How miRNAs can protect humans from coronaviruses COVID-19, SARS-CoV, and MERS-CoV

Viral diseases cause signicant harm to human health and often cause high mortality. In the past twenty years, humanity has undergone infection by SARS-CoV (severe acute respiratory syndrome), MERS-CoV (Middle East respiratory syndrome) and COVID-19 coronaviruses, which spread from animals to humans and from person to person. These diseases have led to large economic losses. To ght coronaviruses and other viruses, it is proposed to use miRNAs, which regulate protein synthesis at the translational level. MirTarget program was used to determine the following binding characteristics: the locations of miRNA binding sites in the 3'UTR, 5'UTR, and CDS; the free energy interaction ∆ G between miRNA and mRNA; the ΔG/ΔGm value, where ΔGm is equal to the free energy binding of miRNA with its full complementary nucleotide sequence; and the nucleotide interaction schemes between miRNAs and mRNAs. Out of 2565 miRNAs, miR-4778-3p, miR-6864-5p and miR-5197-3p were identied as the most effectively interacting with the gRNA of SARS-CoV, MERS-CoV and COVID-19, respectively. Based on the miR-4778-3p, miR-6864-5p and miR-5197-3p sequences, complete complementary miRNA (cc-miR) binding sites in the gRNA coronaviruses were created. The detected binding sites of these cc-miRs did not form intramolecular complexes in the 2D structure of the gRNA of SARS-CoV, MERS-CoV, and COVID-19 with a value of more than 85%. Therefore, the cc-miRs will bind gRNA at these sites without competition. The cc-miRs for SARS-CoV, MERS-CoV, and COVID-19 did not have target genes among the 17508 human coding genes with a ΔG/ΔGm of more than 85%, which implies the absence of side effects of these cc-miRs on the translation of human mRNAs. cc-miRs can be used as therapeutic agents by incorporating them into exosomes or other vesicles and introducing them into the blood or lung by inhalation. The introduction of cc-miR into the blood will suppress the reproduction of the virus in the blood and in all organs into which it can enter. The proposed method of inhibiting the reproduction of coronaviruses can be used for other viruses.

Zou et al., 2020). Therefore, new approaches to the treatment of viral diseases are required. All viruses multiply using a nucleic acid synthesis system and a protein synthesis system in an infected cell. In animal cells, protein synthesis is controlled by miRNA (mRNA-inhibiting RNA).. These nanosized molecules speci cally interact with the mRNAs of target genes and can reversibly suppress translation or contribute to the degradation of mRNA (Bartel, 2018;Krol et al., 2010;Zhdanov, 2018).This biological role of miRNAs can help in the ght against viral reproduction, since the synthesis of viral proteins occurs in the host cell. The use of miRNAs for this purpose requires with the ful llment of a number of conditions. The miRNA must highly speci cally bind to the mRNA of the viral genome (gRNA) or parts of its genome, which can be translated as a whole or in fragments. An arbitrary set of nucleotides in synthetic miRNAs that bind with complete complementarity to the RNA genomes of viruses can have many side effects consisting of the binding of these miRNAs to human mRNAs (Trobaugh,  nucleotide sequences in a special format. This program determines the following binding characteristics: the start of the miRNA binding site on the mRNA; the location of the miRNA binding site (3'UTR, 5'UTR, CDS); the interaction free energy (∆G, kJ/mole); and the nucleotide interaction schemes between miRNAs and mRNAs. The ratio of ΔG/ΔGm (%) was determined for each binding site, where ΔGm is equal to the free energy binding of miRNA with its full complementary nucleotide sequence. The MirTarget program found hydrogen bonds between adenine (A) and uracil (U), guanine (G) and cytosine (C), G and U, A and C. The distances between A and C were equal to 1.04 nanometers, between G and C, and between A and U were equal to 1.03 nanometers, and between G and U equal to 1.02 nanometers. The numbers of hydrogen bonds in the G-C, A-U, G-U and A-C interactions were found to be 3, 2, 1 and 1, respectively (Leontis et al., 2002).

Results
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 nd 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 speci cally. 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 identi ed 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 con dence that cc-miR2 can interact with gRNA without side effects on the human protein-coding genes.
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 con rmed 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;. 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 di cult. 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.

Conclusion
The results show that it is not di cult to select the nucleotide composition of miRNA, siRNA or pi-RNA for target binding to mRNA. The challenge is to predict and verify the absence of side effects of the small RNA on other genes. Unfortunately, this mandatory analysis is performed only at the stage of experimental testing, which will take a long time and is not guaranteed to end successfully. Our proposed method of searching for synthetic miRNA and evaluating its side effects can signi cantly simplify the generation of the miRNA of interest.    Characteristics of miRNA interactions with the gRNA of coronaviruses and human mRNA genes