Design and synthesis of novel nucleotide analogs and protein conjugates for DNA sequencing

Sequencing by Synthesis (SBS), a DNA sequencing methodology based on the DNA polymerase reaction, is a promising paradigm for deciphering large-scale genomes. This thesis describes the design and synthesis of a variety of nucleotide reversible terminators (NRTs) with different characteristics. One set of NRTs possesses a phosphate moiety attached to the 2’ position of the sugar to block further incorporation in polymerase reaction, with the potential for fluorescent tag attachment at the same site or on the base through a cleavable linker for detection. The other set of NRTs possesses an azido-methyl moiety that blocks the 3’-hydroxyl group for detection by surface-enhanced Raman scattering. Each NRT has been tested in proof-of-principle SBS experiments. In addition, a set of 5’-phosphate tagged nucleotides has been developed and tested for nanopore electronic detection. A new set of NRTs, 2’-O-monophosphate 3’-hydroxyl nucleoside 5’-triphosphates (2’-P-NTPs) has been synthesized and its application for SBS has been investigated (chapter 2). These NRTs contain a phosphate at the 2’ position of the sugar ring, which serves as the removable capping group during the polymerase reaction. This moiety is positioned close to the 3’-hydroxyl group so as to block further nucleotide incorporation in the polymerase reaction. It nonetheless should allow improved binding to the polymerase relative to nucleotides with blocking groups at the 3’ position, since polymerases have strict requirements for the 3’-OH binding pocket. 2’-P-NTPs can be incorporated into the growing nucleic acid strand at temperatures ranging from 37oC to 65oC with Stoffel fragment modified 19 (SfM19) polymerase. After incorporation, the phosphate capping moiety on the 2’ position of the DNA extension product can be efficiently removed by enzymatic phosphatase reaction permitting the next incorporation step. Fluorescently labeled 2’-P-NTPs have the potential for sequencing DNA and direct sequencing of RNA-like templates. As an alternative to fluorescence-based SBS, a Raman spectroscopy detection method was developed using an azido moiety (N3) as both a 3’-OH blocking group and a label with an intense, narrow and unique Raman shift at 2125 cm-1, where virtually all biological molecules are transparent (chapter 3). First the four 3’-O-azidomethyl nucleotide reversible terminators (N3-dNTPs) were demonstrated to produce surface enhanced Raman scattering (SERS) at 2125 cm-1. These 4 nucleotide analogues were used as substrates for the polymerase to perform a complete 4-step SBS reaction. SERS was used to monitor the appearance of the azide-specific Raman peak at 2125 cm-1 as a result of polymerase mediated primer extension by a single N3-dNTP and disappearance of this Raman peak upon cleavage of the azido label to permit the next nucleotide incorporation, thereby determining the DNA sequence. Due to the small size of the azido label, the N3-dNTPs are efficient substrates for the DNA polymerase. In the SBS cycles, the natural nucleotides are restored after each incorporation and cleavage, producing a growing DNA strand that bears no modifications and will not impede further polymerase reactions. Thus, with further improvements in SERS for this moiety, this approach has the potential to provide an attractive alternative to fluorescence-based SBS. Chapter 4 describes the design, synthesis and characterization of a new set of 5’-phosphate labeled nano-tag nucleotides (NTNs) for single molecule electronic SBS by nanopore detection. Four modified oligonucleotide polymers that produce distinct electrical current blockade signals in nanopores were designed as the nano-tags. While most of the NTNs flow rapidly through the pore, those complementary to the nucleotide on the DNA template are captured by the polymerase and will have at least 10-fold longer dwell times in the pore, which affords enough time for measuring and discriminating the signals. Since the nano-tags are automatically removed during the polymerase extension reaction in real time, only natural DNA strands are produced. Thus this SBS method should decrease the overall sequencing time and increase the read length.