Development of new DNA sequencing approaches and investigation of vision-related proteins using synthetic chemistry

The fist part of this thesis presents our efforts to investigate vision-related proteins in visual cycle. The synthesis of a seven-membered ring locked analogue of 11-cis-retinol, tethered to a cross-linking moiety on C-15 and a lysine extended biotin on the C-3, was accomplished for its utilization as a probe to isolate retinol binding proteins that may be involved in the reisomerization of retinal from all trans to 11- cis in the visual cycle.;The second part of this thesis focuses on development of new DNA sequencing approaches using the concept of DNA sequencing by synthesis (SBS). In DNA SBS method, four nucleotide reversible terminators (A, C, G, T) are the most critical molecular tools for the efficient DNA polymerase reaction on a massive parallel DNA-sequencing chip. First, we designed and synthesized four nucleotides as reversible terminators by attaching a cleavable fluorophore to the nucleobase and capping the 3'-OH with a small reversible chemical moiety so that they are still recognized by DNA polymerase as substrates. After the cleavable fluorescent nucleotides were incorporated into a growing DNA strand, the fluorophore and the 3'-OH capping group on a DNA extension product, were removed by photocleavage or in a chemical manner. This allows the re-initiation of DNA polymerase reaction and continuation of DNA SBS to the next cycle for incorporation of another fluorescent reversible nucleotide. The efficiency in cleaving the fluorophore and the 3'-OH capping moiety plays a crucial role in DNA SBS approach, and directly determines the read length of this methodology. To improve the efficiency of DNA SBS, we explored a variety of different chemical moieties as a 3'-OH capping group as well as the different cleavable linker bridging the nucleobase and the fluorophore.;The work led to the construction of a 4-color nucleotide library consisting of multiple functionalized nucleotides each with a suitable 3'-OH capping moiety and a cleavable fluorescent fluorophore. Also, we constructed a 3'-O-labeled nucleotide library by design and synthesis of nucleotide reversible terminators with different 3'-OH capping moieties. The above nucleotide libraries have been screened and evaluated as reversible terminators using the DNA polymerase reaction both in solution phase and on a DNA-immobilized chip. Furthermore, photocleavable nucleotide reversible terminators and fluorescent dideoxynucleotides were synthesized for the development of a chip-based Sanger-SBS hybrid DNA sequencing method. Finally, we solved the homopolymeric problems of DNA pyrosequencing by using the 3'-O-modified reversible terminators.