Abstract: Chemical modification is critical for the therapeutic applications of antisense oligonucleotides. Novel 4'-C-MOE and 2'-fluoro-modified monomer 2'-F-4'-C-MOE-araU and its epimeric 2'-F-4'-C-MOE-rU were synthesized from 2'-fluorinated arabinourine (2'-F-araU) and 2'-fluorouridine (2'-F-rU), respectively. Their phosphoramidites were synthesized and successfully incorporated into oligodeoxynucleotides. The mismatch discrimination ability of these unnatural monomers and their effect on thermal stability were evaluated in the context of dsDNA and DNA-RNA chimeras. The thermal denaturation studies showed that the incorporation of 2'-F-4'-C-MOE-araU led to enhanced binding affinity to complementary RNA strand and almost equivalent binding ability to complementary DNA, when compared with 2'-F-4'-C-MOE-rU and 2'-F-araU modified duplexes.Especially a C-H…F-C pseudohydrogen bond was supposed to contribute more binding affinity at uridine-purine steps, meanwhile, 2'-F-4'-C-MOE-araU had almost the same base discriminatory ability as uridine in dsDNA and DNA-RNA chimeras, while 2'-F-4'-C-MOE-rU was found to have only moderate RNA hybridization ability. However, 2'-F-4'-C-MOE-araU at 3'-end of oligonucleotide could not led to more nuclease hydrolytic stability than that with 2'-F-4'-C-MOE-rU modification. These results demonstrated the feasibility of C4'-MOE modification on 2'-F-ANA and the dramatic effects of the 2'-F substituent, which provides a new approach fo r further chemical modification of antisense drugs.