Genome-wide mapping of therapeutically-relevant SARS-CoV-2 RNA structures

Abstract

SARS-CoV-2 is a betacoronavirus with a linear single-stranded, positive-sense RNA genome of ~30 kb, whose outbreak caused the still ongoing COVID-19 pandemic. The ability of coronaviruses to rapidly evolve, adapt, and cross species barriers makes the development of effective and durable therapeutic strategies a challenging and urgent need. As for other RNA viruses, genomic RNA structures are expected to play crucial roles in several steps of the coronavirus replication cycle. Despite this, only a handful of functionally conserved structural elements within coronavirus RNA genomes have been identified to date.Here, we performed RNA structure probing by SHAPE-MaP to obtain a single-base resolution secondary structure map of the full SARS-CoV-2 coronavirus genome. The SHAPE-MaP probing data recapitulate the previously described coronavirus RNA elements (5′ UTR, ribosomal frameshifting element, and 3′ UTR), and reveal new structures. Secondary structure-restrained 3D modeling of highly-structured regions across the SARS-CoV-2 genome allowed for the identification of several putative druggable pockets. Furthermore, ~8% of the identified structure elements show significant covariation among SARS-CoV-2 and other coronaviruses, hinting at their functionally-conserved role. In addition, we identify a set of persistently single-stranded regions having high sequence conservation, suitable for the development of antisense oligonucleotide therapeutics.Collectively, our work lays the foundation for the development of innovative RNA-targeted therapeutic strategies to fight SARS-related infections.

Secondary structure of the SARS-CoV-2 5’-UTR

“…It contains five simple hairpin structures that were named SL1, SL2, SL3, SL4 and SL5 in good agreement with bioinformatic secondary structure predictions for the SARS-CoV-2 [22] and also from other coronaviruses [23]. Our model is also highly similar to the models obtained by probing of the whole SARS-CoV-2 genome in vitro [24] and in vivo [25,26]. However, we found an additional hairpin located between SL4 and SL5 that we named SL4.5.…”

Cryo-electron Microscopy and Exploratory Antisense Targeting of the 28-kDa Frameshift Stimulation Element from the SARS-CoV-2 RNA Genome

“…The urgency of the COVID-19 pandemic, recent advances in targeting RNA 3D structures with ASOs and small molecules, and the identification of the FSE as a potentially well-defined RNA 3D structure in the SARS-CoV-2 genome has generated significant interest in understanding and targeting SARS-CoV-2 ribosomal frameshifting 3,23,27,28,44,45,50,51 . Here, we confirmed the ability of ASOs to invade the SARS-CoV-2 FSE structure and to reduce frameshifting efficiencies in cellfree assays, and we explored the potential for these ASOs to reduce viral replication in cells at submicromolar concentrations.…”

“…1.0 μL 100 μM primer was used per 2.5 μL cDNA, and 19.5 μL PCR master mix (same as PCR assembly, see above). The reaction mixtures were initially denatured at 98℃ for 60 s; then 4x cycles of 98℃ for 10 s, 58℃ for 30 s, and 72℃ .0 μL of equimolar p5/p7 (50 μM per primer) mixture was spiked into each uni-directional PCR reaction product, mixed, spun down, and quickly returned to the thermal cycler to conduct: initial denaturation at 98℃ for 60 s; then 5x cycles of 98℃ for 10 s, 50℃ for 30 s, and 72℃ for 30 s; followed by 15x cycles of 98℃ for 10 s, 60℃ for 30 s, increasing this temperature incrementally each cycle to a final temperature of 70℃ by cycle 15, and 72℃ for 30 s; then 72℃ for 10 mins.Secondary structure modeling was performed for FSE constructs including 110 upstream nucleotides, guided by SHAPE reactivity data collected in this study along with various recently published SHAPE and DMS reactivity datasets27,28,73 . SHAPE reactivity data were collected for the extended FSE constructs with 1M7 as described above; data were averaged across two replicates.…”

The short- and long-range RNA-RNA Interactome of SARS-CoV-2

“…For example, interactions between promoters and enhancers dictate the rate of transcription along the eukaryotic genome (Rowley and Corces, 2018) . Great effort is being made to reveal the structural landscapes of the SARS-CoV-2 genome (Andrews et al, 2020;Huston et al, 2020;Kelly et al, 2020;Lan et al, 2020;Manfredonia et al, 2020;Ryder, 2020;Sanders et al, 2020;Sun et al, 2020) .…”