Sequence-Dependent Fluorescence of Cy3- and Cy5-Labeled Double-Stranded DNA.
Sequence-Dependent Fluorescence of Cy3- and Cy5-Labeled Double-Stranded DNA.
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DOI：
10.1021/acs.bioconjchem.6b00053
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发表时间：
2016-03-16
影响因子：
4.7
通讯作者：
Somoza MM
中科院分区：
文献类型：
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作者：
Kretschy N;Sack M;Somoza MM
Cy 3和Cy 5染料的荧光强度强烈依赖于标记的寡核苷酸的核碱基序列。序列依赖性荧光可以显著地影响从基于核酸的荧光检测的许多常见实验方法（例如测序、PCR、FRET和FISH）获得的数据。为了定量序列依赖性荧光，我们测量了结合到所有1024个可能的双链DNA 5聚体的5′端的Cy 3和Cy 5的荧光强度。还测定了与固定序列双链DNA的5′端结合的这些染料的荧光强度，其中可变序列3′突出端与染料相邻。标记的DNA寡核苷酸使用光定向原位微阵列合成来制备。结果表明，两种染料的荧光强度对所有5个碱基或碱基对都敏感，双链DNA的序列依赖性比单链DNA强，染料对相邻的dsDNA序列和3′-ssDNA突出端都敏感。富含嘌呤的序列导致更高的荧光。该结果可用于估计荧光标记DNA实验中的测量误差，以及通过考虑标记花青染料的核碱基环境来优化荧光信号。
The fluorescent intensity of Cy3 and Cy5 dyes is strongly dependent on the nucleobase sequence of the labeled oligonucleotides. Sequence-dependent fluorescence may significantly influence the data obtained from many common experimental methods based on fluorescence detection of nucleic acids, such as sequencing, PCR, FRET, and FISH. To quantify sequence dependent fluorescence, we have measured the fluorescence intensity of Cy3 and Cy5 bound to the 5′ end of all 1024 possible double-stranded DNA 5mers. The fluorescence intensity was also determined for these dyes bound to the 5′ end of fixed-sequence double-stranded DNA with a variable sequence 3′ overhang adjacent to the dye. The labeled DNA oligonucleotides were made using light-directed, in situ microarray synthesis. The results indicate that the fluorescence intensity of both dyes is sensitive to all five bases or base pairs, that the sequence dependence is stronger for double- (vs single-) stranded DNA, and that the dyes are sensitive to both the adjacent dsDNA sequence and the 3′-ssDNA overhang. Purine-rich sequences result in higher fluorescence. The results can be used to estimate measurement error in experiments with fluorescent-labeled DNA, as well as to optimize the fluorescent signal by considering the nucleobase environment of the labeling cyanine dye.