RT-qPCR as a screening platform for mutational and small molecule impacts on structural stability of RNA tertiary structures

Abstract

The exponential increase in the discovery and characterization of RNA tertiary structures has highlighted their active role in a variety of human diseases, yet often their interactome and specific function remain unknown. Small molecules offer opportunities to both decode these cellular roles and develop therapeutics, however there are few examples of small molecules that target biologically relevant RNA tertiary structures. While RNA triple helices are a particularly attractive target, discovery of triple helix modulators has been hindered by the lack of correlation between small molecule affinity and effect on structural modulation, thereby limiting the utility of affinity-based screening as a primary filtering method. To address this challenge, we developed a high-throughput RT-qPCR screening platform that reports on the effect of mutations and additives, such as small molecules, on the stability of triple helices. Using the 3'-end of the oncogenic long non-coding RNA MALAT1 as a proof-of-concept, we demonstrated the applicability of both a two-step and a one-pot method to assess the impact of mutations and small molecules on the stability of the triple helix. We demonstrated the adaptability of the assay to diverse RNA tertiary structures by applying it to the SARS-CoV-2 pseudoknot, a key viral RNA structure recently identified as an attractive therapeutic target for the development of antivirals. Employment of a functional high-throughput assay as a primary screen will significantly expedite the discovery of probes that modulate the structural landscape of RNA structures and, consequently, help gain insight into the roles of these pervasive structures.

Fig. 1. Schematic representation of small molecule-induced structural modulation of the MALAT1 triple helix assessed via RT-qPCR assay. The MALAT1 triple helix (green/purple) is equipped with a structural SHAPE cassette (orange) to prevent competition between primer binding and triplex formation. According to the assay design, small molecules that destabilize the triple helix construct result in lower frequency of RT stalling and, consequently, in more full-length cDNA synthesis (left). Small molecules that stabilize the triple helix structure result in higher occurrence of RT stalling, ultimately resulting in lower amounts of full cDNA synthesis (right). Reverse transcription reactions are then followed by qPCR for quantification (not shown).

Fig. 2. 2D structure and sequence of MALAT1 WT and U13C mutant.

Fig. 3. Two-step RT-qPCR system. (A) Schematic of the two-step RT-qPCR reaction with the reverse transcription reaction being performed in a thermocycler and then being aliquoted in a 96-well plate for qPCR amplification, which is performed in a light cycler. (B) Amplification curves obtained from 3 independent replicates of RT-qPCR of the U13C MALAT1 mutant and the WT triple helix. Error bars are standard deviation calculated over the three independent experiments. (C) C_t values calculated over three independent experiments for WT and U13C mutant (ΔC_t value = C_t WT − C_t Mut). The mutated destabilized construct amplifies faster than the WT, in agreement with trends reported by Steitz and co-workers.

Fig. 4. One pot RT-qPCR system. (A) Schematic of the one-pot RT-qPCR reaction with reverse transcription reaction being performed in a 96-well format in a light cycler instrument. (B) Raw data obtained from 3 independent replicates of RT-qPCR of the U13C MALAT1 mutant and the WT triple helix. Error bars are standard deviation calculated over the three independent experiments. (C) C_t values obtained for the Mut and WT constructs (ΔC_t value = C_t WT − C_t Mut). The mutated destabilized construct amplifies faster than the WT, in agreement with trends reported by Steitz and co-workers.

Fig. 5. Application of RT-qPCR assays to assess small molecule effect on MALAT1 WT triple helix stability. (A) Structures of two small molecules chosen for assay validation. Both DPF-P20 (a MALAT1 triplex stabilizer) and SM5 (a MALAT1 triplex destabilizer) were previously evaluated in relationship to their effect on triplex enzymatic degradation. (B) Raw data obtained for the two-step RT-qPCR procedure for both small molecules. (C) Small molecule ΔC_t values calculated in reference to DMSO (ΔC_t value = C_t DMSO − C_t SM) from 4 independent replicates are in agreement with their reported effect on MALAT1 triplex enzymatic degradation. (D) Raw data obtained for the one-pot RT-qPCR procedure for both small molecules. (E) Small molecule ΔC_t values calculated in reference to DMSO (ΔC_t value = C_t DMSO – C_t SM) from 4 independent replicates in reference to DMSO are in agreement with their reported effect on MALAT1 triplex enzymatic degradation and in line with the values obtained in the two-step RT-qPCR procedure. All error bars represent the standard deviation calculated over the four independent experiments.

Fig. 6. Application of the RT-qPCR assay to the SARS-CoV-2 pseudoknot. (A) 3D structure of the SARS-CoV-2 frameshifting element pseudoknot structure obtained via NMR (PDB: 7LYJ). (B) 2D representation of the pseudoknot and the relative base-pairing according to the structure of D’Amare and co-workers. (C) Chemical structure of two frameshifting inhibitors merafloxacin and nafamostat. (D) Two-step RT-qPCR adapted to the SARS-CoV-2 pseudoknot identifies frameshifting inhibitors as stabilizing small molecules.