在目的DNA不被酶切成不同片段的情况下，应用光谱方法探测pBR322质粒中十字形结构DNA的形成。吡咯脱氧胞苷（Pyrrolo deoxycytidine，PdC）取代dC掺入到pBR322质粒的特定位点（3195，3222或3281位点）。应用稳态和时间分辨荧光法研究十字形结构DNA的形成。结果显示：（1）稳态荧光特性表明，当PdC掺入到pBR322质粒的3222位点，PdC-pBR322超螺旋荧光强度大约是松弛型PdC-pBR322的4倍；与此同时，其时间分辨荧光寿命大约比松弛型PdC-pBR322长约0.3 ns。当PdC掺入到3195或3281位点时，稳态荧光光谱和荧光寿命（两位点处约1.42 ns）并没有显著变化。（2）随着盐浓度的增大，荧光寿命略微有变化，但不是很明显。通过检测和分析pBR322质粒的荧光光谱和荧光寿命，表明能够形成在非配对环状部分3222位点含有dC的十字形结构。在一定的盐浓度（0-100 mmol/L）条件下，十字形结构能够保持其稳定性。%The specific aim of this work is to probe the formation process of DNA with cruciform extrusion in a pBR322 plasmid using spectroscopic methods while without digesting DNA into pieces. Pyrrolo deoxycitidine(PdC)was incorporated into pBR322 to substitute for dC at specific sites of 3 195,3 222 or 3 281. Steady-state and time-resolved fluorescence were employed to examine the specific properties of PdC in the cruciform region. Results were as followings:1)Steady-state fluorescent properties indicated that the fluorescence intensity of supercoiled PdC-pBR322 was about 4-fold stronger than that of relaxed PdC-pBR322,when PdC was incorporated into pBR322 at position 3 222;meanwhile,its time-resolved lifetime was about 0.3 ns longer than that of relaxed PdC-pBR3322. When PdC was incorporated at position 3 195 or 3 281,there was no significant change in either steady-state fluorescence spectra or time-resolved lifetime(about 1.42 ns at both positions). 2)Lifetimes changed a little with the increasing of salt concentration,but not significantly. In conclusion,by analyzing fluorescence spectra and lifetimes(both in relaxed state and supercoiled plasmid),a cruciform structure with dC at site 3 222 of impaired loop is formed,and the cruciform is stable within this investigated salt concentration range(0-100 mmol/L).
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