Allen D J, Darke P L, Benkovic S J
Department of Chemistry, Pennsylvania State University, University Park 16802.
Biochemistry. 1989 May 30;28(11):4601-7. doi: 10.1021/bi00437a014.
Fluorescent derivatives of short oligonucleotides of defined sequence were prepared by the incorporation of 5-(propylamino)uridine via current phosphoramidite chemistry, followed by derivatization of the propylamine function with mansyl chloride. These oligomers, annealed to complementary oligomers, yielded short duplex DNA fluorescently labeled at a specific base. The fluorescence emission from this labeled duplex increases upon binding to the Klenow fragment of DNA polymerase I (KF) at specific positions within the duplex DNA. By varying the position of the label within the duplex DNA and observing the emission, points of strong enzyme-DNA interactions were elucidated. A similar fluorescent derivative of a deoxynucleoside triphosphate (dNTP), 5-[[[[[[(5- sulfonaphthalenyl)amino]ethyl]amino]carbonyl]- methyl]thio]-2'-deoxyuridine 5'-triphosphate (AEDANS-S-dUTP), was synthesized, whose emission also was increased upon binding to KF. The change in emission intensities between unbound and bound substrates enabled the measurements of KDs for the DNA and dNTP derivative, which were found to be 0.15 nM and 2.9 microM, respectively. Stopped-flow measurements on these species yielded association and dissociation rates for each. Anisotropy measurements of the labeled base at various positions in the duplex yielded values that support the measurements made by observing the emission intensities.
通过当前的亚磷酰胺化学方法，将5-(丙氨基)尿苷掺入，随后用甘露糖氯对丙胺官能团进行衍生化，制备了具有确定序列的短寡核苷酸的荧光衍生物。这些寡聚物与互补寡聚物退火后，产生了在特定碱基处荧光标记的短双链DNA。当该标记的双链体与双链DNA内特定位置的DNA聚合酶I（KF）的Klenow片段结合时，其荧光发射增强。通过改变双链DNA内标记的位置并观察发射情况，阐明了酶与DNA强烈相互作用的位点。合成了一种类似的脱氧核苷三磷酸（dNTP）荧光衍生物，即5-[[[[[[(5-磺基萘基)氨基]乙基]氨基]羰基]-甲基]硫代]-2'-脱氧尿苷5'-三磷酸（AEDANS-S-dUTP），其与KF结合时发射也增强。未结合和结合底物之间发射强度的变化使得能够测量DNA和dNTP衍生物的解离常数（KDs），发现分别为0.15 nM和2.9 μM。对这些物种进行的停流测量得出了各自的缔合和解离速率。对双链体中不同位置的标记碱基进行的各向异性测量得出的值支持通过观察发射强度所做的测量。
