To compile the nucleotide sequence of a fully complementary miRNA (cc-miR) to its binding site in the gRNA of COVID–19, it was necessary to find the cc-miR precursor. Despite the large size of the COVID–19 genome, in comparison with the human protein coding genes, only a few human miRNAs could bind to the viral genome with a ΔG/ΔGm value of 89% (Figure 3). We chose this value as a criterion for the adequacy of determining binding sites based on the requirement that different miRNAs with a length of 22 nt differ in two or more nucleotides, which allows them to act specifically. A decrease in this criterion, for example, by 5%, leads to an exponential increase in the number of putative target genes of a particular miRNA, which results in an uncertainty in the selectivity to the target genes of this miRNA. To create cc-miR, we selected miR–5197–3p, which out of the 2565 known miRNAs (miRBase), binds to the gRNA of the COVID–19 virus with the largest ΔG/ΔGm value of 89% and a miRNA length of 23 nt (Figure 1).
Of the 17,508 human genes available in our gene database, miR–5197–3p bound to the mRNA of only a few genes with similar characteristics (Figure 3). Therefore, the use of miR-5197–3p as a therapeutic agent will cause side effects on the identified genes. Fig. 2 shows the scheme of the interaction of miR–5197–3p with the gRNA.
Note: The schemes above are shown as follows: miRNA; the position at the beginning of the binding site (nt); the value of the free binding energy ∆G (kJ/mole); the ∆G/∆Gm value (%); and the length of the miRNA (nt). Bold letters indicate nucleotides forming non-canonical pairs and nucleotides that do not form hydrogen bonds.
Based on this scheme, a structure for cc-miR2 (cc-miR for the gRNA of COVID–19) with a length of 25 nt was proposed with the replacement of non-canonical nucleotide pairs by canonical ones and the addition of G and U nucleotides at the ends of miR–5197–3p to increase the free interaction energy of cc-miR2 with the gRNA of COVID–19. The interaction characteristics of cc-miR2 with the gRNA of COVID–19 are given in the Figure 1 and the diagram in Fig. 2a. The miRNAs with a length of 25 nt are found among natural miRNAs (miRBase) and can interact with gRNA as part of RISC (RNA-induced silencing complex). The created cc-miR2 was completely complementary to the gRNA and interacted weakly with mRNA of 17508 genes, some of which are presented in Figure 3. This result gives confidence that cc-miR2 can interact with gRNA without side effects on the human protein-coding genes.
Similarly, cc-miRs were designed to bind to the MERS-CoV (cc-miRm) and SARS-CoV (cc-miRs) genomes. Of the 2565 known human miRNAs, miR–6864–5p and miR–4778–5p could interact more strongly than other miRNAs with the gRNA of MERS-CoV and SARS-CoV, respectively (Figure 1, Figure 4). The cc-miRm and cc-miRs do not bind to mRNAs of the 17,508 studied human genes with ∆G/∆Gm values above 85%.
Therefore, these synthetic miRNAs are not expected to have any side effects upon interacting with human genes. The nucleotides of the cc-miR2, cc-miRm, cc-miRs binding sites during intramolecular interactions in the COVID–19, SARS-CoV, and MERS-CoV genomes can bind to other gRNA sites with a value of ∆G/∆Gm less than 85%. Consequently, synthetic cc- miR2, cc-miRm, and cc-miRs will interact with the binding sites in the genomes of the three coronaviruses without competition with other gRNA sites.
Generating cc-miR is an inexpensive and relatively short procedure, similar to the synthesis of primers. In cell culture, the effects of cc-miR and miRNA on coronaviruses need to be tested. The proposed hypothesis can be confirmed in the laboratory with the approval and ability to conduct inexpensive and time-saving tests of the proposed cc-miR as a means of combating coronavirus. Since the size of cc-miR is approximately 10 nm, it can be delivered with blood to many organs as a component of ordinary exosomes in human blood, which measure 30–60 nm (Karothia et al., 2019; Mnyandu et al., 2020; Zhang et al., 2020). The cc-miR contained in exosomes can be introduced into the lungs by inhalation. The proposed method for combating coronaviruses using miRNA does not have side effects and is economically inexpensive. Like all human miRNAs, cc-miR is susceptible to degradation by nucleases, and its removal from the body is not difficult.
A well-known observation that COVID–19 infects children under the age of 15 less than adults over 60 (http://www.cidrap.umn.edu/news-perspective/2003/05/estimates-sars-death- rates-revised-upward) can be explained by the expression of piwi-interacting RNA (pi-RNA) and miRNA, whose concentrations change during ontogenesis: the proportion of pi-RNA decreases with age, and the proportion of miRNA increases (Li et al., 2018). It is possible that the expression of one or more pi-RNAs that can bind to the viral gRNA decreases during ontogenesis, and the virus begins to multiply. Similarly, the concentration of miRNAs capable of binding to the gRNA and are expressed in the early stages of ontogenesis can decrease, and the virus begins to multiply.