Adolph K W
Eur J Biochem. 1975 May 6;53(2):449-55. doi: 10.1111/j.1432-1033.1975.tb04085.x.
The binding of the dye acridine orange to cowpea chlorotic mottle virus (CCMV) and its purified RNA has been studied to obtain the number of dye-binding sites as a function of pH and, through further analysis, to estimate the degree of RNA secondary structure in situ. Acridine organe does not bind to CCMV protein and so the dye binding directly reflects the accessibility and structure of the RNA. The number of dye molecules per nucleotide which can be bound by native virus (pH 4.5, I = 0.1 buffer) is 0.13-0.18, the precise value depending upon the assumption of either heterogeneous binding sites or weak binding forces. The number of binding sites increases by a factor of about 2.7 to 0.34-0.48 when the pH is raised to pH 7.5 and "swelling" of the virus occurs. About 50% of the sites on the free RNA are available to bind dye in the swollen virus. The stacking coefficient, which is a measure of the degree of base pairing in a polynucleotide, has been calculated for the native and swollen virus and for the isolated RNA. The values of the stacking coefficient for the RNA in the virus and following extraction are comparable, which suggests that the structure of the RNA in both cases is similar, and the values are low in magnitude, which indicates the existence of extensive regions of double-helix.
已对吖啶橙染料与豇豆花叶病毒（CCMV）及其纯化RNA的结合进行了研究，以获取染料结合位点数量与pH的函数关系，并通过进一步分析来估计原位RNA二级结构的程度。吖啶橙不与CCMV蛋白结合，因此染料结合直接反映了RNA的可及性和结构。天然病毒（pH 4.5，I = 0.1缓冲液）每核苷酸可结合的染料分子数为0.13 - 0.18，精确值取决于对异质结合位点或弱结合力的假设。当pH升高到pH 7.5且病毒发生“膨胀”时，结合位点数量增加约2.7倍，达到0.34 - 0.48。游离RNA上约50%的位点在膨胀的病毒中可用于结合染料。已计算了天然和膨胀病毒以及分离RNA的堆积系数，该系数是多核苷酸中碱基配对程度的一种度量。病毒中RNA以及提取后RNA的堆积系数值相当，这表明两种情况下RNA的结构相似，且该值较小，这表明存在广泛的双螺旋区域。
