2ʹ-O-methyl nucleotide modified DNA substrates influence the cleavage efficiencies of BamHI and BglII
Zhaoxue Tong Bin Zhao Guojie Zhao Hong Shang Yifu Guan
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Induction of endonucleolytic DNA cleavage is an essential event that links the initiating stimuli to the final effects of cells. The cleavage efficiency and thus the final yield could be affected by many factors, including structures of DNA substrates, composite structures of enzymes–substrates or enzymes–nucleic analogs and so on. However, it is not clear whether a nucleotide derivative-substituted in DNA substrates can influence the efficiency of enzymatic cleavage. To investigate the effect of sugar pucker conformation on DNA–protein interactions, we used 2′-𝑂-methyl modified nucleotides (OMeN) to modify DNA substrates of isocaudemers BamHI and BglII in this study, and used FRET assay as an efficient method for analysis of enzyme cleavage. Experimental results demonstrated that OMeN-substituted recognition sequences influenced the cleavage rates significantly in a position-dependent manner. OMeN substitutions can reduce the cleavage as expected. Surprisingly, OMeN substitutions can also enhance the cleavage rates. The kinetics parameters of 𝑉max and 𝐾m have been obtained by fitting the Michaelis-Menten kinetic equation. These 2′-OMe nucleotides could behave as a regulatory element to modulate the enzymatic activity in vitro, and this property could enrich our understanding about the endonuclease cleavage mechanism and enhance our ability to regulate the enzymatic cleavage efficiency for applications in synthetic biology.
Zhaoxue Tong1 Bin Zhao2 Guojie Zhao1 Hong Shang3 Yifu Guan1
Volume 48, 2023
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