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 Applied Microbiology and Biotechnology https://doi.org/10.1007/s00253-019-10099-4