ABSTRACT
Oligonucleotides containing 2'-O,4'-C-ethylene nucleic acids (ENA) have been proven highly effective for antisense therapeutics. 2'-O,4'-C-Ethyleneguanosine and its phosphoramidite were previously obtained from 3,5-di-O-benzy1-4-C-(p-tolulenesulfonyloxyethyl)-1,2-di-O-acetyl-α-D-erythropentofuranose by glycosylation, but with limited efficiency. Using 3,5-di-O-benzy1-4-C-(2-t-butyldiphenylsilyloxyethyl)-1,2-di-O-acetyl-α-D-erythropentofuranose as an alternative substrate, we developed several methods to obtain 2'-O,4'-C-ethyleneguanosine derivatives with much higher yields than previously reported. These methods were also applicable for the synthesis of 2'-O,4'-C-ethyleneadenosine and 2'-O,4'-C-ethylene-5-methyluridine derivatives. Moreover, we investigated the thermodynamic benefit of DNA strands containing 2'-O,4'-C-ethyleneguanosines during duplex formation with complementary RNA. Only a single modification by the nucleoside resulted in a 10-fold greater binding constant of the DNA/RNA duplex.
Subject(s)
Ethylenes/chemistry , Guanosine/chemistry , Nucleic Acids/metabolism , Glycosylation , Molecular Structure , Organophosphorus Compounds/chemistry , Structure-Activity Relationship , Thermodynamics , Uridine/analogs & derivatives , Uridine/chemistryABSTRACT
PAGE and UV melting analysis revealed that longer LNA-based splice-switching oligonucleotides (SSOs) formed secondary structures by themselves, reducing their effective concentration. To avoid such secondary structure formation, we introduced 7-deaza-2'-deoxyguanosine or 2'-deoxyinosine into the SSOs. These modified SSOs, with fewer secondary structures, showed higher exon skipping activities.
