Design and synthesis of novel nucleotides for enzymatic gene synthesis

• Main Author: Ee, Pin Koon
• Other Authors: Chiba Shunsuke
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2021
• Subjects: Science::Biological sciences::Genetics; Science::Chemistry
• Online Access: https://hdl.handle.net/10356/152672
• Institution: Nanyang Technological University

Traditionally, DNA was synthesized using a four-step chemical method based on phosphoramidite chemistry, allowing the synthesis of DNA strands up to 250-300 base pairs. However, this is insufficient for future application, such as DNA storage and gene synthesis. Alternatives to the phosphoramidite chemistry need to be developed. Enzymatic gene synthesis is being explored as an alternative to achieve the synthesis of long DNA sequences. The Terminal Deoxynucleotidyl Transferase (TdT) is a unique template-independent DNA polymerase that can be used for such enzymatic based gene synthesis. However, unmodified TdT incorporates multiple nucleotides to the 3’ end of the oligonucleotide within a short time. Modification of TdT or nucleotides can be done to synthesize the desired DNA sequence. This study attempts to develop new modified nucleotides to terminate DNA synthesis by TdT. In chapter 2, the synthesis of 3’ perfluoroallyl dTTP serves as a reversible terminator. Through the study, we found that the 3’ blocked nucleotide could not be incorporated by TdT but was readily accepted by sequencing polymerase. Furthermore, the perfluoroallyl group can be removed by basic fluoride reagents, and further incorporation by sequencing polymerase is possible. Since TdT could not accept 3’ modified nucleotides, the use of base-modified nucleotides, known as virtual terminators, was studied (chapter 3). While the use of small modification such as 2- hydroxycinnamyl was insufficient to block, nucleotides conjugated to bulky protein, such as maltosebinding protein (MBP), were not incorporated by TdT. The use of polyethylene glycol (PEG) linkers for conjugation, however, resulted in partial termination of DNA synthesis. The result leads us to conduct a systematic study using PEGylated nucleotides to determine the effect of PEG length on incorporation by TdT (chapter 4). Interestingly, we discovered that the use of various PEG length conjugated to nucleobases changes yield of +1 product. The length of the PEG chains can be finetuned to achieve quantitative yield of the desired product. Further work also suggests that single incorporation of all 4 nucleobases was possible. This work shows that nucleobase modification is a promising way to synthesize genes with just nucleobase modification.