publisher： Oxford University Press

ISSN： 1362-4962

Source： Nucleic Acids Research, Vol.27, Iss.20, 1999-10, pp. : 3976-3985

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Abstract

The synthesis and characterization of RNA mimetics, uniformly modified oligoribonucleotide N3′→P5′ phosphoramidates containing all four natural bases (uracil, cytosine, adenine and guanine) as well as thymidine and 2,6-diaminopurine, are described. These RNA analogs contain N3′→P5′ phosphoramidate internucleotide linkages which replaced natural RNA O3′→P5′ phosphodiester groups. These oligonucleotides were constructed from novel monomeric units (2′-t-butyldimethylsilyl)-3′-(monomethoxyltrityl)-aminonucleoside-5′-phosphoramidites, the preparation of which is also presented. Several mixed base 9–13mer oligoribonucleotide phosphoramidates were synthesized with step-wise coupling yields of 96–98%. Thermal denaturation experiments demonstrated that ribo-N3′→P5′ phosphoramidates form stable duplexes with a complementary RNA strand. Thus, the melting temperature (Tm) of a duplex formed by a 13mer ribo-N3′→P5′ phosphoramidate (84°C) was higher than that observed for the isosequential natural RNA oligomer (64.0°C), or for the 2′-deoxy-N3′→P5′ phosphoramidate counterpart (71.7°C). Moreover, substitution of adenine by 2,6-diaminopurine in an oligoribophosphoramidate pentamer resulted in a very significant increase in the duplex melting temperature (~7°C per base substitution). The RNA phosphoramidates also showed similar rates of hydrolysis by both RNase A and RNase T1 as compared to natural RNA oligomers. The data presented indicate that this class of RNA analogs may be used as structural and functional RNA mimetics.