Structure-based design of antisense oligonucleotides that inhibit SARS-CoV-2 replication

Figures

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  Fig. 1. Concept of 3D-ASO design inspired by RNA pseudoknot structures. a A pseudoknot may be viewed as an ASO bound to a target hairpin. b Secondary structure drawing of the P2b-P3 pseudoknot of human telomerase RNA previously engineered for structural studies26. By omitting the residues shown in silver, we view the structure as a model ASO bound to a Hairpin. Tertiary interactions revealed by the structure are represented by dashed lines and detailed in panels d, e, and f. c NMR structures of the pseudoknot (PDB ID: 1YMO) provide a 3D model of ASO-Hairpin complex. The omitted residues are represented by a silver dashed line. d Minor-groove base triples formed by U5 and G6 of ASO. e A Hoogsteen base pair by U7. f Major-groove base triples by U8, U9 and U10. g 3D-ASO design template A. h ITC binding isotherm. Top: raw data. Bottom: the integrated heat values plotted against ASO:Hairpin molar ratio. i,j Truncation and mutations of the model ASO and Hairpin decrease the affinity, demonstrating the importance of tertiary interactions. Kd values are shown along with standard errors obtained from fitting. k The same ASO may be used to target the basal region of a target hairpin, forming the…

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  Fig. 2. Isosteric tertiary interactions compatible with the design templates. a-g Major-groove base triples. h–k Minor-groove triples. l–o Hoogsteen pairs that can bridge the segments of major-groove and minor-groove base triples.

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  Fig. 3. 3D-ASO designs targeting the SARS-CoV-2 FSE. a Schematic of the genomic RNA of SARS-CoV-2. The TRS and FSE are indicated by horizontal lines. The 3D-ASOs targeting these sites are shown as thick red bars. b The pseudoknot structure in FSE causes ribosome stalling at the slippery site (boxed). The UAA stop codon for ORF1a is shown in black italic. c PRF3p (an ASO with PMO backbone) is designed to disrupt stem 2 and part of stem 1, allowing the ribosome to proceed to the stop codon and dissociate. Dashed lines represent tertiary interactions. d Hoogsteen pair between PRF3p and FSE. e–g Major-groove base triples between PRF3p and FSE base pairs in Stem 3. h Deletion of G19 in PRF3p (PRF3pΔG) results in a 1D-ASO that also disrupts stem 2.

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  Fig. 4. Design of SBD1 targeting the SARS-CoV-2 TRS. a A previously reported ASO called TRS2 was proposed to bind to a hairpin loop25. b SBD1 with PMO backbone, with a variant T15A shown below. c–h Designed tertiary interactions between SBD1 and viral RNA. i, Recent studies strongly suggest an alternative conformation for the 5′-UTR. The hairpin residues in panel b (colored in green) are involved in three stem-loop structures. j SBD1 has to disrupt SL3 but can engage in tertiary interactions with SL2, similar to those shown in panels c, d, and f. k,l A 2D design (Site1p5) is fully complementary to the target sequence. Panels k and l illustrate how Site1p5 is expected to interact with the TRS region in the single-hairpin and alternative conformations, respectively.

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  Fig. 5. Identification of lead 3D-ASO inhibitors. a–e HEK293-hACE2 cells was infected by the SARS-CoV-2 virus. ICC and total RNA extraction were performed at 24 hpi. a ICC images of cells transfected with ASOs at 10 and 20 μM prior to infection, stained for the spike antigen. The scale bars represent 200 μm. b Quantification of spike-positive cells. The data shown are average ± SD. c qRT-PCR quantification of viral RNA (a N-coding sequence). The data were normalized to host GAPDH mRNA. Shown are average ± SD from three (panel c) or four (panels d, e, f) independent repeats. d,e Dose-dependent inhibition by PRF3p and SBD1, respectively, along with their variants. f Scatter plots of MTT cell viability assays. Horizontal bars indicate means and error bars represent SD.

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  Fig. 6. Hoogsteen-pairing residue of PRF3p is important for activity. a,b Cellular SARS-CoV-2 viral inhibition assays comparing PRF3p and PRF3pΔG (a), and PRF3pΔ2 and PRF3pΔ2ΔG (b). c–i Test PRF3p and its variants using the dual luciferase PRF reporter assay and HEK293 cells. c Illustration of the CoV2-FSE reporter construct. PRF3p is designed to bind and disrupt the pseudoknot structure of FSE, interrupt recoding at the slippery site, and cause translation to stop. 2A, also called StopGo, sequences reprogram translation to produce isolated Firefly and Renilla luciferases rather than fusion proteins. d–i PRF reporter activities of indicated ASOs. The data plotted are average ± SD from 4 repeats.