Oligonucleotide gene therapy (OGT) agents (e. g. antisense, deoxyribozymes, siRNA and CRISPR/Cas) are promising therapeutic tools. Despite extensive efforts, only few OGT drugs have been approved for clinical use. Besides the problem of efficient delivery to targeted cells, hybridization specificity is a potential limitation of OGT agents. To ensure tight binding, a typical OGT agent hybridizes to the stretch of 15-25 nucleotides of a unique targeted sequence. However, hybrids of such lengths tolerate one or more mismatches under physiological conditions, the problem known as the affinity/specificity dilemma. Here, we assess the scale of this problem by analyzing OGT hybridization-dependent off-target effects (HD OTE) in vitro , in animal models and clinical studies. All OGT agents except deoxyribozymes exhibit HD OTE in vitro , with most thorough evidence of poor specificity reported for siRNA and CRISPR/Cas9. Notably, siRNA suppress non-targeted genes due to (1) the partial complementarity to mRNA 3'-untranslated regions (3'-UTR), and (2) the antisense activity of the sense strand. CRISPR/Cas9 system can cause hundreds of non-intended dsDNA breaks due to low specificity of the guide RNA, which can limit therapeutic applications of CRISPR/Cas9 by ex-vivo formats. Contribution of this effects to the observed in vivo toxicity of OGT agents is unclear and requires further investigation. Locked or peptide nucleic acids improve OGT nuclease resistance but not specificity. Approaches that use RNA marker dependent (conditional) activation of OGT agents may improve specificity but require additional validation in cell culture and in vivo .

Oligonucleotide gene therapy (OGT) agents suppress specific mRNAs in cells and thus reduce the expression of targeted genes. The ability to unambiguously distinguish cancer from healthy cells can solve the low selectivity problem of OGT agents. Cancer RNA markers are expressed in both healthy and cancer cells with a higher expression level in cancer cells. We have designed a DNA‐based construct, named DNA thresholder (DTh) that cleaves targeted RNA only at high concentrations of cancer marker RNA and demonstrates low cleavage activity at low marker concentrations. The RNA‐cleaving activity can be adjusted within one order of magnitude of the cancer marker RNA concentration by simply redesigning DTh. Importantly, DTh recognizes cancer marker RNA, while cleaving targeted RNA; this offers a possibility to suppress vital genes exclusively in cancer cells, thus triggering their death. DTh is a prototype of computation‐inspired molecular device for controlling gene expression and cancer treatment.

In this paper, we introduce a novel encapsulation system for DNA oligonucleotides. Supramolecular assembly of melamine cyanurate encapsulates DNA at pH 7 and start to release it at pH less than 6.5. We study the assembly and disassembly in time in specially designed reaction-diffusion system. Magnesium ions allow spatial separation of DNA with the highest DNA concentration in the core of melamine cyanurate capsule. Molecular dynamics (MD) simulation shows that DNA acts as a nucleation centre for melamine cyanurate. Dataset of fluorescent images analysed by machine learning algorithms indicates correlation between structure of melamine cyanurate capsules for DNA trapping and concentration of magnesium ions. The concentration of magnesium ions can be recognized with 96% accuracy proving that all environmental conditions are extremely important during the self-assembly and should be considered for laboratory and industrial applications of the suggested approach. Moreover, the This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).