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Autonomous assembly of synthetic oligonucleotides built from an expanded DNA alphabet. Total synthesis of a gene encoding kanamycin resistance

Background: Many synthetic biologists seek to increase the degree of autonomy in the assembly of long DNA (L-DNA) constructs from short synthetic DNA fragments, which are today quite inexpensive because of automated solid-phase synthesis. However, the low information density of DNA built from just f...

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Autores principales: Merritt, Kristen K, Bradley, Kevin M, Hutter, Daniel, Matsuura, Mariko F, Rowold, Diane J, Benner, Steven A
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Beilstein-Institut 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4222377/
https://www.ncbi.nlm.nih.gov/pubmed/25383105
http://dx.doi.org/10.3762/bjoc.10.245
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author Merritt, Kristen K
Bradley, Kevin M
Hutter, Daniel
Matsuura, Mariko F
Rowold, Diane J
Benner, Steven A
author_facet Merritt, Kristen K
Bradley, Kevin M
Hutter, Daniel
Matsuura, Mariko F
Rowold, Diane J
Benner, Steven A
author_sort Merritt, Kristen K
collection PubMed
description Background: Many synthetic biologists seek to increase the degree of autonomy in the assembly of long DNA (L-DNA) constructs from short synthetic DNA fragments, which are today quite inexpensive because of automated solid-phase synthesis. However, the low information density of DNA built from just four nucleotide “letters”, the presence of strong (G:C) and weak (A:T) nucleobase pairs, the non-canonical folded structures that compete with Watson–Crick pairing, and other features intrinsic to natural DNA, generally prevent the autonomous assembly of short single-stranded oligonucleotides greater than a dozen or so. Results: We describe a new strategy to autonomously assemble L-DNA constructs from fragments of synthetic single-stranded DNA. This strategy uses an artificially expanded genetic information system (AEGIS) that adds nucleotides to the four (G, A, C, and T) found in standard DNA by shuffling hydrogen-bonding units on the nucleobases, all while retaining the overall Watson–Crick base-pairing geometry. The added information density allows larger numbers of synthetic fragments to self-assemble without off-target hybridization, hairpin formation, and non-canonical folding interactions. The AEGIS pairs are then converted into standard pairs to produce a fully natural L-DNA product. Here, we report the autonomous assembly of a gene encoding kanamycin resistance using this strategy. Synthetic fragments were built from a six-letter alphabet having two AEGIS components, 5-methyl-2’-deoxyisocytidine and 2’-deoxyisoguanosine (respectively S and B), at their overlapping ends. Gaps in the overlapped assembly were then filled in using DNA polymerases, and the nicks were sealed by ligase. The S:B pairs in the ligated construct were then converted to T:A pairs during PCR amplification. When cloned into a plasmid, the product was shown to make Escherichia coli resistant to kanamycin. A parallel study that attempted to assemble similarly sized genes with optimally designed standard nucleotides lacking AEGIS components gave successful assemblies of up to 16 fragments, but generally failed when larger autonomous assemblies were attempted. Conclusion: AEGIS nucleotides, by increasing the information density of DNA, allow larger numbers of DNA fragments to autonomously self-assemble into large DNA constructs. This technology can therefore increase the size of DNA constructs that might be used in synthetic biology.
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spelling pubmed-42223772014-11-07 Autonomous assembly of synthetic oligonucleotides built from an expanded DNA alphabet. Total synthesis of a gene encoding kanamycin resistance Merritt, Kristen K Bradley, Kevin M Hutter, Daniel Matsuura, Mariko F Rowold, Diane J Benner, Steven A Beilstein J Org Chem Full Research Paper Background: Many synthetic biologists seek to increase the degree of autonomy in the assembly of long DNA (L-DNA) constructs from short synthetic DNA fragments, which are today quite inexpensive because of automated solid-phase synthesis. However, the low information density of DNA built from just four nucleotide “letters”, the presence of strong (G:C) and weak (A:T) nucleobase pairs, the non-canonical folded structures that compete with Watson–Crick pairing, and other features intrinsic to natural DNA, generally prevent the autonomous assembly of short single-stranded oligonucleotides greater than a dozen or so. Results: We describe a new strategy to autonomously assemble L-DNA constructs from fragments of synthetic single-stranded DNA. This strategy uses an artificially expanded genetic information system (AEGIS) that adds nucleotides to the four (G, A, C, and T) found in standard DNA by shuffling hydrogen-bonding units on the nucleobases, all while retaining the overall Watson–Crick base-pairing geometry. The added information density allows larger numbers of synthetic fragments to self-assemble without off-target hybridization, hairpin formation, and non-canonical folding interactions. The AEGIS pairs are then converted into standard pairs to produce a fully natural L-DNA product. Here, we report the autonomous assembly of a gene encoding kanamycin resistance using this strategy. Synthetic fragments were built from a six-letter alphabet having two AEGIS components, 5-methyl-2’-deoxyisocytidine and 2’-deoxyisoguanosine (respectively S and B), at their overlapping ends. Gaps in the overlapped assembly were then filled in using DNA polymerases, and the nicks were sealed by ligase. The S:B pairs in the ligated construct were then converted to T:A pairs during PCR amplification. When cloned into a plasmid, the product was shown to make Escherichia coli resistant to kanamycin. A parallel study that attempted to assemble similarly sized genes with optimally designed standard nucleotides lacking AEGIS components gave successful assemblies of up to 16 fragments, but generally failed when larger autonomous assemblies were attempted. Conclusion: AEGIS nucleotides, by increasing the information density of DNA, allow larger numbers of DNA fragments to autonomously self-assemble into large DNA constructs. This technology can therefore increase the size of DNA constructs that might be used in synthetic biology. Beilstein-Institut 2014-10-09 /pmc/articles/PMC4222377/ /pubmed/25383105 http://dx.doi.org/10.3762/bjoc.10.245 Text en Copyright © 2014, Merritt et al. https://creativecommons.org/licenses/by/2.0https://www.beilstein-journals.org/bjoc/termsThis is an Open Access article under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The license is subject to the Beilstein Journal of Organic Chemistry terms and conditions: (https://www.beilstein-journals.org/bjoc/terms)
spellingShingle Full Research Paper
Merritt, Kristen K
Bradley, Kevin M
Hutter, Daniel
Matsuura, Mariko F
Rowold, Diane J
Benner, Steven A
Autonomous assembly of synthetic oligonucleotides built from an expanded DNA alphabet. Total synthesis of a gene encoding kanamycin resistance
title Autonomous assembly of synthetic oligonucleotides built from an expanded DNA alphabet. Total synthesis of a gene encoding kanamycin resistance
title_full Autonomous assembly of synthetic oligonucleotides built from an expanded DNA alphabet. Total synthesis of a gene encoding kanamycin resistance
title_fullStr Autonomous assembly of synthetic oligonucleotides built from an expanded DNA alphabet. Total synthesis of a gene encoding kanamycin resistance
title_full_unstemmed Autonomous assembly of synthetic oligonucleotides built from an expanded DNA alphabet. Total synthesis of a gene encoding kanamycin resistance
title_short Autonomous assembly of synthetic oligonucleotides built from an expanded DNA alphabet. Total synthesis of a gene encoding kanamycin resistance
title_sort autonomous assembly of synthetic oligonucleotides built from an expanded dna alphabet. total synthesis of a gene encoding kanamycin resistance
topic Full Research Paper
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4222377/
https://www.ncbi.nlm.nih.gov/pubmed/25383105
http://dx.doi.org/10.3762/bjoc.10.245
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