Antisense and sense oligonucleotides targeting the conserved sequences of HCoV-OC43 (NC_006213.1) and SARS-CoV-2 (MT039890.1) were designed (Supplementary Table S1 and Supplementary Figure S1). We designed different OPNA oligomers targeting ORF1a, ORF1b, and the 5′ untranslated region (UTR) sequence.

OPNA oligomers were synthesized by solid-phase peptide synthesis (SPPS) based on Fmoc chemistry according to the method described in our previous patent (Chung et al., 2014, 2017), with minor modifications. The H-Rink Amide-ChemMatrix, which was employed as a solid support, was purchased from PCAS BioMatrix Inc. (Quebec, Canada). Fmoc-PNA monomers with a modified nucleobase were synthesized as described previously (Chung et al., 2014, 2017). Modified nucleobase and natural Fmoc-PNA monomers were used to synthesize the PNA derivatives of this study. PNA oligomers were purified by C18-reverse phase HPLC (water/acetonitrile or water/methanol with 0.1% TFA) and characterized by mass spectrometry including ESI/TOF/MS.

The concentration of 1 μM was decided for the initial screening of the designed OPNA. Tables, including all the details related to the OPNAs of this study, are presented in Supplementary Section.

HCT-8 cells, Caco-2 cells, and Vero E6 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, United States). HCT-8 cells were maintained in RPMI medium supplemented with 10% FBS and penicillin/streptomycin at 37°C for 3 days in a 5% CO2 incubator. Caco-2 cells and Vero E6 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% FBS and penicillin/streptomycin at 37°C for 3 days in a 5% CO2 incubator. OPNA, PF07321331 (Merck, NJ, United States), EIDD-1931(Merck, NJ, United States), chloroquine (Merck, NJ, United States), and kurarinone (Merck, NJ, United States) were treated to the cells at the indicated concentrations. The plasmid (myc-NSP14) was transfected with Lipofectamine 2000 transfection reagents (Invitrogen, MA, United States). The siRNA (N14-R7) (Gu et al., 2020) transfection was performed with Lipofectamine RNAiMAX transfection reagents (Invitrogen, MA, United States), according to the manufacturer’s instructions. The siRNA (5′- AAAGACAUCAGCAUACUCCdTdT-3′) was designed to target the RdRp sequence at 5’-GGAGUAUGCUGAUGUCUUU-3′.

HCoV-OC43 was gathered from the supernatant of HCT-8 cells at 4 to 5 days post-infection. The prepared HCoV-OC43 was used to infect HCT-8 or Caco-2 cells with serum-free medium for 30 min. After 30 min, cells were washed using serum-free fresh medium and maintained in media supplemented with 2% FBS and penicillin/streptomycin. SARS-CoV-2 viruses were propagated in Vero E6 cells and maintained in DMEM supplemented with 2% heat-inactivated fetal bovine serum (FBS; GIBCO, United States). All experimental procedures related to SARS-CoV-2 were performed in a Biosafety Level 3 (BSL-3) facility with the approval of the Agency for Defense Development.

The viral RNA from the cell culture supernatants was isolated using the viral RNA purification QIAamp Kit (Qiagen, Hilden, Germany). The DNA from transfected cells was isolated using TRI Reagent (Merck, NJ, United States), according to the manufacturer’s protocol. Quantitative reverse transcription PCR (RT-qPCR) was performed using the One Step SYBR^® PrimeScriptTM RT-PCR Kit (Takara Bio Inc., Shiga, Japan), according to the manufacturer’s protocol using primer pairs to amplify the HCoV-OC43 nucleoprotein (NP) gene (forward, 5′ -AGCAACCAGGCTGATGTCAATACC-3′, and reverse, 5′ -AGCAGACCTTCCTGAGCCTTCAAT-3′). For amplifying RdRp of SARS-CoV-2, the following primers and probes were used: forward primer, 5’-GTGARATGGTCATGRGTGGCGG-3′; reverse primer, 5’-CARATGTTAAASACACTATTAGCATA-3′; probe, FAM-CAGGTGGAACCTCATCAGGAGATGC-BBQ. For amplifying NSP14 of SARS-CoV-2, the following primers were used: forward primer, 5’-TGGGGCTTTACGGGTAACCT-3′; reverse primer, 5’-AACACGCTTAACAAAGCACTC-3′.

HCoV-OC43 titers were analyzed using TCID₅₀ assay in HCT-8 cells. In brief, 2,000 cells were plated in each well of 96-well plates. Medium from HcoV-OC43-infected cells was used for half-log serial dilution with RPMI and added to the cells. The plates were observed for cytopathic effect (CPE) at 4 or 5 days after infection. The TCID₅₀ results were measured using Viral ToxGlo™ (Promega, United States), according to the manuals. The TCID₅₀ results were calculated using the Spearman and Karber methods.

The half maximal inhibitory concentration (IC₅₀) was analyzed by two different methods as follows: (1) the viral titer in cell supernatants by RT-QPCR and (2) the TCID₅₀ values which were measured using Viral ToxGlo™ (Promega, United States). IC₅₀ values were obtained by non-linear regression analysis using GraphPad Prism^® software V.6.05 for Windows (GraphPad Software Inc., San Diego, CA, United States).

SDS-PAGE and Western blot analysis were performed as described previously (Park et al., 2020). In brief, cells were lysed in a lysis buffer (M-per and a protease inhibitor cocktail) and centrifuged at 3 days after infection or 2 days after transfection. Cell lysates were separated in 12% SDS-PAGE gels and subjected to Western blot analysis. The membranes were incubated with specific antibodies, such as antiviral-NP (Merck, NJ, United States), anti-Myc (Cell Signaling Technology, MA, United States), anti-GFP (Clontech, CA, United States), and anti-β-actin (Merck, NJ, United States).

Chemiluminescent detection was performed with the ECL Detection Reagent (AbClon, Seoul, Republic of Korea) and visualized with a Bio-Imaging analyzer (GE Healthcare, Buckinghamshire, United Kingdom).

HCT-8 cells were seeded in a glass bottom 8-well cell culture slide (SPL, South Korea) and incubated overnight. Next, the FAM-labeled OPNA was treated for 30 min before washing with serum-free RPMI. The cell nuclei were stained with DAPI for 20 min. The slide was observed with a confocal microscope (Leica, Germany), and data analysis was performed using Leica Application Suite X.

HCT-8 cells were seeded in a 96-well clear-bottom white cell culture plate. Next day, OPNAs were treated with RPMI containing 10% FBS and 1% penicillin/streptomycin in a dose-dependent manner. The cell viability was measured using a CellTiter-Glo^® Luminescent Cell Viability Assay Kit on the second day following treatment. An equal volume of reagent was added; plates were shaken for 2 min on an orbital shaker and incubated for 10 min at room temperature. Next, the luminescence signal was detected using the GloMax^® 96 Microplate Luminometer. The values were obtained by non-linear regression analysis using GraphPad Prism^® Software V.6.05 for Windows (GraphPad Software Inc., San Diego, CA, United States).

Data were presented as mean values, and error bars were presented with standard deviation (SD). Data analysis was performed using GraphPad Prism 6. Data were analyzed using two-tailed unpaired Student’s t-test. All statistical analyses were two-sided, and a p-value of <0.05 was considered statistically significant.

The chemical structure of well-studied DNA archetypes, LNA, and PNA is presented in Figure 1A. We have developed novel PNA-based ASOs, so-called OPNAs, designed to sterically block the access of proteins or nucleic acids to the RNA by introducing cationic lipid moiety into the nucleobase of PNA oligomer backbone (Figure 1B). Three lipid modifications that are located on the nucleobase of adenine (A), cytosine (C), and guanine (G) are marked in red with asterisks. In this study, to assess the cell permeability of a designed 14-mer OPNA incorporating 6 modified nucleobases, HCT-8 cells were employed. These cells are commonly used for HCoV-OC43 infection evaluation. Treatment involved the administration of FAM-labeled OPNA or PNA to the cells (Figure 1C). Surprisingly, fluorescence was fully detected only in cells treated with OPNA oligomers, and all cells were detected, resulting in 100% cell permeability. Conventional PNA, however, barely penetrated the cells.

Initially, the genomic sequences of human coronaviruses, HCoV-OC43 and SARS-CoV-2, were aligned, and the generic conserved sequences between them were identified. Next, multiple OPNA ASOs targeting these particular regions were synthesized and evaluated for their antiviral activity against infection with the human coronavirus, HCoV-OC43. The oligomer structure of these OPNA ASOs is presented in Figure 2A.

To find out that the manufactured OPNAs can inhibit viral replication in vitro, experiments were conducted in HCT-8 cells and Caco-2 cells, according to the scheme mentioned in Figure 2B. We combined co-treatment and post-treatment experiments to obtain optimal treatment time course. Cell culture medium was collected at 3 days post-infection (dpi) for the quantification of HCoV-OC43 replication level as described in Materials and Methods. RT-qPCR data showed that the OPNA 5, which targets the NSP14, reduced the viral titer in both cells (Figures 2C,D). Kurarinone (Min et al., 2020) which reportedly inhibited HCoV-OC43 infection and chloroquine (Singh et al., 2021) was used as a positive control. In general, antiviral effects were more pronounced in Caco-2 cells than in HCT-8 cells. Additionally, since the 5’ UTR region plays an important role in the life cycle of the virus, we designed and synthesized the OPNAs against this region to investigate their antiviral effect. In vitro screening of ASO candidates targeting the 5’ UTR region exhibited similar antiviral impact. Especially, OPNA 530, 531, and 533 lowered viral gene expression to half which is comparable with the inhibitory effect of the previously described EIDD1931 (parent nucleoside of molnupiravir) (Rosenke et al., 2021) and PF07321332 (viral 3CL protease inhibitor developed by Pfizer) (Owen et al., 2021) (Figure 2E). The above data confirmed the antiviral effect of OPNA ASOs against coronavirus in our in vitro cell system.

A similar strategy was used to design sense OPNA oligomers targeting the HCoV-OC43 genome. Sense OPNA oligomers which are complementary to the ASOs were developed, as shown in Figure 3A, and their viral inhibitory effect was evaluated according to the scheme presented in Figure 3B. The antiviral activity of sense OPNA oligomers was monitored daily after OPNA treatment for 3 days post-infection (Figures 3C–E). According to the RT-qPCR results, sense OPNA oligomers (OPNA 501, 502, and 506) comparably diminished virus replication in HCT-8 cells. Interestingly, sense OPNA 11, which is complementary to OPNA 5, was found to be particularly effective in suppressing the virus in the early stages of infection, reducing viral titer to less than 50% at dpi. These results suggested that both sense and antisense OPNA oligomers are capable of inhibiting HCoV-OC43 in HCT-8 cells.

As the sense OPNA oligomer 11 exhibited the highest antiviral impact, it was selected for further modifications to enhance its efficiency. A variety of modifications were explored including oligomer size, the number and position of each modification, and lastly attaching a cap to the original OPNA 11 structure (Figure 4A). In the case of the number of modifications, the most effective OPNA 11 was the one containing five modifications. Nevertheless, different numbers and types of modifications or changes in oligomer size did not seem to positively enhance the effect of OPNA 11. Elongating the OPNA from 17 mer (OPNA 11) to 20 mer (OPNA 514 and 515) resulted in a similar reduction level in OC43 expression level (Figures 4B,C).

In addition to the fethoc cap that was initially introduced in OPNA 11, other options including acetyl or H groups were substituted, and relative antiviral activity was measured. Apparently, the OPNAs with a small number of modifications and the fethoc cap better enhanced the antiviral feature of OPNA than acetyl or H (OPNA 525 vs. 526 vs. 11). However, when the number of modifications was 2 (OPNA 520 vs. 524) or 5 (OPNA 11 vs. 525 vs. 526), the different types of cap had a consistent effect without much difference (Figure 4D).

Increasing the number of modifications and extending the oligomer at the 5′ end showed room for improvement in the performance of OPNA 11 (OPNA 514 vs. 11). Therefore, we further modified the OPNA 11 in this order and evaluated its antiviral ability in HCoV-OC43-infected HCT-8 cells (Figure 4E). OPNA 456 reduced viral replication most significantly (approximately 80%). In addition, OPNA 5, a complementary sequence to OPNA 11, had relatively good antiviral activity among the antisense OPNAs (Figure 2), so we modified it to compare its effectiveness with OPNA 11. However, as shown in the activity data in Figure 4F, the OPNA 5-based modifications (OPNAs 527 and 528) were unable to compete with OPNA 11 (Figure 4F). Overall, our data suggest that the antiviral effect can be modulated by controlling the length, number of modifications, position, and cap of the OPNA. Based on the optimization results, OPNAs 11 and 546 have been modified to exert maximum antiviral effect on our system.

Since OPNAs targeting the 5’ UTR region, including 530, 531, 533, and 536, showed good antiviral activity (Figure 2E), based on them, we designed modified OPNAs to investigate their antiviral ability. According to the data presented in Figure 5A, extending the length of OPNA 530 by 4mer, or adjusting the position of the modification, did not improve the inhibitory impact of the OPNA as both OPNA 530 and 549 had almost similar effects (Figure 5A). In contrast, the corresponding effects of the modified OPNA 531 (OPNA 566) suppressed viral expression more dramatically with approximately 25% more potency. This improved performance was most likely related to the substitution of A* with G* at the 3′ end of the OPNA oligomer (Figure 5B). Regarding the OPNA 533, altering the length and number of modifications resulted in a preferable antiviral effect which was comparable to the inhibitory level of OPNA 11 (~50%) (Figure 5C). Unexpectedly, however, introducing the G-clamp modification (C**), which is known to enhance specificity and thermal stability of the formed duplex structure (Rajeev et al., 2002), rather reduced the viral suppressive activity of OPNA 536 dramatically (Figure 5D). Collectively, by adjusting different elements of modification, the most effective OPNA targeting the 5’ UTR region was discovered.

The OPNA 7, an antisense OPNA oligomer, targeting the pseudoknot slippery site and OPNA 506, a sense OPNA oligomer, targeting the same region were developed and examined for antiviral efficiency in Figures 2A, 3A, respectively. Nonetheless, virus reduction levels were not observed. One reason might be that the 14-mer length of these OPNA oligomers was too short for the 20mer that was once thought to be the ideal size for function. We, therefore, sought to design a sense and three antisense oligos specific to different positions within the pseudoknot slippery site while maintaining the oligo size at 20-mer. Although the 537 to 540 oligos were able to reduce the OC43 levels, there seemed to be no benefit to target the other sites, with OPNA 537 having the greatest inhibitory effect on viral titers (Figure 6A). Consequently, OPNA 537 was subjected to a combination of length and modification adjustments, and the in vitro antiviral effect of these oligos was assessed (Figures 6B,C). The RT-qPCR results indicated that the virus inhibitory effect of OPNA 563 and 564 was similar to that of 537, with an almost 55% reduction (Figure 6B). Finally, among the OPNA 538-based modifications, OPNA 556 represented the most promising results with higher potency compared with the template oligo 538 (Figure 6C). This enhanced antiviral activity was achieved by incorporating the G-clamp (Rajeev et al., 2002; Ortega et al., 2007) (C**) structure, as shown in Figures 5D, 6C. Taken together, the above results suggest that pseudoknot and slippery sites are good candidate targets for ASO and that effective antisense OPNA oligos can be obtained by controlling the length and base modification of OPNAs, targeting these sites.

Time-of-addition assay (Min et al., 2020) was performed to determine and compare the effect of four selected OPNAs in HCT-8 cells. The experiments were conducted with four treatment methods along with 30 min virus infection scheme (Figure 7A). Using the two-time treatment approach, only 2 OPNA oligomers (11 and 546) showed noticeable antiviral activity (Figure 7B). Incubation of virus and OPNA in one tube for 30 min prior to infection resulted in a strong decrease in HCoV-OC43 and inhibited the expression of the nucleoprotein (NP), which was confirmed by RT-qPCR and Western blot, respectively. After 3 days of infection, the virus titer and NP protein levels in OPNA 11, 531, 546, and 566-treated cells drastically decreased, nearly eliminating viral replication (Figure 7C). Unlike the two-time treatment strategy with the 531 and 566, which failed to significantly reduce virus titer, co-treating these OPNA oligomers with virus at the time of infection proved effective (Figure 7D), and more interestingly, the OPNA 531 exhibited the highest antiviral activity among the other four oligos through post-treatment (Figure 7E). Overall, the pre-incubation treatment strategy proved to be the most efficient method in our system for significantly lowering viral titer and NP protein production.

Following the identification of four potent OPNAs that effectively inhibit HCoV-OC43 titer and its NP protein expression, we measured their IC₅₀ levels according to the virus titer and TCID₅₀ methods, respectively (Figures 8A,B). To determine the IC₅₀ of selected OPNA oligomers, they were treated to HCoV-OC43-infected cells in a dose-dependent manner along with PF07321332. Three days post-infection, the cell supernatant was collected to analyze virus titer by RT-qPCR. The IC₅₀ values for OPNA 11, 546, 531, 566, and PF07321332 were 290.5 nM, 172.1 nM, 700.5 nM, 491.5 nM, and 86.1 nM, respectively (Figure 8A). Meanwhile, the TCID₅₀ was measured according to the CPE caused by the virus at 5 days after infection. The TCID₅₀ levels are presented in Figure 8B. Based on the TCID₅₀, the calculated IC₅₀ values were 4.31 μM for OPNA 11, 10.86 μM for OPNA 546, 7.36 μM for OPNA 531, 4.50 μM for OPNA 566, and 0.16 μM for PF07321332 (Figure 8B). For IC₅₀ determination, the TCID₅₀ method was much more sensitive than that of RT-qPCR. In addition, we confirmed the cell viability of OPNA using the CellTiter-Glo^® Luminescent Cell Viability Assay Kit, and OPNAs exhibited no cell toxicity as shown in Figure 8C.

In addition, NP protein levels were measured using Western blotting to ascertain whether OPNA treatment reduced viral protein levels. As shown in Figure 8D, escalating doses of OPNA substantially decreased NP protein levels, particularly above 250 nm.

As the OPNAs were designed to bind to the SARS-CoV-2 NSP14 sequence, their specificity was evaluated by measuring the NSP14 protein level after ectopic expression of the Myc-NSP14 construct (Figure 8E). Additionally, pEGFP was used as the transfection control. The transfected DNA was isolated using TRI reagent, and the NSP14 gene levels were determined using semi-quantitative PCR (Figure 8E). As predicted, the expression of NSP14 protein was significantly reduced in samples treated with OPNA 5, 11, and 546 compared with untreated or PF07321332-treated samples, suggesting that OPNAs inhibit the transcription or translation of target genes. In addition, we confirmed that OPNA 11 had no effect on other SARS-CoV-2 genes that do not contain the target sequence of OPNA11 (Figure 8F). These results indicate that, similar to the general mechanism of ASO, OPNAs bind specifically to SARS-CoV-2 NSP14 gene or RNA and inhibit translation.

Next, the antiviral ability of a combination of OPNAs targeting multiple sites was examined when treated simultaneously. Experiments were conducted by treating 1 μM of OPNA 566 (targeting the 5’ UTR of HCoV-OC43 virus), OPNA 546 (targeting NSP14), OPNA 556 (targeting pseudoknot slippery site), and OPNA 501 (targeting RdRp) (Figures 9A,B). When the infected cells were treated with a mixture of oligos, the virus titer and NP protein levels decreased dramatically by more than 8-fold.

To demonstrate that the designated OPNAs are applicable to SARS-CoV-2 (Figure 9C), as the final stage of oligo screening in our study, a mixture of OPNAs targeting the sites similar to that of HCoV-OC43 was synthesized and treated 3 h before infection and 1 h after infection, and the viral titers in the culture supernatant were measured daily from 1 to 3 dpi (Figure 9D). The maximum antiviral effect was observed 1 day after infection. Similar outcomes were observed when a siRNA specific for SARS-CoV-2 was applied (Figures 9E–G). The treatment with a viral 3CL protease inhibitor did not inhibit viral titers at 2 or 3 dpi, whereas OPNA had a significantly greater anti-SARS-CoV-2 effect (Owen et al., 2021). Of note, the cells were not treated with efflux inhibitor in this experiment. Eventually, the immunofluorescence data presented in Figure 9H clearly demonstrated that the combination of OPNAs targeting multiple regions significantly reduced CPE 3 days after SARS-CoV-2 infection, demonstrating their effectiveness in controlling viral infection.