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Programmed assembly of long DNA synthons: design, mechanism, and online monitoring

Authors:
Aliaksei Yantsevich at National Academy of Sciences of Belarus
Aliaksei Yantsevich
Veronika Shchur at National Academy of Sciences of Belarus
Veronika Shchur
Sergey A Usanov at National Academy of Sciences of Belarus
Sergey A Usanov
Michail A Shapira
Michail A Shapira
Michail A Shapira
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Abstract and Figures

Synthesis of custom de novo DNA sequences is highly demanded by fast-growing field of synthetic biology. Usually DNA sequences with length more than 1 kb are assembled from smaller synthetic DNA fragments (synthons) obtained by PCR assembly. The ability to synthesize longer synthons sufficiently reduces efforts and time for DNA synthesis. We developed a novel rational oligonucleotide design and programmed approach for the assembly of synthetic DNA synthons up to 1550 bp. The developed procedure was thoroughly investigated by synthesis of cholesterol oxidase gene from Streptomyces lavendulae (1544 bp). Our approach is based on combined design, oligonucleotide concentration gradient, and specialized assembly program that directs assembly reaction to full-length gene in a stepwise manner. The process includes conventional thermodynamically balanced assembly, thermodynamically balanced inside-out elongation, and further amplification. The ability of DNA polymerase to perform programmed assembly is highly influenced by the presence of 5′ → 3′-exonuclease activity. Oligonucleotide probing of PCR assembly products allowed us to shed light on the nature of high molecular weight spurious by-products and to understand the mechanism of their formation. For the first time, we applied light scattering techniques for tracking of oligonucleotide annealing, analysis of gene assembly products, and even for real-time monitoring of gene assembly process.
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METHODS AND PROTOCOLS
Programmed assembly of long DNA synthons: design, mechanism,
and online monitoring
Veronika V. Shchur
1
&Yuliya P. Burankova
1
&Michail A. Shapira
1
&Dmitry V. Klevzhits
1
&Sergei A. Usanov
1
&
Aleksei V. Yantsevich
1
Received: 24 May 2019 /Revised: 7 August 2019 /Accepted: 21 August 2019
#Springer-Verlag GmbH Germany, part of Springer Nature 2019
Abstract
Synthesis of custom de novo DNA sequences is highly demanded by fast-growing field of synthetic biology. Usually DNA
sequences with length more than 1 kb are assembled from smaller synthetic DNA fragments (synthons) obtained by PCR
assembly. The ability to synthesize longer synthons sufficiently reduces efforts and time for DNA synthesis. We developed a
novel rational oligonucleotide design and programmed approach for the assembly of synthetic DNA synthons up to 1550 bp. The
developed procedure was thoroughly investigated by synthesis of cholesterol oxidase gene from Streptomyces lavendulae (1544
bp). Our approach is based on combined design, oligonucleotide concentration gradient, and specialized assembly program that
directs assembly reaction to full-length gene in a stepwise manner. The process includes conventional thermodynamically
balanced assembly, thermodynamically balanced inside-out elongation, and further amplification. The ability of DNA polymer-
ase to perform programmed assembly is highly influenced by the presence of 5′→3′-exonuclease activity. Oligonucleotide
probing of PCR assembly products allowed us to shed light on the nature of high molecular weight spurious by-products and to
understand the mechanism of their formation. For the first time, we applied light scattering techniques for tracking of oligonu-
cleotide annealing, analysis of gene assembly products, and even for real-time monitoring of gene assembly process.
Keywords Gene synthesis .Concatemer .Dynamic light scattering .Polymerase chain reaction .Cholesterol oxidase
Introduction
During the last few decades, the progress in the methodology
of the DNA synthesis created fundamentally new opportuni-
ties for molecular biology. These opportunities formed the
basis for a new scientific discipline —synthetic biology.
The ability to synthesize custom DNA molecules along with
the insight in structure-function relations for biosystems opens
unlimited possibilities for biotechnology in the nearest future.
Nowadays, artificial DNA sequences or synthetic genes are a
powerful tool in molecular biology and biotechnology field.
Synthetic genes have already found applications for protein
engineering (Cox et al. 2007; Plesa et al. 2018), metabolic
engineering, species-specific sequence optimization (Kudla
et al. 2009; Lichtenberg et al. 2009), and even whole synthetic
genomes (Gibson et al. 2008; Smith et al. 2003). All currently
described methods of DNA synthesis are based on ligase
chain reaction (LCR) (Smith et al. 2003; Strizhov et al.
1996) or polymerase chain reaction (PCR) (Stemmer et al.
1995) that allows to assemble pools of oligonucleotides, ob-
tained by phosphoramidite chemistry. LCR techniques are
rarely used in practice.
The most commonly used artificial DNA synthesis tech-
niques currently rely on PCR-mediated assembly of target
DNA sequence (PCA) from oligonucleotides with length
40–100 bases. Usually the process consists of two stages:
assembly of target sequence from a pool of oligonucleotides
and enrichment of target gene from a crude PCR mixture by
additional amplification by flanking primers.
There are a lot of developed designs for PCA starting from
the procedure described by Stemmer in 1995 (Stemmer et al.
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00253-019-10099-4) contains supplementary
material, which is available to authorized users.
*Sergei A. Usanov
usanov@iboch.by
*Aleksei V. Yantsevich
al.yantsevich@gmail.com
1
Institute of Bioorganic Chemistry, National Academy of Sciences of
Belarus, Acad. Kuprevicha Street, 5/2, 220141 Minsk, Belarus
https://doi.org/10.1007/s00253-019-10099-4
Applied Microbiology and Biotechnology (2019) 103:9103–9117
/Published online: 12 September 2019
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
... Besides that, unmodified PCA has been simplified by combining the gene assembly and PCR reaction into one reaction [11][12][13]. Moreover, a combination of PCA and TBIO techniques has been shown to enable the synthesis of genes up to 1550 bp [14]. ...
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