The miRNA COMPLEXES AGAINST CORONAVIRUSES COVID-19, SARS-CoV, and MERS-CoV

doi:10.21203/rs.3.rs-19592/v1

The possibility of using miRNA (mRNA-inhibiting RNA) to inhibit infections caused by the coronaviruses COVID-19, SARS-CoV, and MERS-CoV has been shown. Using bioinformatics approaches, completely complementary miRNA (cc-miRNA) complexes were predicted to be able to bind and inhibit the translation of coronavirus proteins and the replication of COVID-19, SARS-CoV, and MERS-CoV genomes. For complexes of seven cc-miRc for COVID-19, seven cc-miRs for SARS-CoV, and eight cc-miRm for MERS-CoV, the interactions with the RNA genomes (gRNAs) of the corresponding coronaviruses was evaluated. The free energy of the interactions of cc-miRNAs with binding sites was significantly higher than the free energy of the interactions with other regions in gRNA, which ensures high selectivity of the binding of cc-miRNAs. Weak binding of cc-miRNAs to the mRNAs of 17508 human genes was shown, which suggests the absence of side effects of the cc-miRNAs in humans. A feature of this method is the simultaneous inhibition of translation and replication by several cc-miRNAs binding from the 5' end to the 3' end of gRNA. The use of several cc-miRNAs to suppress infections allows each of them to be used at a lower concentration to avoid side effects when one cc-miRNA is introduced into humans at a high concentration.

General Biochemistry

Bioinformatics

Virology

Biotechnology and Bioengineering

Molecular Biology

miRNA

human

CORONAVIRUSE

COVID-19

SARS-CoV

MERS-CoV

miRNAs (mRNA-inhibiting RNAs) are involved in the regulation of gene expression at the translational level [1]. These nanosized molecules with a length of 6–8 nm can bind to mRNAs with all 19–28 nucleotides, providing high selectivity for interaction with a single gene or several target genes [2,3]. Based on this property of miRNA, attempts have been made to regulate the expression of viral genes using natural miRNAs or by creating synthetic siRNAs [4,5,6]. In some cases, this approach gave a positive result in vitro but was not applicable in vivo for several reasons, including the lack of evidence that miRNA and siRNA do not have side effects in humans or experimental animals [7,8,9,10]. The basis of our approach to the use of miRNAs in the fight against coronaviruses COVID–19, SARS-CoV, and MERS-CoV is to create completely complementary miRNA (cc-miRNA) that will highly specifically inhibit the translation of viral proteins by strong interactions with the RNA genomes (gRNA) of viruses. Such cc-miRNAs associated with the gRNA of the virus will inhibit genome replication. Thus, two goals are achieved by using cc-miRNA: to stop the synthesis of proteins of the virus that has entered the cell and to inhibit the reproduction of its genome. Such actions of cc-miRNAs on the virus should be highly specific and not have side effects on any human genes.

The first task of this study was to identify human miRNAs that would have the greatest effect on the expression of the genomes of the coronaviruses COVID-19, SARS-CoV, and MERS-CoV at the translational level. Then, cc-miRNAs were created that could efficiently bind to the gRNA nucleotide sequence at the 5'-end to avoid wasting the resources of the recipient cell on the synthesis of all proteins encoded by the viral genome. Furthermore, cc-miRNAs were also created for other parts of gRNA to enhance translational inhibition by applying two or more cc- miRs. To inhibit genome replication, we searched for cc-miRNAs with binding sites at the 3' end of gRNA to block replication at the beginning of the process. To assess the side effects of cc- miRNAs in humans, the characteristics of the interactions of cc-miRNAs with the mRNAs of 17508 human genes in our database were determined. In addition, intramolecular interactions of the cc-miRNA binding site with gRNA regions were taken into account.

Creation of the cc-miRc complex for the gRNA of COVID-19

Despite the large gRNA of COVID-19, in comparison with human protein-coding genes, only a few human miRNAs with a ΔG/ΔGm of 90% or more could bind to the COVID-19 genome. We chose this ΔG/ΔGm value as a performance criterion based on the requirement that different miRNAs with a length of 22 nucleotides (nt) are different by two or more nucleotides, which allows them to bind specifically. For example, a decrease in this criterion by 5% leads to an increase in the putative target genes of a particular miRNA by a factor of many, which leads to a large number of false target genes of the miRNA. To create cc-miRc (completely complementary miRNA of COVID-19), we chose ID02510.3p-miR, ID00448.3p-miR, miR-3154, miR-7114-5p, miR-5197-3p, ID02750.3p-miR,

and ID01851.5p-miR, which bind with the gRNA of COVID-19 with ΔG/ΔGm equal to 89% or more. Furthermore, the length of these miRNAs was increased to 25–27 nt at the 5′ and 3′ ends of the miRNAs, and noncanonical C-A and G-U pairs were replaced by canonical U-A and G-C pairs to increase the free energy of the interactions of cc-miRc with the gRNA of COVID-19. Table 1 shows the characteristics of the fully complementary interactions of the nucleotides of seven cc-miRc with gRNA. Lengths of 25–28 nt and more have been found among natural miRNAs (miR-1273a, miR-1273d, miR-1272, miR-1292-5p, miR-3143, miR-1226-5p, miR-

7161-3p) and therefore, as part of the RISC (RNA-induced silencing complex), can interact with gRNA. In the gRNA of COVID-19, which has a length of 29903 nt, intramolecular hydrogen bonds are formed that involve cc-miRc binding sites (bs-cc-miRc), which can impede the interaction of cc-miRc with their bs-cc-miRc. The free energy of the intramolecular interactions of bs-cc-miRc is -39 kJ/mole ÷ -43 kJ/mole lower than the ∆G of their interactions with cc-miRc (Table 1), which indicates their weak influence on the binding of cc-miRc with bs-cc-miRc. The created cc-miRc interacted with the mRNAs of 17508 genes with free energy -19 kJ/mole ÷ -22 kJ/mole, that lower than cc-miRc with gRNA (Table 1). This result suggests that each cc-miRc can, when used at adequate concentrations, interact with gRNA without side effects on the human protein-coding genes.

Table 1| Characteristics of the interactions of cc-miRc with gRNA COVID-19 and mRNAs of human genes

RNA

miRNA

Start of site, nt

Region of RNA

∆G, kJ/mole

∆G/∆Gm,

%

Length, nt

gRNA

cc-miR1c

193

5'UTR

-149

100

27

gRNA

bs-cc-miR1c

544

CDS

≤-106

≤71

27

mRNAs

cc-miR1c

≤-126

≤85

27

gRNA

cc-miR2c

16390

CDS

-144

100

26

gRNA

bsi-cc-miR2c

3157

CDS

≤-104

≤75

26

mRNAs

cc-miR2c

≤-122

≤85

26

gRNA

cc-miR3c

17116

CDS

-146

100

27

gRNA

bs-cc-miR3c

253

5'UTR

≤-104

≤71

27

mRNAs

cc-miR3c

≤125

≤86

27

gRNA

cc-miR4c

18101

CDS

-146

100

27

gRNA

bs-cc-miR4c

ND

CDS

≤-102

≤69

27

mRNAs

cc-miR4c

≤125

≤86

27

gRNA

cc-miR5c

21893

CDS

-132

100

25

gRNA

bs-cc-miR5c

20296

CDS

≤-93

≤71

25

mRNAs

cc-miR5c

≤-112

≤85

25

gRNA

cc-miR6c

28359

CDS

-140

100

25

gRNA

bs-cc-miR6c

15100

CDS

≤-100

≤71

25

mRNAs

cc-miR6c

≤-119

≤85

25

gRNA

cc-miR7c

28883

CDS

-146

100

27

gRNA

bs-cc-miR7c

23076

CDS

≤-104

≤71

27

mRNAs

cc-miR7c

≤-124

≤85

27

Note. cc-miR1c - cc-miR7c are cc-miRc created on the basis of ID02510,3p-miR (1), ID00448,3p-miR (2), miR-

3154 (3), miR-7114-5p (4), miR-5197-3p (5), ID02750.3p-miR (6), ID01851.5p-miR (7), respectively.

The interaction schemes of the seven cc-miRc complexes with the corresponding bs-cc-miRc on the gRNA of COVID-19 are shown in Fig. 1. These interaction schemes of cc-miRc with binding sites in gRNA were predicted by the MirTarget program, and quantitative characteristics of interactions are shown in Table 1. In the interaction schemes of bs-cc-miRc with gRNA, there were only completely complementary bindings of all nucleotides, and there were no non- canonical A-C and G-U pairs, which was confirmed by a ∆G/∆Gm value of 100%.

Creation of the cc-miRm complex for the gRNA of MERS-CoV

The size of the gRNA of MERS-CoV is 30119 nt, which is several times larger than the average size of the mRNA of human genes. Only a few human miRNAs with a ΔG/ΔGm value of 89% or more could bind with the MERS-CoV genome. To create cc-miRm (completely complementary miRNA of MERS-CoV) for MERS-CoV, we chose miR-3976, ID02684.5p-miR, miR-3591-3p, ID02892.3p-miR, ID02389.3p-miR, miR-1271-3p, and ID00939.5p-miR, which bind with gRNA of MERS-CoV with a ΔG/ΔGm value of 89% or less. Furthermore, the length of these miRNAs was increased to 25–27 nt at the 5′ and 3′ ends of the miRNAs, and non-canonical A-C and G-U pairs were replaced by canonical U-A and G-C pairs to increase the free energy of the interaction of cc-miRm with the gRNA of MERS-CoV. Table 2 shows the characteristics of the fully complementary interactions of the nucleotides of seven cc-miRm with gRNA. Lengths of 25–27 nt, as mentioned above, are found among natural miRNAs and therefore, as part of the RISC, these miRNAs can interact with gRNA. Intramolecular hydrogen bonds are formed in the gRNA of MERS-CoV with the participation of bs-cc-miRm binding sites, which can impede the interaction of cc-miRm with their bs-cc-miRm. The free energy of the intramolecular interactions of bs-cc-miRm is -30 kJ/mole ÷ -46 kJ/mole, that lower than the ∆G of their interaction with cc-miRm (Table 2), which indicates the weak influence of their intramolecular interactions on the binding of cc-miRm with bs-cc-miRm. The created cc-miRm interacted with mRNA of 17508 genes with free energy -20 kJ/mole ÷ -22 kJ/mole lower than cc-miRm with gRNA (Table 2). This result indicates that each cc-miRm, used at adequate concentrations, can interact with gRNA without side effects on the human protein-coding genes.

Table 2| Characteristics of the interactions of cc-miRm with the gRNA of MERS-CoV and mRNAs of human genes

RNA

miRNA

Start of site, nt

Region of RNA

∆G, kJ/mole

∆G/∆Gm,

%

Length, nt

gRNA

cc-miR1m

123

CDS

-142

100

27

gRNA

bs-cc-miR1m

24648

CDS

≤-112

≤79

27

mRNAs

cc-miR1m

CDS

≤-120

≤85

27

gRNA

cc-miR2m

515

CDS

-144

100

26

gRNA

bs-cc-miR2m

8742

CDS

≤-102

≤71

26

mRNAs

cc-miR2m

≤-122

≤85

26

gRNA

cc-miR3m

3164

CDS

-151

100

27

gRNA

bs-cc-miR3m

12492

CDS

≤-105

≤70

27

mRNAs

cc-miR3m

≤-129

≤86

27

gRNA

cc-miR4m

10236

CDS

-138

100

25

gRNA

bs-cc-miR4m

236

CDS

≤-99

≤72

25

mRNAs

cc-miR4m

≤-117

≤85

25

gRNA

cc-miR5m

13796

CDS

-136

100

26

gRNA

bs-cc-miR5m

12614

CDS

≤-102

≤75

26

mRNAs

cc-miR5m

≤-116

≤85

26

gRNA

cc-miR6m

24307

CDS

-142

100

26

gRNA

bs-cc-miR6m

9997

CDS

≤-103

≤73

26

mRNAs

cc-miR6m

≤-120

≤85

26

gRNA

cc-miR7m

28342

CDS

-146

100

27

mgRNA

bs-cc-miR7m

13480

CDS

≤-111

≤79

27

mRNAs

cc-miR7m

≤-126

≤86

27

Note. cc-miR1m - cc-miR7m are cc-miRm created on the basis of miR-3976 (1), ID02684.5p-miR (2), miR-3591-3p (3), ID02892.3p-miR 94), ID02389.3p-miR (5), miR-1271-3p (6), ID00939.5p-miR (7), respectively.

The interaction schemes of the complex of seven cc-miRm with the corresponding bs-cc- miRm on the gRNA of MERS-CoV are shown in Fig. 2. These interaction schemes of cc-miRm with binding sites in gRNA were predicted by the MirTarget program, and quantitative characteristics of interactions are shown in Table. 2.

Creation of the cc-miRs complex for the gRNA of SARS-CoV

Despite the large gRNA of SARS-CoV, which is 29751 nt long, compared with that of human protein-coding genes, only a few human miRNAs with ΔG/ΔGm of 89% or more could bind to the SARS-CoV genome. To create cc-miRs, we chose ID00322.5p-miR, miR-20b-3p, miR-497-3p, ID01820.3p-miR, miR- 505-3p, ID00749.3p-miR, ID03254.5p-miR, and ID00271.5p-miR, which bound with the gRNA of SARS-CoV with a ΔG/ΔGm value equal to 89% or less. The length of these miRNAs was increased to 25–28 nt at the 5′ and 3′ ends of the miRNAs, and non-canonical C-A and G-U pairs were replaced by canonical U-A and G-C pairs to increase the free energy of the interaction of cc-miRs with the gRNA of SARS-CoV. Table 3 shows the characteristics of the fully complementary interactions of the nucleotides of eight cc-miRs with gRNA. Intramolecular hydrogen bonds involving bs-cc-miRs are formed in the gRNA of SARS-CoV, which can impede the interaction of cc-miRs with their bs-cc-miRs. The free energy of the intramolecular interactions of bs-cc-miRs is -34 kJ/mole ÷ -44 kJ/mole lower than the ∆G of their interaction with cc-miRs (Table 3), which indicates a weak effect of the intramolecular interactions on the binding of cc-miRs to bs-cc-miRs. The created cc-miRs interacted with the mRNAs of 17508 genes with free energy -19 kJ/mole ÷ -24 kJ/mole lower than cc-miRs with gRNA (Table 3). This result suggests that each cc-miRs at concentrations comparable with the concentrations of endogenous miRNA can interact with gRNA without side effects on the human protein-coding genes.

Table 3| Characteristics of the interactions of cc-miRs with the gRNA SARS-CoV and mRNAs of human genes

RNA

miRNA

Start of site, nt

Region of RNA

∆G, kJ/mole

∆G/∆Gm,

%

Length, nt

gRNA

cc-miR1s

91

5'UTR

-153

100

27

gRNA

bs-cc-miR1s

746

CDS

≤-113

≤74

27

mRNAs

cc-miR1s

≤-133

≤87

27

gRNA

cc-miR2s

574

CDS

-157

100

28

gRNA

bs-cc-miR2s

16058

CDS

≤-113

≤72

28

mRNAs

cc-miR2s

≤-133

≤85

28

gRNA

cc-miR3s

1849

CDS

-140

100

27

gRNA

bs-cc-miR3s

15403

CDS

≤-106

≤76

27

mRNAs

cc-miR3s

≤-120

≤86

27

gRNA

cc-miR4s

2058

CDS

-136

100

26

gRNA

bsi-cc-miR4s

11658

CDS

≤-99

≤73

26

mRNAs

cc-miR4s

≤-117

≤86

26

gRNA

cc-miR5s

12482

CDS

-144

100

27

gRNA

bs-cc-miR5s

20270

CDS

≤-103

≤72

27

mRNAs

cc-miR5s

≤-123

≤86

27

gRNA

cc-miR6s

19145

CDS

-142

100

27

gRNA

bs-cc-miR6s

18037

CDS

≤-99

≤70

27

mRNAs

cc-miR6s

≤-121

≤85

27

gRNA

cc-miR7s

21142

CDS

-142

100

27

gRNA

bs-cc-miR7s

17101

CDS

≤-104

≤73

27

mRNAs

cc-miR7s

≤-121

≤85

27

gRNA

cc-miR8s

27316

CDS

-142

100

27

gRNA

bs-cc-miR8s

21548

CDS

≤-104

≤73

27

mRNAs

cc-miR8s

≤-121

≤85

27

Note. cc-miR1s - cc-miR7s are cc-miRs created on the basis of ID00322.5p-miR (1), miR-20b-3p (2), miR-497-3p (3), ID01820.3p-miR (4), miR-505-3p (5), ID00749.3p-miR (6), ID03254.5p-miR (7), ID00271.5p-miR (8), respectively.

The interaction schemes of the complex of eight cc-miRm with the corresponding bs-cc- miRm on the gRNA of SARS-CoV are shown in Fig. 3. These interaction schemes of cc-miRm with binding sites in gRNA were predicted by the MirTarget program, and quantitative characteristics of interactions are shown in Table. 3.

Synthesis and delivery of cc-miRNAs into humans

The synthesis of cc-miRNAs is an inexpensive procedure - in terms of cost, it corresponds to the synthesis of primers. Our hypothesis can be tested in laboratories with the right and ability to conduct inexpensive and short-term tests proposed by cc-miRNAs as a means of combating COVID-19, SARS-CoV, and MERS-CoV coronaviruses. Since the size of cc-miRNAs is approximately 9 nm, they can be delivered via circulation to many organs as part of ordinary exosomes in human blood measuring 30–150 nm [11,12,13]. As part of exosomes, cc-miRNAs can be introduced into the lung by inhalation. The proposed method of combating coronavirus does not have toxicity or side effects. cc-miRNAs are susceptible to degradation by nucleases, similar to all human miRNAs, and the removal of cc-miRNAs from the body is not difficult. In the absence of side effects, cc-miRNAs can be used as a therapeutic agent.

The nucleotide sequences of the cc-miRNAs were created based on known miRNAs and on their binding sites in the gRNA of COVID-19, SARS-CoV, and MERS-CoV. The binding characteristics of cc-miRNAs and their complementary nucleotide sequence (as a pre-miRNA) on mRNAs of human genes were determined to identify side effects of cc-miRNAs on human gene expression. The use of a complex of several miRNAs to suppress coronavirus infection allows the inhibition of viral protein synthesis and gRNA replication; reduces the toxicity of each cc-miRNA by allowing the use of concentrations equal to those of endogenous miRNA; quickly spreads via circulations to many organs; and is excreted from organisms as are all endogenous miRNAs. The proposed method does not require a large number of reagents and is not time consuming. The cc-miRNAs can be used as a therapeutic agents for coronavirus infections with COVID-19, SARS-CoV, and MERS-CoV.

The nucleotide sequences of 2565 miRNAs were downloaded from miRBase (http://mirbase.org, Release 22.1). Another 3307 miRNAs were obtained from the article by Londin et al. [14]. The nucleotide sequences of human genes and coronavirus COVID-19, SARS-CoV, and MERS-CoV genomes were obtained from GenBank (http://www.ncbi.nlm.nih.gov). A search for the target genes of miRNAs was performed using the MirTarget program [15,16,17]. This program determines the following binding characteristics: the start of the miRNA binding site on the mRNA; the locations of the miRNA binding sites in the 3'UTR, 5'UTR, or CDS; the interaction free energy (∆G, kJ/mole); and 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 of the binding of miRNA with its fully complementary nucleotide sequence. The MirTarget program looks for hydrogen bonds between adenine (A) and uracil (U), guanine (G) and cytosine (C), G and U, and A and C [18]. The distances between A and C are equal to 1.04 nanometers, between G and C, and A and U are equal to 1.03 nanometers, between G and U are equal to 1.02 nanometers [17]. The numbers of hydrogen bonds in the G-C, A-U, G-U and A-C interactions were 3, 2, 1 and 1, respectively [16,17].

Acknowledgments

All authors designed the research and wrote the paper.

Funding

This study was supported by a grant (AP05132460) from the Ministry of Education and Science, Kazakhstan Republic, SRI of Biology and Biotechnology Problems, al-Farabi Kazakh National University.

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ThemiRNACOMPLEXESAGAINSTCORONAVIRUSESCOVID19SARSCoVandMERSCoV.pdf

The miRNA COMPLEXES AGAINST CORONAVIRUSES COVID-19, SARS-CoV, and MERS-CoV

Status:

Version 1

Abstract

Figures

Introduction

Results

Creation of the cc-miRc complex for the gRNA of COVID-19

Table 1| Characteristics of the interactions of cc-miRc with gRNA COVID-19 and mRNAs of human genes

Creation of the cc-miRm complex for the gRNA of MERS-CoV

Table 2| Characteristics of the interactions of cc-miRm with the gRNA of MERS-CoV and mRNAs of human genes

Creation of the cc-miRs complex for the gRNA of SARS-CoV

Table 3| Characteristics of the interactions of cc-miRs with the gRNA SARS-CoV and mRNAs of human genes

Synthesis and delivery of cc-miRNAs into humans

Conclusion

Methods

Declarations

References

Supplementary Files

Status:

Version 1