Thomas T J, Baarsch M J, Messner R P
Department of Medicine, University of Minnesota, Minneapolis 55455.
Anal Biochem. 1988 Feb 1;168(2):358-66. doi: 10.1016/0003-2697(88)90330-2.
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.
我们使用酶免疫分析法研究了在氯化钠存在下聚（dG-dC）·聚（dG-dC）和聚（dG-m5dC）·聚（dG-m5dC）从B-DNA到Z-DNA的转变。将多核苷酸以不同浓度的氯化钠包被在微量滴定板上，并用单克隆抗Z-DNA抗体Z22处理。随后用碱性磷酸酶偶联的多价小鼠免疫球蛋白和酶底物对硝基苯磷酸处理平板。使用酶标仪对酶-底物反应引起的显色进行定量。我们的结果表明，该抗体不识别处于B-DNA构象的多核苷酸，而是与Z-DNA构象强烈结合。在抗衡离子的中间浓度下获得了一条平滑的转变曲线。从转变曲线中，我们确定了B-DNA到Z-DNA转变中点处抗衡离子的浓度。聚（dG-dC）·聚（dG-dC）和聚（dG-m5dC）·聚（dG-m5dC）的中点浓度分别为2.3 M和0.74 M氯化钠。使用免疫方法，我们还研究了在天然存在的多胺存在下聚（dG-m5dC）·聚（dG-m5dC）从B-DNA到Z-DNA的转变。多胺的中点浓度如下：腐胺为2.5 mM；亚精胺为34 μM；精胺为1.8 μM。通过酶免疫分析法测定的中点值与通过圆二色性和紫外吸收光谱测量法测定的结果非常一致。这些结果表明，将预先存在的DNA构象或构象混合物固定在固体支持物上，然后使用单克隆抗DNA抗体对DNA构象进行滴定，是研究DNA构象动力学的一种极好方法。
