Development of Artificial Genetic Systems for Therapeutic Applications

Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are among the most remarkable biomolecules occurring in nature, since they store and transfer all genetic information using only four letters (G, C, A & T or U). In the last 60 years, since the discovery of the structure of nucleic acids, numerous chemically modified nucleosides and nucleotides have been produced and incorporated into oligomeric sequences either by chemical or enzymatic synthesis. These molecules were primarily designed to increase the complexity and stability of natural biopolymers for the development of nucleic acid-based drugs and, lately, for various biotechnological purposes. While many synthetic biopolymers are currently available, none of those possessing all four modified bases has been found to be functional in vivo.

Here, we pose an intriguing and challenging question: “What would happen if we could evolve a novel fully-modified genetic system within a cell?” These redesigned biomolecules with improved stability in serum could potentially open new horizons in the development of advanced therapeutics. The biomolecule template with all four artificial nucleobases could mimic the genetic functions of DNA and RNA in a cell. The fully morphed genetic alphabet could also encourage investigations on the in vitro evolution of nucleic acids enriching their complexity and functional diversity. The replacement of the original biomolecules by a third type of nucleic acid might foster the evolution of chemically redesigned cells and safe genetically modified organisms.

Based on this premise, this doctoral thesis aims at exploring artificial nucleic acids with altered nucleobase or sugar moieties as versatile analogs of natural biopolymers. Non-canonical nucleotide analogs, containing 7-substituted-7-deaza- or 8-substituted-purines as nucleobases pairing with 5-substituted pyrimidines, were chosen to construct an unprecented genetic information system in vivo (DZA). Another alternative biopolymer, namely, 1,5-anhydrohexitol nucleic acid (HNA), was selected for the development of specific inhibitors against the therapeutically relevant target Vascular Endothelial Growth Factor (VEGF).

These studies intend to develop chemically modified nucleic acids for their subsequent application as a stable and safe tool for therapeutics and biotechnology. These redesigned nucleic acids can be used as genetic elements to code for functional proteins with desired functions (DZA) or specifically inhibit target biomolecules in a host organism (HNA). Alternative genetic molecules with increased stability in the biological environment could improve the existing therapeutics as well as assist in the development of new advanced nucleic acid drugs. Moreover, modified nucleic acid modules may open new perspectives in the understanding of the enzymatic pathways by interacting with the target molecules.

In the first project (Chapters 2-4), a chemically redesigned DNA analog, in which all four nucleobases are functionalized (denoted as DZA), was investigated. In Chapter 2, the synthetic DZA biomolecules composed of 5-Cl-dU, 7-deaza-dA, 5-F-dC, and 7-deaza-dG were used to transfer the genetic information in living organisms. By using a gene encoding resistance to the antibiotic trimethoprim, we demonstrated that a fully morphed DNA was successfully replicated in vitro and served as genetic template in vivo.

In Chapter 3, we describe in more details the choice of the 5-Cl-dU:7-deaza-dA base pair as an excellent alternative to the natural dT:dA. The role of 5-Cl-dU as a possible component of a chemically modified genome has been discussed regarding its influence on duplex stability and DNA polymerase incorporation properties. The search for its counterpart among different deoxyadenosine analogs showed that a stable duplex formation as well as the synthesis of long constructs, more than 2 kb, were successful with the 5-Cl-dU:7-deaza-dA combination in the presence of Taq DNA polymerase.

In the study described in Chapter 4, we focused on the selection of a second dG:dC base pair alternative to complete the four-base artificial genetic alphabet. The search for optimal combinations of the synthetic purine-pyrimidine base pairs was based on their enzymatic biocompatibility. We demonstrated that the naturally occurring four DNA letters A, T, G, and C can be successfully replaced by 7-deazapurines and 5-substituted pyrimidines during in vitro replication of random oligonucleotide libraries as well as extended 1.5 kb genetic templates. The most fruitful combinations include: 7-deaza-dG or 7-F-deaza-dG pairing with 5-F-dC or 5-Me-dC together with the 5-Cl-dU:7-deaza-dA base pair. The in vitro replication of a 0.5 kb fragment was also achieved with a nucleotide set containing dI:5-Br-dC and 5-Cl-dU:7-deaza-dA, although it was not successful in the synthesis of a short random DZA library. Furthermore, we demonstrated that the modified segments can selectively block restriction enzymes, and still be able to bypass the bacterial replication machinery. These evidences make the fully morphed nucleic acids highly promising for synthetic biology and biotechnological applications. We demonstrated that DZA segments could be used to produce diverse DNA libraries and extended PCR fragments, as well as to control restriction enzyme cleavage or synthesize functional genetic templates.

In Chapter 5, the generation of HNA aptamers was described. We successfully evolved high-affinity HNA-RNA hybrid aptamers against rat VEGF164 starting from a completely random library using just seven rounds of in vitro selection. The selected aptamers exhibited affinity in the picomolar range to their target protein and possessed a remarkable stability against DNase I.

The last chapter (Chapter 6) summarizes the obtained results and includes prospects and future plans. Overall our results demonstrate that chemically redesigned biomolecules different from natural DNA/RNA can code and accurately transfer the genetic information in living cells, as well as providing stable inhibitors against therapeutic targets. At the forefront of research in synthetic biology and therapy, our next goal is to explore whether these synthetic molecules can be used for more complicated tasks such as the development of advanced therapy and a genetic manipulation tool.