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This Article outlines the optimized chemical synthesis and preliminary biochemical characterization of a new oligonucleotide analogue called thiophosphoramidate morpholinos (TMOs). Their rational design hinges upon integrating two well-studied pharmacophores, namely, phosphorothioates (pS) and morpholinos, to create morpholino–pS hybrid oligonucleotides. Our simple synthesis strategy enables the easy incorporation of morpholino–pS moieties and therapeutically relevant sugar modifications in tandem to create novel oligonucleotide (ON) analogues that are hitherto unexplored in the oligotherapeutics arena. Exclusively TMO-modified ONs demonstrate high stability toward 3′-exonuclease. Hybridization studies show that TMO chimeras consisting of alternating TMO and DNA–pS subunits exhibit higher binding affinity toward complementary RNA relative to the canonical DNA/RNA duplex (∼10 °C). Oligonucleotides that consist entirely of TMO linkages also show higher RNA binding affinity but do not recruit ribonuclease H1 (RNase H1). Chimeric TMO analogues demonstrate high gene silencing efficacy, comparable to that of a chimeric 2′-OMe–pS/pO control, during in vitrobioassay screens designed to evaluate their potential as microRNA inhibitors of hsa-miR-15b-5p in HeLa cells.