**2.1 The concept of the DNA-FACE™ method for directional DNA fragment amplification and protein concatemers construction**

The rapid development of synthetic biology has generated a high demand for synthetic "artificial" genes that do not exist in Nature. Whatever their application, the construction of such synthetic genes may require the use of repetitive DNA fragments. However, one of the major limitations of the chemical synthesis of DNA is the difficulty in assembling repeated segments into longer DNA sequences. The ability to construct DNA molecules of any sequence or size is crucial for numerous applications, especially in the areas of biomedical and biotechnological research.

The biosynthesis-based strategies, that can ensure control over joining repeated DNA segments (multimerization or concatemerization), which would enable head-to-tail arrangement of the monomers of DNA, RNA, and peptides, require the development of special DNA manipulation methods. Otherwise, the obtained arrangements of monomeric units would be random and result in a mixture of head-to-head, tail-to-tail orientations of DNA fragments within the assembled multimeric DNA. Such randomized monomer arrangement could render any DNA construct useless for any rational applications, as it would disable a constructed DNA molecule from performing its desired function of encoding the genetic information about specific RNA and protein. For example, even a single undesired tailto-tail segment within a constructed DNA multimer causes a nonsense amino acid sequence translation within this segment or even further downstream, an appearance of stop codons with inverted DNA segment, or a prematurely terminated translation of the constructed gene. Thus, controlling the mode of multimerization is pivotal in downstream processes after DNA multimerization. Furthermore, the controlled head-to tail-arrangement of the multimerized DNA segments provides stability of the recombinant DNA plasmid-vector and allows for a constructed operon expression.

Several alternative strategies for the construction of concatemeric genes have been developed so far [15–29]. However, most of the established technologies suffered from several problems, such as (*i*) limitations on the sequence of the DNA segment serving as the monomer; (ii) technical difficulties in joining of the DNA segments; (*iii*) an excessively complicated reaction, leading to the necessity of tedious DNA manipulations (*iv*) an inadequate copy number of the monomers within the formed multimer; (*v*) inability to repeat another round of the DNA fragment multimerization, if a desired monomer copy number within the resulting concatemeric DNA was not obtained. There were also two critically important problems; (*vi*) completion of a DNA concatemer formation without the ability to express coded RNA and proteins; (vii) codon discontinuity in the newly created ORF, which would prevent its expression and the production of the final result (a polypeptide/protein, containing multiple, linked together, bioactive peptides with programmed functions, without off-frame segments) [15–26].

A simple and efficient method was developed by us to make concatemeric "artificial" proteins or to emulate the old novel – "Frankenstein" proteins, composed of multiple functional parts, dedicated to suit a particular task. The DNA-FACE™ technology enables both homoconcatemers and heteroconcatemers formation, which highly enhances the pre-programmed functionality of the resulting "artificial" proteins (**Figure 1**). The technology allows for insertion of the synergistically acting bioactive

#### **Figure 1.**

*Potential protein products are obtainable with the DNA-FACE™ technology.*

peptides into the nascent concatemeric "Frankenstein" protein. The examples of such bioactive peptides are: different epitopes or antigen domains incorporated during a vaccine construction or combinations of various pro-regenerative peptides/protein segments. The technology is based on the custom vector-enzymatic system, which employs: (*i*) atypical Type IIS restriction endonucleases (REases). These Type IIS REases possess unique features: the ability to recognize 4-7 base pairs (bp) DNA sequence and to cleave at a fixed distance outside this sequence. Out of the known Type IIS REases, the DNA-FACE™ uses SapI (or its isoschizomers), which generate 3-nt protruding DNA ends; (*ii*) DNA ligase and (*iii*) dedicated amplification-expression vectors. DNA-FACE™ offers a significant improvement from earlier strategies [17]. It highly improves the construction of DNA concatemers, additionally allowing for the formation of continuous, multimeric ORFs as well as concatemeric proteins, with the desired monomer copy number and polymer/co-polymer length. **Figure 2** shows schematically the DNA fragment amplification reaction and its potential for employing multiple amplification cycles.
