生物化学

A covalently closed loop structure provides circular RNA (circRNA) with more stability than conventional RNAs in linear form, making circRNA an emerging tool in RNA therapeutics. The qualification and quantification of circRNA after production is critical for its design and effectiveness assessments, particularly when the following applications could be affected by byproduct RNAs. Despite PCR-based methods effectively detecting low-abundance circRNA, they are unsuitable for assessing uncircularized RNA in a mass production fraction to maintain quality control. Here, we present a straightforward protocol for evaluating uncircularized byproduct RNAs from circRNA production. This method enrolls the template-independent RNA polymerase activity to add adenine tails (polyA) to the 3' ends of a linear RNA, making it easy to distinguish trace byproducts or uncircularized RNA from a pool of mass circRNA products. With conventional linear RNA and RNase R-treated circRNA as the positive and negative controls, the purity of a circRNA preparation could be readily resolved. Regardless of circRNA production strategies, this protocol provides a reliable and practical way to ensure the consistent quality of homemade circRNAs or to recheck circRNA quality from commercial manufacturing.

Understanding the function of oligonucleotides on a molecular level requires methods for studying their structure, conformational changes, and internal dynamics. Various biophysical methods exist to achieve this, including the whole toolbox of Electron Paramagnetic Resonance (EPR or ESR) spectroscopy. An EPR method widely used in this regard is Pulsed Electron-Electron Double Resonance (PELDOR or DEER), which provides distances in the nanometer range between electron spins in biomolecules with Angstrom precision, without restriction to the size of the biomolecule, and in solution. Since oligonucleotides inherently do not contain unpaired electrons, these have to be introduced in the form of so-called spin labels. Firstly, this protocol describes how nitroxide spin labels can be site-specifically attached to oligonucleotides using “Click” chemistry. The reaction provides little byproducts, high yields, and is conveniently performed in aqueous solution. Secondly, the protocol details how to run the PELDOR experiment, analyze the data, and derive a coarse-grained structure. Here, emphasis is placed on the pitfalls, requirements for a good dataset, and limits of interpretation; thus, the protocol gives the user a guideline for the whole experiment i.e., from spin labeling, via the PELDOR measurement and data analysis, to the final coarse-grained structure.

Graphical abstract:

Schematic overview of the workflow described in this protocol: First, the spin-labeling of RNA is described, which is performed as a "Click"-reaction between the alkyne-functionalized RNA strand and the azide group of the spin label. Next, step-by-step instructions are given for setting up PELDOR/DEER distance measurements on the labeled RNA, and for data analysis. Finally, guidelines are provided for building a structural model from the previously analyzed data.

RNA secondary structures are highly dynamic and subject to prompt changes in response to the environment. Temperature in particular has a strong impact on RNA structural conformation, and temperature-sensitive RNA hairpin structures have been exploited by multiple organisms to modify the rate of translation in response to temperature changes. Observing RNA structural changes in real-time over a range of temperatures is therefore highly desirable. A variety of approaches exists that probe RNA secondary structures, but many of these either require large amount and/or extensive processing of the RNA or cannot be applied under physiological conditions, rendering the observation of structural dynamics over a range of temperatures difficult. Here, we describe the use of a dually fluorescently labelled RNA oligonucleotide (containing the predicted hairpin structure) that can be used to monitor subtle RNA-structural dynamics by Förster Resonance Energy Transfer (FRET) at different temperatures with RNA concentration as low as 200 nM. FRET efficiency varies as a function of the fluorophores’ distance; high efficiency can thus be correlated to a stable hairpin structure, whilst a reduction in FRET efficiency reflects a partial opening of the hairpin or a destabilisation of this structure. The same RNA sequence can also be used for Circular Dichroism spectroscopy to observe global changes of RNA secondary structure at a given temperature. The combination of these approaches allowed us to monitor RNA structural dynamics over a range of temperatures in real-time and correlate structural changes to plant biology phenotypes.

Graphic abstract:

Monitoring temperature-dependent RNA structural dynamics using Förster Resonance Energy Transfer (FRET)