Molecular engineering of novel nucleotide analogues for DNA sequencing by synthesis

The first part of the thesis presents the design and synthesis of nucleotide reversible terminators for Pyrosequencing. Pyrosequencing is a method to sequence DNA by detecting the pyrophosphate group that is generated when a nucleotide is incorporated into the growing DNA strand in polymerase reaction. However, this method has an inherent difficulty in accurately deciphering the homopolymeric regions of the DNA templates. We developed a new method to solve this problem using nucleotide reversible terminators. These nucleotide analogues are modified with a reversible chemical moiety attached to their 3'-OH position to temporarily terminate the polymerase reaction. In this way, only one nucleotide is incorporated into the growing DNA strand even in homopolymeric regions. After detection of the pyrophosphate for sequence determination, the 3'-OH of the primer extension products is regenerated through different deprotection methods. Using ally, 2-nitrobenzyl group, t-butyldithiolmethyl, or azidomethyl as the reversible moiety to cap the 3'-OH of the four nucleotides, we have synthesized two sets of 3'-O-modified nucleotides, 3'-O-allyl-dNTPs, 3'-O-(2-nitrobenzyl)-dNTPs, 3'-O-t-butyldithiolmethyl-dNTPs and 3'-O-azidomethyl-dNTPs, as reversible terminators for pyrosequencing. The capping moiety on the 3'-OH of the DNA extension product is efficiently removed after pyrophosphate detection by either a chemical method or photolysis. To sequence DNA, self-priming templates containing homopolymeric regions are immobilized on sepharose beads, and then Extension-Signal Detection-Deprotection cycles are conducted on the DNA beads to unambiguously decipher the sequence of DNA templates. Our results establish that this reversible-terminator-pyrosequencing approach can be potentially developed into a powerful methodology to accurately determine DNA sequences.;The second part of the thesis presents the design and synthesis of terminal phosphate fluorescence labeled nucleotide reversible terminators for DNA Sequencing By Synthesis. Instead of attaching the fluorephore on the base, the fluorephore is linked to the tetraphosphate or pentaphosphate of the nucleotide anologues to facilitate the signal detection in sequencing; another modification is the 3'-OH cleavable moiety as a capping group to temporarily terminate the polymerase reaction after the modified nucleotide is incorporated. In this method, the DNA template is immobilized on beads. After the polymerase reaction, the triphosphate attached dye is released. Upon phosphatase digestion to remove the phosphate group from the dye, the free dye will yield the corresponding fluorescence which indicates the incorporation of the right nucleotide. After the extension, the capping moiety is removed through a deprotection step to regenerate the 3'-OH group, thus the next cycle's extension can be initiated. To demonstrate the feasibility of this approach, I have synthesized phosphate labeled nucleotide reversible terminators, [7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one)] DDAO-3'-O-allyl-dT4P and DDAO-3'-O-azidomethyl-dC4P and showed that these modified nucleotides can be used to accurately decipher DNA template.;The third part of the thesis demonstrates a DNA sequencing-by-synthesis method using detection by Infrared Spectroscopy. In this method, the 3'-OH of the nucleotides were capped with azidomethyl group to temporarily terminate the polymerase reaction after complementary base incorporation. The azidomethyl capping moiety on 3'-OH also servers as a unique reporter group, which gives a specific infrared absorbance at 2115 cm-1 to indicate the complementary base incorporation. After IR signal detection, the capping moiety, azidomethyl, can be removed to resume the polymerase reaction. The IR signal of four consecutive incorporations of the modified nucleotides was demonstrated to verify the feasibility of this method to sequence DNA.;The fourth part of the thesis describes a new convenient, fast, reliable, radioisotope-free and fluorescence-free method for polymerase kinetic studies. In this method, MALDI-TOF Mass Spectrometry was used to detect the amount of extension DNA product accumulated during the polymerase reaction. The K D and Kpol of ddCTP incorporation by Klenow polymerase (exo-) determined by the MALDI-TOF MS method was remarkably close to the published data obtained with the conventional radioisotope based approach, verifying the reliability of the new MALDI-TOF MS method. Moreover, the kinetics for the reversible terminators 3'-O-allyl-dATP, 3'-O-allyl-dCTP, 3'-O-allyl-dGTP, and 3'-O-allyl-dTTP, which have great potential application in genomic sequencing, is revealed for the first time using the MALDI-TOF MS based method. The results also showed that the cofactors Mn2+ and Mg2+, as well as incubation temperature, greatly influence 3'-O-allyl-dNTP incorporation by Therminator II polymerase.