In order to understand the function of RNA molecules on a molecular level, methods are needed that provide information on their structures and conformational changes. The first level of RNA structural description is the identification of the base sequence. The second level of organization, the secondary structure, is mainly governed by Watson-Crick and Wobble base pairing, which leads to double-stranded helices interrupted by single-stranded regions in internal or hairpin loops.6 Nowadays, in silico methods are available to predict the secondary structure but research is still ongoing. Due to computational complexity, standard folding programs disregard structural features as pseudoknots, loop-loop interactions or k-way junctions. However, the available in silico methods provide a good first insight into the RNA structure. One widely used approach for computational prediction of RNA secondary structure is based on the thermodynamics of the molecule.70,71 The free energy of the real, native structure is supposed to be the one of the minimum free energy and is computed through thermodynamic parameters via softwares such as Mfold,72 RNAfold,73 or RNAstructure.74 The softwares provide a secondary structure with base pair probabilities and can be used to design experimental measurements or support interpretations. Alternative approaches rely on stochastic models predicting the highest possibility of base pairing or algorithms comparing structural conservations of homologous RNA sequences i.e., sequences of RNAs (tRNA or rRNA) belonging to different organism.70,71
A reliable tool to deduce the secondary structure under wet lab conditions is chemical probing. Chemical probing detects single stranded and double stranded elements within RNA strands and allows prediction of the respecitve secondary structure. The RNA structural elements are characterized either through introduction of chemical adducts or through techniques leading to strand scission. The products can be identified by e.g., direct 5′-labeling with the radioacitve 32P or by reverse transcription. The introduction of chemical adducts is carried out with either base specific reactive alkylation agents or with sequence-independ 2′-hydroxyl acylation reagents. The former can be performed by e.g., dimethylsulfate, kethoxal or carbodiimides. The latter, which is commonly used and the so-called selective hydroxyl acylation analyzed by primer extension (SHAPE) method, is carried out with electrophile reagents such as IM7, NAI, or FAI, which are selective for flexible positions within the RNA structure. Ongoing research on novel chemical designs of the aforementioned acylation reagents enable RNA modifications also within cells. The chemical adducts generally block the RNA polymerase so that the truncated RNA strands can be analyzed. Another form of chemical probing relies on
13 strand scission. The so-called In-line probing utilized slightly basic conditions which promotes the intrinsic phosphate backbone cleavage reactivity of the 2′ hydroxyl group.
The induced ‘spontaneous’ cleavage reactions are more pronounced on flexible positions within the RNA structure and allow in turn predictions of the RNA structure.
Strand scission can also be performed by RNase enzymes cutting at specific binding sites such as singles stranded or double stranded regions within the RNA structure.75,76 Currently, progress is made to obtain secondary structures via chemical probing in cell.75 In order to unravel the full three-dimensional structure of RNA molecules additional methods are needed and a selection of them is described in the following.
Since the Nobel prize in chemistry was awarded to J. Dubochet, J. Frank and R.
Henderson in 2017, cryo-electron microscopy (cryo-EM) has emerged as a tool to “move biochemistry into a new era”.77,78 Technologic breakthroughs enabled developing a method called single-particle cryo-EM. It is based on an electron beam that is applied to a frozen sample. A large number of 2D images of randomly oriented particles is recorded and computationally combined to a 3D reconstruction. The samples are placed inside a vacuum chamber.79 To avoid dehydration of the biomolecules in the vacuum, different methods of sample preparation have been developed.80 Freezing the sample at a specific point in time allowed time-dependent measurements.81 Thus, a main drawback that cryo-EM cannot detect dynamic motions has been eliminated. Another widely assumed opinion that RNA molecules are too small to achieve an acceptable contrast of individual particles or conformational too heterogeneous to obtain enough images of all sites to reconstruct a model is currently refuted.79 For example, different RNA molecules, ranging from 119 - 338 nt, from ribozymes to riboswitches, with and without ligands, were successfully resolved.82
Nevertheless, the Protein Data Bank,83 the database for 3D structural data for proteins, nucleic acids and their complexes, is dominated by X-Ray structures (Figure 9).83 X-Ray crystallography provides detailed atomic information without size restriction from diffraction patterns derived from well-ordered, good quality crystals.82 It is an excellent method to gain information about rigid proteins and stable complexes.
However, obtaining crystals is challenging especially regarding RNA structures that are often highly flexible.
In addition, X-Ray crystallography requires pure and
Figure 9. Statistic of the total number of entries available in the Protein Data Bank. Data are obtained from reference 83 on July 9, 2020.
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homogeneously folded RNA in large scale.84 Different crystallization conditions regarding concentration, temperature, precipitant, and salt need to be tested.85 Besides the difficulties in crystallization, the crystal represents only a static picture of the system. In this regard, X-Ray Free Electron Lasers (XFELs) relying on the “diffraction before destruction” approach, may help.86 Powerful pulses are applied on a large amount of crystals as small as 0.1-10 µm.87 Before the radiation damage destroys the crystal, a diffraction pattern is collected. With this approach, four reaction states of the adenine riboswitch with and without ligand binding were obtained.88 Hence, also dynamics and kinetics are resolvable and methods are currently under development to do so, without the need growing large crystals.89
One method that does not require crystals but is still reliant on considerable large amounts of homogeneous RNA, is Nuclear Magnetic Resonance (NMR) spectroscopy.
NMR spectroscopy is the main tool to elucidate structure, dynamics and interactions of nucleic acids in solution and on different timescale.90–92 NMR spectroscopy investigates the interaction of nuclear spins in an applied magnetic field. Due to spectral crowding, NMR measurements are until now restricted to approximately 100 nt.91–93
A size unlimited method with respect to the oligonucleotide is Förster Resonance Energy Transfer (FRET). FRET is based on the attachment of two fluorophores to the surface of the biomolecule.94,95 Due to a broad user application, there are well-studied dyes available with high quantum yields like Alexa Fluor or Atto Dye.94 Through non-radiative energy transfer between the donor and the acceptor fluorophore, the dipole-dipole interaction allows the determination of distances up to 10 nm.95,96 Bleaching and stacking of the dyes is a drawback that one needs to bear in mind.94,95 A concern often raised is related to the large size of the dyes and their long, flexible linkers, which both may have an influence on the biomolecule and the distances measured. However, a recent study of 20 laboratories demonstrated that FRET is a reliable and reproducible method that is applicable on the single-molecule level (smFRET). They showed that a set of DNA duplexes with dyes separated by 11, 15 and 23 nt corresponding to distances between 46 - 84 Å could be measured with total uncertainty of less than 6 Å although different experimental set-ups were used.94 Also for RNA molecules, several investigations have been published.97–102 Last but not least, recent studies shown that time-resolved measurements on the picosecond time scale 103–106 as well as in cell measurements at room temperature are possible with FRET.96,104
Complementary to these methods, electron paramagnetic resonance (EPR) spectroscopy provides insight into the dynamics and structures of biomolecules without
15 size restriction and in solution.107–114 In order to apply EPR spectroscopic methods to RNA, the RNA needs to bear one or more unpaired electrons. These electron spin centers can be paramagnetic metal ions or organic radicals, the so-called spin labels.
EPR spectroscopic methods enable to measure the dipolar coupling between two or more electron spins that can be translated into distance distributions.108,110,114–117
Extensive efforts have been made to facilitate measurements under physiological, in cell conditions.118–136 Beyond these distance measurements, EPR-based hyperfine spectroscopy offers the opportunity to unravel the structure of a binding site with atomistic resolution. The number and affinity of metal ion binding sites can be unraveled by means of quantitative continuous wave (cw) EPR measurements, provided that the metal ion is either intrinsically paramagnetic or can be substituted by a paramagnetic one.108,137–146
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