SHAPE-based chemical probes for studying preQ1-RNA interactions in living bacteria

Abstract

Interrogating RNA-small molecule interactions inside cells is critical for advancing RNA-targeted drug discovery. In particular, chemical probing technologies that both identify small molecule-bound RNAs and define their binding sites in the complex cellular environment will be key for establishing the on-target activity necessary for successful hit-to-lead campaigns. Using the small molecule metabolite preQ₁ and its cognate riboswitch RNA as a model, herein we describe a chemical probing strategy for filling this technological gap. Building on well-established RNA acylation chemistry employed by in vivo click selective 2'-hydroxyl acylation analyzed by primer extension (icSHAPE) probes, we developed an icSHAPE-based preQ₁ probe that retains biological activity in a preQ₁ riboswitch reporter assay and successfully enriches the preQ₁ riboswitch from living bacterial cells. Further, we map the preQ₁ binding site on probe-modified riboswitch RNA by mutational profiling (MaP). As the need for rapid profiling of on- and off-target small molecule interactions continues to grow, this chemical probing strategy offers a method to interrogate cellular RNA-small molecule interactions and support the future development of RNA-targeted therapeutics.

Keywords: RNA; SHAPE; SHAPE-MaP; chemical probes; preQ1; small molecules; target engagement.

Figure 1.

RNA acylation-based strategies for analyzing and discovering RNA-small molecule interactions. (A) Varying probe designs. (B) Workflow for using SHAPE-based acylation probes for in-cell investigation of target engagement and binding site mapping of RNA-small molecule interactions.

Figure 2.

Bioactivity of non-activated preQ₁ probe 6b in a Lrh-preQ₁-GFPuv reporter assay. (A) Fluorescence emission and (B) fold-repression following treatment of Lrh-preQ₁-GFPuv-transformed JW2765 E. coli cells with 6b or preQ₁ (10 μM).

Figure 3.

Chem-CLIP using icSHAPE-based probes for target engagement analysis using qRT-PCR. [8a] and [8b] = 50 μM. DMSO was used as a negative control. Competition studies were performed by co-treating with 10 μM preQ₁. Statistical significance was determined using an unpaired, two-tailed Student’s t test. *** p = 0.0009.

Figure 4.

SHAPE-MaP analysis of icSHAPE preQ₁ probe binding to the Lrh-preQ₁ riboswitch in bacterial cells. (A) SHAPE mutation rates across the Lrh-preQ₁-GFPuv transcript following treatment with 10 μM or 50 μM 8b. (B) 8b reactivities shown on the predicted structure of the Lrh-preQ₁-GFPuv transcript.

Figure 5.

Average normalized FAI-N₃ SHAPE reactivity from three replicates across the Lrh-preQ₁-GFPuv transcript 5′ end following in-cell treatment with DMSO (top), preQ₁ (5 μM) (middle), or preQ₁ probe 6b (50 μM) (bottom). 8b-reactive sites are circled.

Scheme 1.

Synthesis of icSHAPE-based probes. Reagents and conditions: a. (i) NBS, AIBN, CCl₄, 50 °C; (ii) NaN₃, DMF, 23 °C, 83% over 2 steps; b. 4-(N-Boc-amino)phenylboronic acid pinacol ester, Pd(PPh₃)₄, K₂CO₃, 1,4-dioxane, 55 °C, 46%; c. (i) LiOH•H₂O, 1,4-dioxane:H₂O (3:1), 23 °C; (ii) allyl bromide, K₂CO₃, DMF, 0 °C to 23 °C, 55% over 2 steps; d. (i) TFA:CH₂Cl₂ (1:4), 0 °C; (ii) glutaric anhydride, DIPEA, DMAP, DMF, 23 °C; 82% over 2 steps; e. benzylamine or preQ₁, DIPEA, HATU, DMF, 23 °C, 33% for 6b; f. Pd(PPh₃)₄, pyrrolidine, DMF, 23 °C, 43% for 7a (over steps e. and f.) and 68% for 7b; g. CDI, DMSO, 23 °C, 76% for 8a and 41% for 8b.