Exploring G and C-quadruplex structures as potential targets against the severe acute respiratory syndrome coronavirus 2

Abstract

In this paper we report the analysis of the 2019-nCoV genome and related viruses using an upgraded version of the open-source algorithm G4-iM Grinder. This version improves the functionality of the software, including an easy way to determine the potential biological features affected by the candidates found. The quadruplex definitions of the algorithm were optimized for 2019-nCoV. Using a lax quadruplex definition ruleset, which accepts amongst other parameters two residue G- and C-tracks, hundreds of potential quadruplex candidates were discovered. These sequences were evaluated by their in vitro formation probability, their position in the viral RNA, their uniqueness and their conservation rates (calculated in over three thousand different COVID-19 clinical cases and sequenced at different times and locations during the ongoing pandemic). These results were compared sequentially to other Coronaviridae members, other Group IV (+)ssRNA viruses and the entire realm. Sequences found in common with other species were further analyzed and characterized. Sequences with high scores unique to the 2019-nCoV were studied to investigate the variations amongst similar species. Quadruplex formation of the best candidates was then confirmed experimentally. Using NMR and CD spectroscopy, we found several highly stable RNA quadruplexes that may be suitable theranostic targets against the 2019-nCoV.

CNBP Binds and Unfolds In Vitro G-Quadruplexes Formed in the SARS-CoV-2 Positive and Negative Genome Strands

“…SARS-CoV-2 presents the lowest number of selected PQSs in the +gRNA (only ≈8% of the PQSs originally predicted), probably indicating a negative selection of PQSs capable of forming stable G4s in this virus. This is in agreement with the previously reported data indicating that SARS-CoV-2 displays general PQSs poverty when compared to the virus realm, its PQS density being in the lower end of results from the Coronaviridae family, which itself is in the lower end of the (+) ssRNA Group IV [ 34 ] and the PQS frequency in SARS-CoV-2 is significantly lower than expected from its base composition [ 33 ]. On the contrary, the SARS-CoV-2 −gRNA presents the highest percentage of selected PQSs from the initially predicted PQSs by QGRS Mapper (≈26%), and a similar tendency is observed for bat-SL-CoVZC45, bat-SL-CoVZXC21, and MERS-CoV, while a lower percentage is observed for RaTG13 and SARS-CoV.…”

“…During the COVID-19 pandemic, mainly along the last half of 2020, several works analyzed the SARS-CoV-2 RNA genome to seek PQSs. All of them analyzed the +gRNA [ 30 , 31 , 32 , 33 , 34 , 35 , 36 ], while a few made a superficial overview of PQSs on the −gRNA [ 32 , 33 , 36 ]. Although −gRNA and −sgRNAs are minority in respect of their positive-sense RNA counterparts and represent only about 1% of viral RNA [ 9 , 10 ], negative-sense RNAs are key intermediates functioning as templates for +gRNA replication and +sgRNAs transcription.…”

“…Consequently, −gRNA and −sgRNAs may contain G4s with putative regulative functions on these processes. In addition, most of the previous PQSs analyses on SARS-CoV-2 genome have used different bioinformatics prediction tools, some of them designed for DNA-G4, and a variety of criteria for selecting the best PQS candidates, mainly including higher prediction scores [ 30 , 31 , 32 , 33 , 34 , 35 , 36 ] combined with lower potential thermodynamic stability of secondary structures competitive with G4s [ 32 ], uniqueness in SARS-CoV-2 and conservation among variants of SARS-CoV-2 [ 34 ], and conservation among human coronaviruses [ 36 ]. Although there is divergence in G4 prediction tools and selection criteria, none of the predictions have found PQSs with four tracts of three consecutive guanines (with the potential of forming three-tetrads G4s), and have only found PQSs with four tracts of two consecutive guanines (with the potential of forming two-tetrads G4s).…”

“…Critical roles for viral G4s have been described in human viruses, including immunodeficiency virus (HIV), herpes simplex virus (HSV), Epstein-Barr virus (EBV), human papillomavirus (HPV), hepatitis B virus (HBV), Nipah virus, hepatitis C virus (HCV), Zika virus, and Ebola virus [ 27 , 28 , 29 ], and some G4-specific compounds have shown powerful antiviral activity by targeting G4 structures [ 28 , 29 ]. Very recent reports have initiated the search for G4s in the genomes of human coronaviruses, including SARS-CoV-2 [ 30 , 31 , 32 , 33 , 34 , 35 , 36 ]. Beyond the prediction of PQSs, some works have demonstrated the formation of a few G4s in vitro by PQSs found in the +gRNA [ 30 , 34 , 35 , 36 ] and one of them was demonstrated to be formed within cultured human cells and controls the translation efficiency of the nucleocapsid N protein [ 35 , 36 ].…”

“…Very recent reports have initiated the search for G4s in the genomes of human coronaviruses, including SARS-CoV-2 [ 30 , 31 , 32 , 33 , 34 , 35 , 36 ]. Beyond the prediction of PQSs, some works have demonstrated the formation of a few G4s in vitro by PQSs found in the +gRNA [ 30 , 34 , 35 , 36 ] and one of them was demonstrated to be formed within cultured human cells and controls the translation efficiency of the nucleocapsid N protein [ 35 , 36 ].…”