Abstract
Neutralized DNA (nDNA) is an emerging class of DNA oligonucleotides chemically synthesized with site-specific internucleoside methyl phosphotriester (MPTE) linkages, changing the negatively charged DNA phosphodiester backbone to a neutral MPTE backbone. nDNA was used as sensitive probes for nucleotide sequencing; however, the application of nDNA for microRNAs (miRNA) inhibition and ex vivo RNA hybridization remains largely unexplored. In this study, we determined that partially methylated nDNA probes inhibited reverse transcription of oncogenic miRNA miR-21 in synthetic human plasma more efficiently than canonical DNA/highly methylated nDNA probes and with an efficiency comparable with locked nucleic acid probes at nM concentrations. Moreover, in situ hybridization using a miR-21-expressing colorectal cancer cell HCT116 stained with partially methylated nDNA probes revealed a greater efficacy than HCT116 stained with canonical DNA probes. Given that the N4 nDNA probe is immune-negative and endonuclease-resistant, partially methylated nDNA could be further developed to have significant applications in biotechnology and medicine.
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