Is MMS the key to RNA analytics?

Introduction

Riboswitches are small, well-folded mRNA sequences that regulate protein production by controlling the translation of specific mRNA segments. Each riboswitch has a unique small molecule that induces structural changes upon binding, activating the riboswitch. In this study, X-ray crystallography data and Microfluidic Modulation Spectroscopy (MMS) was used to characterise the structural changes of the SAM-I riboswitch upon ligand binding and determine the apparent dissociation constant (Kd). MMS provides a comprehensive understanding of RNA-ligand binding and structural changes, offering potential for developing novel small molecule RNA regulators for therapeutic purposes.

Background

RNA molecules play versatile roles beyond mediating gene expression. RNA riboswitches are critical regulatory components that govern gene expression in response to small molecule ligands. Understanding RNA-ligand binding through riboswitches can reveal gene regulation mechanisms for targeted therapeutics.

The S-adenosylmethionine (SAM)-I riboswitch regulates gene expression in response to intracellular SAM concentrations. SAM is a key metabolite involved in various cellular processes, including transmethylation, transsulfuration, and polyamine synthesis. The SAM-I riboswitch comprises an aptamer domain for SAM recognition and a downstream expression platform governing gene expression. Upon SAM binding, the aptamer domain undergoes conformational rearrangements, transmitting signals to modulate transcriptional or translational outputs.

Study Overview

Structural studies of the SAM-I riboswitch have provided insights into RNA-ligand interactions. Techniques such as X-ray crystallography, NMR spectroscopy, and cryo-electron microscopy have detailed the SAM-binding pocket and allosteric communication pathways. However, these techniques only provide either structural information or binding information on the RNA-ligand complex.

In this study, MMS was used to study the structural changes of the SAM-I riboswitch upon binding. MMS probes the nucleic acid bases in the Amide-I IR range to examine base pairing and stacking during RNA folding and unfolding. This technique provides real-time background correction, making it useful for quality control and compatible with various formulation buffers. The Apollo MMS system, equipped with a high-power Quantum Cascade Laser, was used for this study, offering greater sensitivity compared to traditional FTIR and CD methods.

Methods

SAM-I riboswitches (apo and ligand-bound) were obtained from Arrakis (Waltham, MA). The RNA samples were buffer-exchanged to their formulation buffer and run in triplicate on the Apollo MMS system at a concentration of 0.67 mg/mL (22 μM). A backing pressure of 5 psi was used to move the samples into the flow cell, where they were modulated at 1 Hz between sample and reference buffer for background subtraction. Differential absorbance was measured between 1580–1765 cm^-1. Data processing involved converting raw absorbance to absolute absorbance, normalising by concentration and optical pathlength, and analysing the second derivatives of the spectra.

MMS spectra (Similarity plot) is used to quantitate similarity between samples.

Results and Discussion

MMS results indicated subtle but detectable structural changes due to ligand binding on the SAM-I riboswitch. Specific peaks in the spectra corresponded to changes in base pairing and stacking, with notable changes observed at 1690, 1640–1670, and 1604 cm^-1. These peaks were primarily assigned to guanine, uracil/cytosine, and adenine, respectively. A dose-dependent titration of SAM into the SAM-I riboswitch demonstrated gradual spectral changes, suggesting guanine base-pairing and interactions with adenine residues.

The spectral changes observed were significant, indicating conformational changes in the RNA due to ligand binding. A plot of the spectral difference showed that changes plateaued at around 20 μM SAM concentration, suggesting a 1:1 ligand-to-riboswitch molar ratio. This study demonstrated MMS as a quick and effective tool to detect structural changes and determine Kd values for RNA-ligand binding.

Conclusions

This work highlighted MMS as a viable orthogonal assay for detecting RNA structural changes in the presence of small molecule ligands. It also demonstrated the potential of MMS to determine the apparent Kd of RNA-ligand binding in the micromolar range, sensitively distinguishing structural changes in the RNA riboswitch due to ligand binding.

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_{1. Huang, R., & Colins, V. (2024, May 8). Structural Characterization of RNA and Detection of RNA-ligand Binding Using Microfluidic Modulation Spectroscopy. Redshiftbio.com. Retrieved May 23, 2024, from https://www.redshiftbio.com/resources/structural-characterization-of-rna-and-detection-of-rna-ligand-binding-using-microfluidic-modulation-spectroscopy}

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