2'-O,4'-C-Ethylene-bridged nucleic acid (ENA) is a sugar-modified oligonucleotide with an ethylene bridge between the 2'-oxygen and 4'-carbon of ribose. ENA not only has as high binding affinity to complementary RNA as conventional bridged/locked nucleic acid, but also has much higher nuclease resistance in plasma, which makes it a promising candidate for antisense therapeutics. This unit presents detailed protocols for the synthesis and characterization of ENA nucleosides and oligonucleotides. © 2018 by John Wiley & Sons, Inc.