Site-selective DNA hydrolysis by combining Ce(IV)/EDTA with monophosphate-bearing oligonucleotides and enzymatic ligation of the scission fragments.
By using two oligonucleotide additives that bear a monophosphate group at the termini through various linkers, gap structures were formed at predetermined positions in substrate DNA, and the monophosphate groups were placed at both edges of these gaps. At pH 7.0 and 37 degrees C, the phosphodiester linkages in the gap sites were efficiently and selectively hydrolyzed by Ce(IV)/EDTA complex (EDTA = ethylenediamine-N,N,N',N'-tetraacetate). The linkages in the middle of the gaps were predominantly hydrolyzed. Compared with DNA scission using oligonucleotide additives that bear no terminal monophosphate, the present scission was much faster (22-fold for a 3-base gap and 14-fold for a 5-base gap) and more site selective. Introduction of one monophosphate group to either edge of the gaps was also effective for promotion of both site selectivity and scission rate. The monophosphate group(s) at the gap site recruits the Ce(IV) to the target site and magnifies the difference in intrinsic reactivity between the target site and the others. Even at higher reaction temperatures, the site selectivity remained satisfactorily high. Furthermore, the fragments formed by the site-selective scission were connected with various oligonucleotides by using DNA ligase, producing desired recombinant DNAs.
Contribution from the Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan.
Wen
W
Chen
Yoshihito
Y
Kitamura
Jing-Min
JM
Zhou
Jun
J
Sumaoka
Makoto
M
Komiyama
D001483:Base Sequence; D002563:Cerium / Q000737:chemistry*; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D011088:DNA Ligases / Q000737:chemistry* / Q000378:metabolism; D004492:Edetic Acid / Q000737:chemistry*; D019793:Fluorescein / Q000737:chemistry; D006868:Hydrolysis; D007700:Kinetics; D009841:Oligonucleotides / Q000737:chemistry*; D010755:Organophosphates / Q000737:chemistry; D013379:Substrate Specificity
D009841:Oligonucleotides; D010755:Organophosphates; D002563:Cerium; D004247:DNA; D004492:Edetic Acid; D011088:DNA Ligases; D019793:Fluorescein