5'-DMT-protected double-stranded DNA: Synthesis and competence to enzymatic reactions.

The functionalization of 5'-OH group in nucleic acids is of significant value for molecular biology. In the current work we discovered that acid-labile 4,4'-dimethoxytrityl protecting group (DMT) of oligonucleotides (ONs) is stable under PCR conditions and does not interfere with activity of DNA polymerases. So application of 5'-DMT-protected ONs could allow producing both symmetric and asymmetric 5'-DMT-blocked double-stranded DNA (dsDNA) fragments. We demonstrated that the presence of thiol compounds (mercaptoethanol and dithiothreitol) in PCR mixture is undesirable for the stability of DMT-group. DMT-ONs can be successfully used during polymerase chain assembly of synthetic genes. We tested 5'-DMT dsDNA in blunt-end DNA ligation reaction by T4 DNA ligase and found that it could not be ligated with 5'-phosphorylated DNA fragments, namely linearized plasmid vector pJET1.2/blunt. Possible reason for this is steric hindrance created by bulky and rigid DMT-group, that prevents entering enzyme active site. We also demonstrated that 5'-DMT modification of dsDNA does not affect activity of T5 5',3'-exonuclease towards both ssDNA and dsDNA. Further screening of the exonucleases, sensitive to 5'-DMT-modification or search of ways to separate long 5'-DMT-ssDNA and 5'-OH-ssDNA could allow finding application of 5'-DMT-modified oligo- and polynucleotides.

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

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.

Oligonucleotide Preparation Approach for Assembly of DNA Synthons.

An effective oligonucleotide preparation approach for the thermodynamically balanced, inside-out (TBIO) PCR-based assembly of long synthetic DNA molecules (synthons) is described in the current work. We replaced the necessity to purify individual oligonucleotides with just one purification procedure per approximately 500 base pairs (bp) of duplex DNA. So for an enhanced green fluorescent protein (EGFP) gene of 717 bp, we synthesized 24 oligonucleotides with a length of 50 bases and performed just two solid-phase extraction (SPE) purification procedures. It was found that the capacity of ZipTip microextractors, usually used for sample desalting in proteomics, perfectly corresponds to the gene synthesis scale (40-60 pmol). The robustness of the approach was validated with a 65-mer oligonucleotide design of the same gene. The modification of the oligonucleotide concentration gradient from the original TBIO scheme substantially increased the purity of the PCR product. We proposed a mechanism for the formation of supramolecular structures, which often occur during TBIO assembly. By using the proposed workflow, any laboratory with a standard facility for molecular biology manipulation, a 16-channel oligonucleotide synthesizer, and a conventional thermocycler has the ability to prepare one gene with a length of about 700 bp per day.