Immunological detection of B-DNA to Z-DNA transition of polynucleotides by immobilization of the DNA conformation on a solid support.
We studied the B-DNA to Z-DNA transition of poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) in the presence of NaCl using an enzyme immunoassay. The polynucleotides were coated on microtiter plates at varying concentrations of NaCl and treated with a monoclonal anti-Z-DNA antibody, Z22. The plates were subsequently treated with alkaline phosphatase conjugated polyvalent mouse immunoglobulins and the enzyme substrate, p-nitrophenyl phosphate. The color development due to the enzyme-substrate reaction was quantitated using a microplate autoreader. Our results show that the antibody does not recognize the polynucleotides in the B-DNA conformation and binds strongly to the Z-DNA conformation. A smooth transition curve is obtained at intermediate concentrations of the counterions. From the transition curves, we determined the concentration of the counterions at the midpoint of B-DNA to Z-DNA transition. The midpoint concentrations for poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) are 2.3 and 0.74 M NaCl, respectively. Using the immunological method, we also examined the B-DNA to Z-DNA transition of poly(dG-m5dC).poly(dG-m5dC) in the presence of naturally occurring polyamines. The midpoint concentrations of the polyamines are as follows: putrescine, 2.5 mM; spermidine, 34 microM; spermine, 1.8 microM. The midpoint values determined by the enzyme immunoassay are in good agreement with those determined by circular dichroism and ultraviolet absorption spectroscopic measurements. These results demonstrate that immobilization of a preexisting conformation or a mixture of conformations of DNA on a solid support followed by a titration of the DNA conformations using a monoclonal anti-DNA antibody is an excellent method to study the conformational dynamics of DNA.
Thomas
T J
TJ
Department of Medicine, University of Minnesota, Minneapolis 55455.
Baarsch
M J
MJ
Messner
R P
RP
D000911:Antibodies, Monoclonal; D004247:DNA / Q000032:analysis* / Q000276:immunology; D007124:Immunoenzyme Techniques; D009690:Nucleic Acid Conformation; D011073:Polyamines / Q000494:pharmacology; D011119:Polynucleotides; D013056:Spectrophotometry, Ultraviolet
D000911:Antibodies, Monoclonal; D011073:Polyamines; D011119:Polynucleotides; D004247:DNA