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.