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RNA structure probing

M. R. Brgger-Jensen, M. Lckmann, V. Valintlis

Abstract Information about biological RNA function is stored inside native RNA structure. While most RNA structures are determined by X-ray or NMR spectroscopy, there are still many RNAs left of which the structure cannot be predicted by biophysical methods. Different chemical probing techniques are developed to determine secondary and tertiary RNA structure[1]. Here we would like to introduce the most common probing techniques grouped by what information on the RNA structure it provides. A combination of different probing experiments is needed for the best prediction of secondary RNA structure. Moreover, in recent years promising techniques were developed allowing probing of many different RNAs in one sample [2, 3]. to proteins, the RNA structure is divided into primary (RNA sequence), secondary (base pairing) and tertiary (complex higher order interactions) structure. While the primary sequence can nowadays be easily obtained (next generation sequencing), information about secondary and tertiary RNA structure requires usage of more sophisticated methods. By sequence alone, the 3D structure of a complex RNA molecule cannot be predicted accurately (computational de novo prediction) [1]. Biophysical highresolution methods, such as X-ray crystallography and NMR spectroscopy are routinely used to determine RNA 3Dstructure. However, these methods can neither be applied to all RNAs nor in all cellular conditions [4]. A variety of lower-resolution chemical methods (chemical probing) can often be used to gain RNA structure information. Assessing the susceptibility of functional groups to specific reagents constitutes a versatile strategy for obtaining insights into their solvent accessibility and, by extension, into their proximity to the substrate surface and

Word count: 2200 Introduction Knowing the 3D structure of an RNA molecule is fundamental to understand its function, especially for catalytic properties and interactions with other molecules. Similar

Figure 1: Major groups of information on RNA structure provided by different probing experiments. [1]

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local structural context [5]. Here, we want to give an overview of the varied approaches to determine RNA structure based on chemical mapping. Depending on the reagent and setup used, information about basically four different structure properties (see Figure 1) can be obtained from the experiments. First, baseselective probes yield structural information about the base stacking, hydrogen bonding and electrostatic environment immediately adjacent to the base. Second, hydroxyl radicals which can be generated in the solvent, are able to cleave the RNA backbone and therefore give information about the solvent accessibility of different parts of the RNA. Third, spontaneous self-cleavage and accessibility for acylating reagents show local dynamics of the nucleotides. Finally, bifunctional reagents that are able to react with two spatially close nucleotides provide through-space information regarding RNA structure [1].

towards probes distinguishes paired and unpaired nucleotides in the sequence. Probes can recognize specific bases or be sequenceindependent. Many classes of probes are used chemicals, radicals, protein and metal ion nucleases, but it is very important for the probe to be efficient and highly specific to RNA structure. Modification places [see Figure 2] in the RNA strand can later be identified with reverse transcription (RT) followed by cDNA sequencing or directly detected by autoradiography of cleaved endlabelled RNA. Probes have very different specificity structure specific enzymes, such as nonsequence specific for dsRNA RNase V1 or S1 nuclease recognising only single stranded RNA. Experiment results should be analysed keeping in mind that first cleavage may induce conformational changes in RNA that potentially provide new targets (secondary cuts) for RNase. The majority of chemicals are base specific; moreover the base selectivity of dimethyl sulfides (DMS) and diethylpyrocarbonates (DEPC) can be adjusted with pH for noncanonical base pair (A+C) identification. DMS is a very versatile probe, it is able to penetrate cell membrane and is used in vivo probing. Only after a combination of different probing experiments and computational data analysis a reasonable RNA secondary structure model can be suggested [6]. Enzymes and chemicals are extensively used for searching and mapping ligand binding sites in RNA sequence in two ways. RNA footprinting experiments reveal sequence regions protected from cleavage by ligandRNA binding or by RNA structure itself. An opposite RNA probing approach is interference, where nucleotide modifications 2012 Page 2

Base-selective probing A variety of chemical probes have been widely used to define secondary RNA structure. Reactivity or non-reactivity of RNA

Figure 2: RNA modification sites for base-selective (left) and sequence-independent (right) probes. It is not shown, but DEPC and CMCT also react with guanosine. [1]

RNA structure probing

are identified, leading to RNA function loss or changes in protein binding [6]. Coupling probing and new generation sequencing technologies was a big step towards faster and genome-wide RNA secondary structure determination in the same experiment. Two recent approaches, FragSeq and PARS, work in similar way structure specific nucleases cleave RNA into fragments, which are later ligated to adapters and sequenced. The major difference is that in FragSeq experiment only ssRNA regions are cut with P1 RNase, while PARS uses two RNases V1 for dsRNA and S1 for ssRNA regions [2, 3].

Cleavage by OH

Product separation on gel

Solvent accessibility Hydroxyl radical probing (HRP) is mostly used for determining an RNA folding pathway. This method is known for ability to give solvent accessibility information on each nucleotide in an RNA molecule. Footprinting with hydroxyl radicals reveal protected RNA regions because of protein association or RNA folding on itself (auto-footprinting). Probing with hydroxyl radicals involves additional step to create hydroxyls in solution, e.g. by the Fenton reaction (Fe(II) + H2O2 Fe(III) +OH + OH ), using ascorbic acid for Fe(II) regeneration and EDTA for binding to RNA and Fe(II). The improvement of this reaction, called fast Fenton, can be very easily implemented with inexpensive reagents and three-syringe quench flow mixers in labs, while other method for fast radical generation by synchrotron (X-rays) needs more sophisticated instruments. Moreover, it was shown that radical induced cleavage results

Figure 3: RNA folding analysis with hydroxyl radicals. Radical induced cleavage can be made in millisecond resolution giving information on RNA folding steps A-D. Unfolded RNA (A) is cleaved uniformly , while RNA native structure (D) inhibits cuts where sequence is buried inside 3D RNA structure. Edited image [8].

are very similar between three different radical generating techniques [7]. HRP data is often used in combination with other probing techniques to reconstruct detailed folding pathways and 3D structures for increasingly large and complicated RNA molecules. These include synthetic ribozymes, and group I and group II ribozymes, from yeast, the Azoarcus cyanobacterium and Tetrahymena thermophile [8]. Moreover, an advance in HRP analysis has been reported, assigning qualitative accessibility information to every nucleotide. Hydroxyl radical reactivity is correlated with backbone solvent accessibility, which is inversely proportional to the number of through-space neighbours. Reactivity is used in discrete molecular dynamics simulation, where every nucleotide is represented by three

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beads base, sugar and phosphate, to refine a 3D RNA model [9].

SHAPE In SHAPE (Selective 2-hydroxyl acylation analysed by primer extension) the RNA is treated with electrophiles which react with the 2hydroxyl group on the RNA backbone and forms a 2-O-ester adduct. The electrophiles, are independent of the type and order of bases in the backbone and will form adducts on unstructured (typically single-stranded) regions in the RNA. The electrophilic attack on the 2hydroxyl group can be performed in a few minutes and can be applied to RNA of any size. After random acylation and RT a variety of different length cDNAs is made. The cDNAs are then quantified by electrophoresis. A primer extension and electrophoresis is also performed on unreacted RNA. After normalization and by subtracting the two distributions the SHAPE reactivity for each nucleotide can be determined. As the SHAPE reactivity represents the propensity of a 2-O-ester

Local nucleotide dynamics In-line probing In-line probing is a way to investigate local nucleotide flexibility in RNA without the use of chemicals. The unstructured regions in RNA are more flexible than the structured regions (helical, double stranded, etc.) and they will cleave at the 2hydroxyl group when left in an alkaline environment (typically pH 9). The RNA is radioactive labelled and the resulting bands of the degraded RNA can be visualized on a gel. In general, in-line probing results are often used in comparison to other RNA footprinting or interference experiment results [10].

Figure 4: Mutate-and-map strategy explained on riboswitch from Vibrio vulfinicus. Each base on a sequence is mutated to its complement base. A 2-dimensional SHAPE data for each mutation are shown in Z-score (number of standard variations from the mean accessibility). Signals from the mutated bases form a diagonal line from top left to bottom right. If a mutated base was involved in a base pair, the other base will become accessible to electrophilic attack on the 2hydroxyl group and show a reading outside the diagonal line. Basepairs forming a helical structure will therefore be visual as lines of signals on each side of the diagonal line [12].

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adduct to be formed on each nucleotide it is a measure of local nucleotide flexibility. But be aware that some base pairs which would form in the living bacteria do not form when the rRNA is isolated from the bacteria [11]. By inserting mutations in native RNA sequence and performing SHAPE experiments, it is possible to obtain valuable information about RNA structure. The so called mutate-and-map strategy does this by adding the SHAPE electropherograms of the RNA mutations in a two-dimensional plot (see Figure 4 above) [12]. SHAPE can be combined with thermodynamic parameters for RNA to make more precise analysis of secondary structure. The RNAstructure program finds the lowest free energy RNA structure by using empirical thermodynamic parameters fit against a large database of model structures with known stability. The term GSHAPE(i) = m ln[SHAPE reactivity(i) + 1] + b is created. The slope (m) and the intercept (b) was parametrized against 23S rRNA by using the secondary structure determined by comparative sequence analysis. At a positive slope, the SHAPE reactivity is high and baseparing is unlikely to occur. As m and b increase, the accuracy for which the secondary structure is predicted by RNA structure increases nearly 20% [11].

extremely valuable since they provide strong restraints that reduce conformational space search for computational modelling. In general, current methods to analyse through-space interactions involving chemical mapping are based either on a site-specific cleavage that is initiated by a spatially close nucleotide or on bifunctional reagents that connect short-distanced nucleotides. Three prominent methods for chemical contact mapping of RNAs are Multiplexed Hydroxyl Radical Cleavage Analysis (MOHCA), bifunctional crosslinking and site-directed cleavage. MOHCA is a gel-based probing technique that uses EDTA and phosphothioate groups to identify the cleavage and probe position. These groups are attached to modified nucleotides. First, a cleavage agent is randomly inserted into the structure. Then, the positions of radical hydroxyl cleavages are read in a two-dimensional gel against the positions of the probe, producing a set of pairwise distance constraints. The distance between a cleavage site and the probe is proportional to the intensity of the cleavage. The analysis of MOHCA data has recently been automated [4]. Although its set-up is straightforward, MOHCA is based on the usage of a relatively bulky reagent that may influence the overall structure of the analysed RNA [1]. Site-directed cleavage also uses modified nucleotides to which a cleavage agent (usually Fe(II)-EDTA) is attached. In contrast to MOHCA, the cleavage agent is not inserted randomly but systematically, using its property of binding to specific motives for example to bulged, three base-pair helix sites which can be generated on every position of

Through-space neighbours Apart from the base-paired secondary structure, many RNA molecules form higherordered tertiary structures which are essential for function (e.g. catalytic activity of ribozymes). In the absence of crystallographic data, information about these interactions is

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the RNA using mutagenesis. Consequently, a new mutant RNA has to be created for every experiment. Finally, the fragments can be separated easily using PAGE or capillary electrophoresis [13] Bifunctional crosslinking agents, such as nitrogen mustard (bis(2-chloroethyl)methylamine), bridge nitrogen atoms of proximal bases and therefore give valuable distance relationships. The analysis of the fragments is done as follows: Nuclease treatment provides a wide range of products consisting of two hydrolytic fragments conjugated by the cross-linker, bifunctional adducts in which both conjugated bases were present on the same fragment or monofunctional adducts corresponding to failed cross-links. Afterwards, mass mapping and tandem sequencing (MS3D) are used to characterize the products of probing reactions and identify the sequence position of conjugated nucleotides [14]. Future outlook Chemical probing of RNA molecules is a varied field, in which many developments have taken place over the last years. It has turned out that probing techniques are especially useful to study larger, more dynamic RNAs and their folding intermediates. Consequently, the combination of different mapping approaches and techniques often allows deep insight into RNA molecules that are sufficiently long to represent most systems of interest in a timeresolved way. A general trend is that many experiments and mechanisms become easier to apply and to read out. Therefore, several high-throughput approaches, like PARS or FragSeq have been established.

Computational de novo structure prediction of RNA molecules still lacks accuracy, since the minimum free energy models are often incorrect. Also suboptimal models that a folding algorithm provides should be regarded and used for further analysis. Including experimental data in the computational structure prediction significantly increases the prediction accuracy. Above all, information about tertiary interactions provides strong restraints that reduce computational effort. For the future, the use of soft techniques to act under more native conditions will be a great challenge to determine native RNA structures.

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