Enzymatic Synthesis of Chemical Nuclease Triplex-Forming Oligonucleotides with Gene-Silencing Applications

Triplex-forming oligonucleotides (TFOs) are short, single-stranded oligomers that hybridise to a specific sequence of duplex DNA. TFOs can block transcription and thereby inhibit protein production, making them highly appealing in the field of antigene therapeutics. In this work, a primer extension protocol was developed to enzymatically prepare chemical nuclease TFO hybrid constructs, with gene-silencing applications. Click chemistry was employed to generate novel artificial metallo-nuclease (AMN)-dNTPs, which were selectively incorporated into the TFO strand by a DNA polymerase. This purely enzymatic protocol was then extended to facilitate the construction of 5-methylcytosine (5mC) modified TFOs that displayed increased thermal stability. The utility of the enzymatically synthesised di-(2-picolyl)amine (DPA)-TFOs was assessed and compared to a specifically prepared solid-phase synthesis counterpart through gel electrophoresis, quantitative PCR, and Sanger sequencing, which revealed similar recognition and damage properties to target genes. The specificity was then enhanced through coordinated designer intercalators-DPQ and DPPZ-and high-precision DNA cleavage was achieved. To our knowledge, this is the first example of the enzymatic production of an AMN-TFO hybrid and is the largest base modification incorporated using this method. These results indicate how chemical nuclease-TFOs may overcome limitations associated with non-molecularly targeted metallodrugs and open new avenues for artificial gene-editing technology.

Automated summary

In this study, a enzymatic protocol was developed for the preparation of chemical nuclease-TFO hybrid constructs, which includes the incorporation of novel artificial metallo-nuclease (AMN)-dNTPs into the TFO strand. The specificity of enzymatically synthesised DPA-TFOs was improved through coordinated designer intercalators, and high-precision DNA cleavage was achieved. This research demonstrates the potential of chemical nuclease-TFOs in overcoming the limitations of non-molecularly targeted metallodrugs and advancing artificial gene-editing technology.