Synthesis and application of four fluorescence labeled nucleotides through disulfide as reversible terminators in DNA sequencing by synthesis

DNA sequencing is one of the important means of biological research. The development of accurate and high-throughput DNA-sequencing methods has a strong impact on biomedical research. Sequencing by synthesis (SBS) approaches is one of the most important techniques of DNA sequencing. A series of SBS methods have been investigated and implemented. In SBS methods, the principal thing is to synthesize four fluorescently labelled cyclic reversible terminators as the sequencing reagents. These cyclic reversible terminators need to connect the fluorescein through a cleavable linker. In chemical biology, disulfide bridges, which are known to be efficiently and rapidly cleaved by mild reducing agents, have been used in a wide range of applications including drug delivery, functional and structural proteomics, DNA and tumor imaging. The disulfide-based cleavable linker is commonly used due to its rapid cleavage and straightforward synthesis. In this work, starting from 5-iodo-2'-deoxyuridine, 5-iodo-2'-deoxycyridine, 7-deazo-7-iodo-2'-deoxyadenosine and 7-deazo-7-iodo-2'-deoxyguanosine, four kinds of fluorescence labelled necleotides with four different bases through disulfide were synthesized and characterized. The synthesized fluorescence labelled nucleotides can be recognized and accepted by an identified DNA polymerase, incorporated faithfully into DNA strand, followed by cleaved completely by DTT within 1 min. These results demonstrate that the fluorescence labelled nucleotides have potential value as reversible terminators in DNA sequencing by synthesis.