Synthesis and Characterization of Thiophosphoramidate Morpholino Oligonucleotides and Chimeras

Abstract

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 vitro bioassay screens designed to evaluate their potential as microRNA inhibitors of hsa-miR-15b-5p in HeLa cells.

Nuclear compartmentalization of TERT mRNA and TUG1 lncRNA is driven by intron retention

“…TMOs are oligonucleotides in which the bases (thymine, cytosine, adenine, and guanine) are attached to morpholine, and these nucleosides are joined through thiophosphoramidate internucleotide linkages (Fig. 7a ) 60 . They show increased hybridization stability towards complementary RNA (10 °C increased melting temperature compared to an unmodified control duplex of identical sequence).…”

“…Prior to thiomorpholino oligonucleotide (TMO) synthesis, appropriately protected morpholino nucleosides of adenine, guanine, thymine and cytosine and their corresponding phosphorodiamidites were synthesized as reported elsewhere 60 . All TMOs were synthesized using an Applied Biosystems Model 394 Automated DNA Synthesizer using conventional DNA synthesis reagents that were purchased from Glen Research, VA.…”

Self-transfecting GMO-PMO and PMO-GMO chimeras enable gene silencingin vitro and in vivozebrafish model and NANOG Inhibition Induce the Apoptosis in Breast and Prostate Cancer Cells

“…In the case of GMO-PMO/PMO-GMO chimeras, no such delivery vehicle is required and showed the antisense efficacy as low as 750 nM dose in in vitro and required as minimum as four guanidinium linkages. Though, backbone modified PMO and their chimeras [59,60] or PMO-DNA chimera [61] or chimeric oligonucleotides containing morpholino thymidine analogues [62,63] or with a single guanidinium morpholino unit incorporated DNA [64] has been reported earlier however, to the best of our knowledge, GMO-PMO/PMO-GMO chimera is the first report from our group. Considering the simplicity, the possibility of rational design, relatively inexpensive and easy synthesis, GMO-PMO/PMO-GMO self-transfecting chimera, in principle could be useful as tools to target any gene including virus and bacteria towards the development of antisense therapy and could be a practical approach.…”

Xeno nucleic acids (XNAs) having non-ribose scaffolds with unique supramolecular properties

“…This yielded a non-charged XNA. PMO has a higher affinity for RNA than for DNA, 21 and the melting temperature of PMO/RNA is higher than that of DNA/ RNA of the same sequence. 21,22 A PMO/RNA duplex is not as stable as a 2 0 -O-methyl-RNA/RNA duplex, however.…”

“…PMO has a higher affinity for RNA than for DNA, 21 and the melting temperature of PMO/RNA is higher than that of DNA/ RNA of the same sequence. 21,22 A PMO/RNA duplex is not as stable as a 2 0 -O-methyl-RNA/RNA duplex, however. 23 PMO has remarkably high nuclease resistance, low toxicity compared with phosphorothioate oligomers, and higher water solubility than non-charged peptide nucleic acids (PNAs).…”