Sensitive and specific miRNA in situ hybridization using partially methylated phosphotriester antisense DNA probes

Neutralized DNA (nDNA) is an emerging class of DNA oligonucleotides chemically synthesized with site-specific internucleoside methyl phosphotriester linkages, changing the negatively charged DNA phosphodiester backbone to a neutral methyl phosphotriester backbone. The reduction of inter-strand charge repulsion of nucleotide duplexes results in stronger binding between nDNA and other nucleic acids, and as such, nDNA has been used as a sensitive antisense probe for sequencing nucleotides. From a thermodynamic perspective due to steric effects, a hybrid duplex between DNA and partially methylated nDNA should possess higher specificity than a duplex between DNA with fully methylated nDNA, while retaining binding affinity. However, the application of nDNA for ex vivo RNA hybridization at low transcript abundance remains completely unexplored. Here, we determined that partially methylated nDNA (N4 nDNA; with 4 methylated nucleotides) probes inhibited reverse transcription of oncogenic miRNA miR-21 more efficiently than canonical DNA probes or highly methylated nDNA probes (all probes share the same sequence) and with an efficiency rivaling LNA probes. Subsequently, we performed in situ hybridization analysis using a miR-21-expressing colorectal cancer cell line (HCT116). HCT116 stained with N4 nDNA probes revealed a greater detection intensity and specificity than HCT116 stained with canonical DNA probes. Consistently, enzyme-linked immunosorbent assays revealed that miRNA hybridization efficiency of N4 nDNA probes was greater than that of canonical DNA probes at cellular transcript levels. Given that N4 nDNA probe is immune-negative and DNase I-resistant, partially methylated nDNA could be further developed to have significant applications in biotechnology and medicine.