Abstract:In this study, we synthesised a morpholino nucleoside-uridine (MNA-U) phosphoramidite and evaluated the potential of a MNA-modified antisense oligonucleotide (AO) sequences to induce exon 23 skipping in mdx mouse myotubes in vitro towards extending the applicability of morpholino chemistry with other nucleotide monomers. We designed, synthesised, and compared exon skipping efficiencies of 20 mer MNA-modified 2 -O-methyl RNA mixmer AO on a phosphorothioate backbone (MNA/2 -OMePS) to the corresponding fully modified 2 -O-methyl RNA AO (2 -OMePS) as a control. Our results showed that the MNA/2 -OMePS efficiently induced exon 23 skipping. As expected, the 2 -OMePS AO control yielded efficient exon 23 skipping. Under the applied conditions, both the AOs showed minor products corresponding to exon 22/23 dual exon skipping in low yield. As these are very preliminary data, more detailed studies are necessary; however, based on the preliminary results, MNA nucleotides might be useful in constructing antisense oligonucleotides.

Systematic evolution of ligands by exponential enrichment (SELEX) is an established procedure for developing short single-stranded nucleic acid ligands called aptamers against a target of choice. This approach has also been used for developing aptamers specific to whole cells named Cell-SELEX. Aptamers selected by Cell-SELEX have the potential to act as cell specific therapeutics, cell specific markers or cell specific drug delivery and imaging agents. However, aptamer development is a laborious and time-consuming process which is often challenging due to the requirement of frequent optimization of various steps involved in Cell-SELEX procedures. This review provides an insight into various procedures for selection, aptamer enrichment, regeneration and aptamer-binding analysis, in addition to a very recent update on all aptamers selected by Cell-SELEX procedures.

Low-density lipoprotein receptor (LDL-R) is a cell surface receptor protein expressed in a variety of solid cancers, including lung, colon, breast, brain, and liver, and therefore it opens up opportunities to deliver lysosome-sensitive anticancer agents, especially synthetic nucleic acid-based therapeutic molecules. In this study, we focused on developing novel nucleic acid molecules specific to LDL-R. For this purpose, we performed in vitro selection procedure via systematic evolution of ligands by exponential enrichment (SELEX) methodologies using mammalian cell-expressed human recombinant LDL-R protein as a target. After 10 rounds of selections, we identified a novel DNA oligonucleotide aptamer, RNV-L7, that can bind specifically to LDL-R protein with high affinity and specificity (K D = 19.6 nM). Furthermore, flow cytometry and fluorescence imaging assays demonstrated efficient binding to LDL-R overexpressed human cancer cells, including Huh-7 liver cancer cells and MDA-MB-231 breast cancer cells, with a binding affinity of 200 nM. Furthermore, we evaluated the functional potential of the developed LDL-R aptamer RNV-L7 by conjugating with a previously reported miR-21 targeting DNAzyme for inhibiting miR-21 expression. The results showed that the miR-21 DNAzyme-RNV-L7 aptamer chimera efficiently reduced the expression of miR-21 in Huh-7 liver cancer cells. As currently there are no reports on LDL-R aptamer development, we think that RNV-L7 could be beneficial toward the development of targeted cancer therapeutics.

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