# Biochemical engineering of 5hmdC-DNA using a Tet3 double-mutant

## TL;DR

This paper introduces a new method to efficiently generate DNA containing the epigenetic marker 5hmdC, which is important for gene regulation and cancer diagnostics.

## Contribution

A novel Tet3 double-mutant method is developed to produce pure 5hmdC-DNA efficiently and specifically.

## Key findings

- The Tet3 mutant converts 5mdC to a mixture of 5hmdC and 5fdC, followed by a reduction step to yield pure 5hmdC.
- The method works on PCR products, synthetic oligos, and entire genomes with high specificity and efficiency.
- The generated 5hmdC-DNA is suitable for epigenetic research, diagnostics, and protein binding studies.

## Abstract

5-Hydroxymethyl-2′-deoxycytidine (5hmdC) is an important epigenetic marker involved in gene regulation and DNA demethylation. It has potential use as a biomarker for cancer and other diseases due to its significant depletion in various cancers and disease models. This research aimed to develop a reliable and efficient method for generating 5hmdC-containing DNA, addressing limitations in existing techniques. We created a Tet3 stalling mutant that converts 5-methyl-2′-deoxycytidine (5mdC) into a mixture of 5hmdC and 5-formyl-2′-deoxycytidine (5fdC), followed by a reduction step to convert 5fdC to 5hmdC, ensuring a pure 5hmdC state within the CpG context. This method can convert any PCR product, synthetic oligos, and entire genomes into 5hmdC-modified DNA. The principal results demonstrate high specificity and efficiency, providing a robust tool for epigenetic research, cancer diagnostics, and protein binding assays. Additionally, our technique offers 5hmdC-DNA for functional studies and as standards for diagnostic assays.