Ultra-sensitive detection of DNA N6-adenine methyltransferase based on a 3D tetrahedral fluorescence scaffold assisted by symmetrical double-ring dumbbells

Abstract

DNA methylation is an epigenetic modification that plays a vital role in X chromosome inactivation, genome imprinting, and gene expression. DNA methyltransferase establishes and maintains a stable methylation state in genomic DNA. Efficient and specific DNA methyltransferase testing is essential for the early diagnosis and treatment of cancer. In this study, we designed an ultra-sensitive fluorescent biosensor, based on a 3D tetrahedral fluorescent scaffold assisted by symmetrical double-ring dumbbells, for the detection of DNA-[N 6-adenine]-methyltransferase (Dam MTase). Double-stranded DNA was methylated by Dam MTase and then digested by DpnI to form two identical dumbbell rings. The 3D tetrahedral fluorescent scaffold was synthesized from four oligonucleotide chains containing hairpins. When the sheared dumbbells reacted with the 3D tetrahedral fluorescent scaffold, the hairpins opened and a fluorescence signal could be detected. The strategy was successful over a wide detection range, from 0.002 to 100 U mL^-1 Dam MTase, and the lowest detection limit was 0.00036 U mL^-1. Control experiments with M.SssI methyltransferase and HpaII methylation restriction endonuclease confirmed the specificity of the method. Experiments with spiked human serum and the 5-fluorouracil inhibitor proved the suitability of the method for early cancer diagnosis.

Keywords: 3D tetrahedral fluorescent scaffold; Dam methyltransferase; Fluorescence signal amplification; Symmetrical double-ring dumbbell.