A simple fluorescent method for detecting mismatched DNAs using a MutS-fluorophore conjugate.
A fluorescent method was developed for the detection of unpaired and mismatched DNAs using a MutS-fluorophore conjugate. The fluorophore, 2-(4'-(iodoacetoamido)anilino) naphthalene-6-sulfonic acid (IAANS), was site-specifically attached to the 469 position of Thermus aquaticus (Taq.) MutS mutant (C42A/T469C). The fluorophore labeled residue located at the dimer interface of the protein undergoes a drastic conformational change upon binding with mismatched DNA. The close proximity of the two identical fluorescent molecules presumably causes the self-quenching of the fluorophore, since fluorescence emission of the biosensor decreases with increasing concentrations of mismatched DNA. The order of binding affinity for each unpaired and mismatched DNA obtained by this method was DeltaT (Kd=52 nM)>GT (62 nM)>DeltaC (130 nM)>CT (160 nM)>DeltaG (170 nM)>DeltaA (250 nM)>CC (720 nM)>AT (950 nM). This order is comparable to the previous results of the gel mobility shift assay. Thus, this method can be a simple, useful tool for elucidating the mechanism of DNA mismatch repair as well as a novel probe for detecting of genetic mutation.
Cho
Minseon
M
Department of Chemistry and Division of Molecular and Life Sciences (BK21), Pohang University of Science and Technology, San 31, Hyojadong, Pohang, Gyungbuk 790-784, Republic of Korea.
Chung
Suhman
S
Heo
Seong-Dal
SD
Ku
Jakang
J
Ban
Changill
C
D015374:Biosensing Techniques; D004247:DNA / Q000032:analysis* / Q000737:chemistry*; D005456:Fluorescent Dyes; D051719:MutS DNA Mismatch-Binding Protein / Q000235:genetics; D009282:Naphthalenesulfonates; D017354:Point Mutation; D013824:Thermus
D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; C011488:IAANS; D004247:DNA; D051719:MutS DNA Mismatch-Binding Protein