Modern approaches to artificial gene synthesis: aspects of oligonucleotide synthesis, enzymatic assembly, sequence verification and error correction

Synthetic biology is a rapidly developing field aimed at engineering of biological systems with predictable properties. Synthetic biology accumulates the achievements of modern biological sciences, programming and computational model­ing as well as engineering technologies for creation of biologi­cal objects with user-defined properties. Evolution of synthetic biology has been marked by a number of technological developments in each of the mentioned fields. Thus, significant reduction in cost of DNA sequencing has provided an easy access to large amounts of data on the genetic sequences of various organisms, and decreased the price of the DNA sequence synthesis, which, analogous to Moore’s law, resulted in an opportunity to create a lot of potential genes without the time – consuming and labor – intensive traditional methods of molecular biology. Development of system biology has allowed forming a deeper understanding of the functions and relationship of natural biological models, as well as of the computational models describing processes at the cell and system levels. Combination of these factors has created an op­portunity for conscious changes of natural biological systems. In this review the modern approaches to oligonucleotide gene assembly synthesis are discussed, including such aspects as protocols for gene assembly, sequence verification, error cor­rection and further applications of synthesized genes

RNase T1 mimicking artificial ribonuclease

Recently, artificial ribonucleases (aRNases)—conjugates of oligodeoxyribonucleotides and peptide (LR)4-G-amide—were designed and assessed in terms of the activity and specificity of RNA cleavage. The conjugates were shown to cleave RNA at Pyr-A and G–X sequences. Variations of oligonucleotide length and sequence, peptide and linker structure led to the development of conjugates exhibiting G–X cleavage specificity only. The most efficient catalyst is built of nonadeoxyribonucleotide of unique sequence and peptide (LR)4-G-NH2 connected by the linker of three abasic deoxyribonucleotides (conjugate pep-9). Investigation of the cleavage specificity of conjugate pep-9 showed that the compound is the first single-stranded guanine-specific aRNase, which mimics RNase T1. Rate enhancement of RNA cleavage at G–X linkages catalysed by pep-9 is 108 compared to non-catalysed reaction, pep-9 cleaves these linkages only 105-fold less efficiently than RNase T1 (kcat_RNase T1/kcat_pep-9 = 105)

QCM-based rapid analysis of DNA

We demonstrated that it is possible to break apart double-stranded DNA using a thickness shear mode resonator. This procedure measures the force of bond rupture. The measured rupture forces were in the range of 10–40 pN. The procedure was tested experimentally with model complementary and non-complementary oligonucleotides. In the case of the DNA mismatch we observed a signal at a driving voltage of 0.8 V (corresponding to a shear force of 11 pN), while in the case of complementarity the signal at the voltage of 3 V (36 pN) appeared. This procedure may be the ground for the development of new types of biosensors measuring the force of affine interactions (probe/biomolecular target) on the surface of the sensor element