A Suite of Therapeutically-Inspired Nucleic Acid Logic Systems for Conditional Generation of Single-Stranded and Double-Stranded Oligonucleotides

Abstract

Several varieties of small nucleic acid constructs are able to modulate gene expression via one of a number of different pathways and mechanisms. These constructs can be synthesized, assembled and delivered to cells where they are able to impart regulatory functions, presenting a potential avenue for the development of nucleic acid-based therapeutics. However, distinguishing aberrant cells in need of therapeutic treatment and limiting the activity of deliverable nucleic acid constructs to these specific cells remains a challenge. Here, we designed and characterized a collection of nucleic acids systems able to generate and/or release sequence-specific oligonucleotide constructs in a conditional manner based on the presence or absence of specific RNA trigger molecules. The conditional function of these systems utilizes the implementation of AND and NOT Boolean logic elements, which could ultimately be used to restrict the release of functionally relevant nucleic acid constructs to specific cellular environments defined by the high or low expression of particular RNA biomarkers. Each system is generalizable and designed with future therapeutic development in mind. Every construct assembles through nuclease-resistant RNA/DNA hybrid duplex formation, removing the need for additional 2′-modifications, while none contain any sequence restrictions on what can define the diagnostic trigger sequence or the functional oligonucleotide output.

Specificity of oligonucleotide gene therapy (OGT) agents

“…The system is comprised of a 3-input AND gate and a NOT gate that are constructed by coupling the sense hybrid (activated by the connective tissue growth factor (CTGF) that used as an RNA trigger) with the antisense hybrid (designed to repress the strand exchange in the presence of a trigger sequence derived from the Kirsten rat sarcoma proto-oncogene (KRAS) mRNA). Both hybrids and the CTGF trigger are required for the dsRNA release, while the presence of the KRAS trigger inhibits strand exchange 22 .…”

“…They used several RNA hairpins as in the work of Masu et al 121 and a simple logic for the formation of DsiRNA or shRNA in response to the detection of the mRNA target followed by processing the complex by intracellular enzymes yielding active siRNA 124 . In 2019, Zakrevski et al developed four logic gates for trigger-inducible or repressible siRNA release (Figure 6 ) 22 . The undoubted efficiency of this strategy in extracellular experiments was not supported by cell experiments.…”

“…This low OGT specificity can cause HD OTEs in vivo. Designing OGT with low affinity to unintended RNA molecules remains a desirable but challenging task [9,[20][21][22][23][24][25][26].…”

Editorial for the Special Issue on “Nucleic Acid Architectures for Therapeutics, Diagnostics, Devices and Materials”

“…This special issue assembles 11 original articles (with seven research manuscripts and four reviews) which nicely outline several advances made in the field of RNA and DNA nanotechnology. Unified by the versatile use of the intriguing biopolymers, these manuscripts explore the various facets of nucleic acid nanostructures such as design, production, and characterization of RNA and DNA nanoassemblies [1,2,3,4], rational design of functional molecular machines [5,6,7,8], immunorecognition of nucleic acid nanoparticles [8], in vivo delivery of therapeutic nucleic acids [9,10], and nucleic acid-based biosensors [11]. We anticipate this special issue to be accessible to a wide audience, as it explores not only the biological aspects of nucleic acid nanodesigns, but also different methodologies of their production, their interactions with other classes of biological molecules, physicochemical characteristics, and possible applications.…”

“…Towards that end, Zakrevsky et al describe the use of logic gates to mimic complex molecular biological phenomena with the potential for use in cells [7]. In their research, the authors computationally design and characterize DNA/RNA hybrid-based logic gates suited for the conditional release of single- and double-stranded nucleic acids, thus displaying the possibility of therapeutic action.…”