A novel strategy for regulation of an enzymatic
DNA modification reaction has been developed by employing a designed nanoscale
DNA scaffold.
DNA modification using
enzymes often requires bending of specific
DNA strands to facilitate the reaction. The DNA methylation
enzyme EcoRI
methyltransferase (
M.EcoRI)
bends double helix
DNA by 55 degrees-59 degrees during the reaction with flipping out of the second
adenine in the GAATTC sequence as the methyl transfer reaction proceeds. In this study, two different double helical tensions, tense and relaxed states of double helices, were created to control the methyl transfer reaction of
M.EcoRI and examine the structural effect on the methylation. We designed and prepared a two-dimensional (2D)
DNA scaffold named the "
DNA frame" using the
DNA origami method that accommodates two different lengths of the double-strand
DNA fragments, a tense 64mer double strand and a relaxed 74mer double strand. Fast-scanning atomic force microscope (AFM) imaging revealed the different dynamic movement of the double-strand DNAs and complexes of
M.EcoRI with 64mer and 74mer double-strand DNAs.
After treatment of the double strands in the
DNA frame with
M.EcoRI and the subsequent digestion with restriction
enzyme EcoRI (R.EcoRI), AFM analysis revealed that the 74mer double-strand
DNA was not effectively cleaved compared with the 64mer double-strand
DNA, indicating that the methylation preferentially occurred in the relaxed 74mer double-strand
DNA compared with that in the tense 64mer double strand. Biochemical analysis of the methylation and specific digestion using a real-time PCR also supported the above results. These results indicate the importance of the structural flexibility for bending of the duplex
DNA during the methyl transfer reaction with
M.EcoRI. Therefore, the DNA methylation can be regulated using the structurally controlled double-strand DNAs constructed in the
DNA frame nanostructure.