Design and synthesis of novel nucleotides for enzymatic gene synthesis
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...
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sg-ntu-dr.10356-1526722021-10-05T07:44:18Z Design and synthesis of novel nucleotides for enzymatic gene synthesis Ee, Pin Koon Chiba Shunsuke School of Physical and Mathematical Sciences Illumina Singapore Pte Ltd Teo Yin Nah Shunsuke@ntu.edu.sg, ynteo1@illumina.com Science::Biological sciences::Genetics Science::Chemistry 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. Doctor of Philosophy 2021-09-10T03:38:05Z 2021-09-10T03:38:05Z 2020 Thesis-Doctor of Philosophy Ee, P. K. (2020). Design and synthesis of novel nucleotides for enzymatic gene synthesis. Doctoral thesis, Nanyang Technological University, Singapore. https://hdl.handle.net/10356/152672 https://hdl.handle.net/10356/152672 10.32657/10356/152672 en This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0). Nanyang Technological University |
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Science::Biological sciences::Genetics Science::Chemistry Ee, Pin Koon Design and synthesis of novel nucleotides for enzymatic gene synthesis |
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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. |
author2 |
Chiba Shunsuke |
author_facet |
Chiba Shunsuke Ee, Pin Koon |
format |
Thesis-Doctor of Philosophy |
author |
Ee, Pin Koon |
author_sort |
Ee, Pin Koon |
title |
Design and synthesis of novel nucleotides for enzymatic gene synthesis |
title_short |
Design and synthesis of novel nucleotides for enzymatic gene synthesis |
title_full |
Design and synthesis of novel nucleotides for enzymatic gene synthesis |
title_fullStr |
Design and synthesis of novel nucleotides for enzymatic gene synthesis |
title_full_unstemmed |
Design and synthesis of novel nucleotides for enzymatic gene synthesis |
title_sort |
design and synthesis of novel nucleotides for enzymatic gene synthesis |
publisher |
Nanyang Technological University |
publishDate |
2021 |
url |
https://hdl.handle.net/10356/152672 |
_version_ |
1713213282099134464 |