Method for promoting RNA crystal growth and improving diffraction resolution and application thereof

Abstract

The invention belongs to the field of nucleic acid crystallographic structures, and particularly discloses a method for promoting RNA crystal growth and improving diffraction resolution and application thereof. The resolution limit of the RNA crystal in X-ray diffraction is remarkably improved by selectively using a GU base pair to replace a Woson-Krike base pair or reversely replacing the Woson-Krike base pair in an RNA sequence. According to the method, high-resolution structures of eight unique RNA crystals are successfully obtained, and it is summarized that GU base replacement is introduced and placed in the middle of a P1 (a first 5 '-double helix stem) of RNA or in the middle of a peripheral stem part of any inactive center, so that the effect can be achieved. According to the scheme, a simple and effective method is provided from the perspective of RNA sequence editing, and the sequence diversity is increased by changing the types of the base pairs, so that the X-ray diffraction resolution of the RNA crystal is remarkably improved. The method is of great significance in analyzing the complex structure of RNA molecules and revealing the function, catalytic mechanism, ligand recognition and intermolecular interaction of the RNA molecules.

Description

Technical Field

[0001] The invention belongs to the field of nucleic acid crystallographic structure, and specifically relates to a method for promoting RNA crystal growth and improving diffraction resolution and an application thereof. Background Art

[0002] RNA molecules are central components of many cellular processes. In addition to being carriers of genetic information during protein synthesis, RNA molecules also display a variety of catalytic and regulatory functions. The versatility of RNA molecules in cells is conferred by their three-dimensional structure, whose precise folding plays a vital role in living organisms.

[0003] X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryo-electron microscopy (cryo-EM) are the main tools in RNA structural biology. As of July 3, 2024, records in the Protein Data Bank (PDB) indicate that of the 1,861 pure RNA structures, approximately 62.4% were determined by X-ray crystallography, 31.6% by NMR, and 6% by cryo-EM. NMR spectroscopy is of particular value in exploring the dynamic properties of RNA structures in solution, while cryo-EM has unique advantages in solving the structures of large RNA molecules and protein complexes without the need for crystallization.

[0004] So far, X-ray crystallography is the most successful method for determining RNA structure. Its core lies in revealing the three-dimensional conformation of RNA with high precision by analyzing X-ray diffraction images. The high-resolution RNA structure obtained by X-ray crystallography analysis helps to elucidate the function of RNA, catalytic mechanism, ligand recognition and interaction of RNA molecules. In addition, these structural information are indispensable in promoting the development of RNA targeted therapy, promoting the advancement of gene editing technology, and analyzing the molecular mechanism of RNA-related diseases. The resolution of crystal data obtained by crystal X-ray diffraction is a key indicator of the overall quality and accuracy of the derived structural model. The data resolution is directly related to the number of independent measurements that can be used to determine the atomic parameters of the molecule (atomic coordinates x, y, z and at least one thermal vibration parameter). The higher the resolution, the higher the number of independent measurements and the higher the accuracy of the model. The resolution is usually expressed in To measure, the smaller the number, the higher the resolution (less than or equal to Researchers have encountered many challenges in high-resolution RNA crystallography. The molecular dynamics and conformational fluctuations of RNA molecules make it extremely difficult to obtain high-quality crystals. Secondly, the phase problem remains a core problem in this field. The accurate acquisition of phase information is crucial for deriving precise atomic structures from diffraction patterns, but the acquisition process is still full of technical difficulties.

[0005] There are many techniques in the art for obtaining RNA crystals with good diffraction. For example, RNA binding proteins such as U1A protein, Fab antibodies or tRNA scaffolds are used to promote the crystallization process. These molecular scaffolds can stabilize the RNA structure, thereby increasing the possibility of forming crystals suitable for high-resolution analysis. In addition, strategies such as cation substitution and dehydration can be used to improve the quality of RNA crystals. Although these methods have potential, they have not been fully utilized in RNA crystallography due to operational complexity. Among the methods for obtaining high-resolution RNA crystal structures, one method has been widely used, namely, to increase RNA sequence diversity, which focuses on changing the length of the P1 stem (the first 5' double helix stem) or modifying unstable loops to generate stable tetranucleotide loops (Tetraloop) such as GAAA or UUCG. However, the pursuit of RNA crystals with good diffraction requires continuous exploration of new strategies. Different from traditional methods, the present invention proposes a new strategy, namely, manipulating the presence of GU base pairs in RNA sequences.

[0006] GU (guanine-uracil) base pairs are often observed in RNA structures and have unique chemical, thermodynamic and structural properties. In various RNA molecules, GU base pairs play important roles in folding, ribozyme catalysis and interaction with proteins. However, the substitution and reverse substitution of GU with Watson-Crick base pairs are rarely used in academia, which deserves further exploration. Summary of the invention

[0007] The present invention aims to overcome the deficiencies of the prior art and provide a method and application thereof for promoting the growth of RNA crystals and improving the diffraction resolution. By changing the base types and increasing the sequence diversity, the X-ray diffraction resolution of RNA crystals is significantly improved, providing an effective means for analyzing the complex structure of RNA molecules.

[0008] The technical solution adopted by the present invention is to provide a method for promoting RNA crystal growth and improving diffraction resolution, including forward substitution and / or reverse substitution of RNA GU base pairs and Watson-Crick base pairs.

[0009] Further, the forward substitution includes AU-GU substitution, UA-UG substitution, GC-GU substitution and / or CG-UG substitution. GU pairs and UG pairs are collectively referred to as GU base pairs below; unlike Watson-Crick base pairs, GU pairs and UG pairs do not have the property of spatial overlap, so G or U can try to replace or be replaced, resulting in the above-mentioned several substitution situations, which are collectively referred to as forward substitution of GU base pairs below.

[0010] Furthermore, the position of the forward substitution is located in the middle of the pairing region of the RNA stem.

[0011] Furthermore, the RNA stem includes the first 5' double helix stem of the RNA and / or the stem of any inactive center.

[0012] Furthermore, the reverse substitution includes GU-AU substitution, UG-UA substitution, GU-GC substitution and / or UG-CG substitution.

[0013] Existing X-ray diffraction crystallography relies on initial screening through raw sequences, followed by optimization of screening conditions after crystals are formed, and collection of diffraction data to solve RNA structures. However, this process often fails to produce satisfactory results, such as low crystal quality or poor X-ray data.

[0014] In order to solve this problem, the strategy proposed in this scheme is to increase sequence diversity and significantly improve the probability of obtaining high-quality crystals. In one or more embodiments of the present invention, the RNA sequence is modified, and a pair of AU or GC base pairs are replaced with GU base pairs in the middle position of P1 (the first 5' double helix stem), P2 and P3 stems (inactive central stems are numbered from the 5' end), or on the double helix stems where the pseudoknot or hairpin loop binds to the protein auxiliary factor, thereby increasing the sequence diversity, thereby promoting the growth of RNA crystals and significantly improving the diffraction resolution of the crystals. In another embodiment of the present invention, after the naturally occurring GU base pairs in the RNA sequence are replaced with GC base pairs, the sequence diversity is also increased, thereby significantly improving the diffraction resolution of the crystals.

[0015] Another object of the present invention is to provide a method for promoting RNA crystal growth and improving diffraction resolution for use in RNA crystal structure analysis. This solution improves the X-ray diffraction resolution of RNA crystals, and researchers can reveal the three-dimensional conformation of RNA with high precision by analyzing X-ray diffraction images, which helps to clarify the function of RNA, catalytic mechanism, ligand recognition and interaction of RNA molecules. In addition, this structural information is indispensable in promoting the development of RNA targeted therapy, promoting the advancement of gene editing technology, and analyzing the molecular mechanism of RNA-related diseases.

[0016] Another object of the present invention is to provide a method for solving the phase problem of RNA crystals, including forward substitution and binding of barium ions. In an embodiment of the present invention, some GU base pairs are bound to barium ions, and the barium ions, as heavy atoms, provide phase information of RNA crystals and improve X-ray diffraction results. This embodiment proves that GU base pairs have multiple effects in RNA crystallography. Although they do not always improve resolution, they can promote the binding of heavy atoms and provide a new idea for solving the phase problem.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention increases sequence diversity and improves crystal quality by implementing forward and reverse substitution of GU base pairs with Watson-Crick base pairs, especially replacing base pairs in the middle position of the paired part of the RNA stem, thereby significantly improving the X-ray diffraction resolution. In addition, RNA crystal phase information is provided by combining barium ions with the forward substituted GU base pairs. This scheme provides a simple and efficient tool from the perspective of RNA sequence editing. By changing the base types and increasing sequence diversity, the X-ray diffraction resolution of RNA crystals is improved, which is conducive to analyzing the complex structure of RNA molecules and is of great significance for clarifying the function, catalytic mechanism, ligand recognition and interaction of RNA molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a scatter plot of the resolution changes of 8 RNA crystal structures before and after base pair substitution. In the figure, different colors are used to represent the change in resolution: dots represent the situation where the resolution is improved after replacing other base pairs with GU pairs; squares represent the situation where the change in GU pairs has little effect on the resolution; diamonds represent the situation where the resolution is increased after GU is restored to GC.

[0020] Figure 2 Includes a flowchart of RNA crystals and their structure determination (left) and a schematic diagram of GU base pair editing (right).

[0021] Figure 3 We show that LINE-1 ribozyme and SAM-I riboswitch achieve higher resolution after replacing GC base pairs with GU base pairs.

[0022] Figure 4 It was shown that the resolution of Halotrichia maritima kink-turn-7 (HmKt-7) and HmKt7-19ntX3 was significantly improved after replacing GC base pairs with GU base pairs.

[0023] Figure 5 We show that the 2'-dG-III ribosomal switch and Broccoli RNA aptamer achieve higher resolution by replacing AU base pairs with GU base pairs.

[0024] Figure 6 We show that the OR4K15 ribozyme was transformed to a higher resolution by a GU to GC substitution.

[0025] Figure 7The main structural changes of a conventional A-form RNA helix in the presence of a standard wobble U·G pair are shown. U moves into the major groove between two Watson-Crick base pairs. The usual torsion angle in an RNA helix is ​​33°; in a U·G pair, the torsion angle in the 5' direction of U increases, while the torsion angle in the 3' direction decreases. These changes in torsion angles result in significant interchain stacking between the G in the U·G pair and the 3'-end residue of U (connected by a black box). In the 5' direction of U, stacking occurs within the chains on both sides.

[0026] Figure 8 Superimposed models of the SAM-I-GC riboswitch (red, PDB ID: 4B5R) and GU (cyan, PDB ID: 5FJC) are shown. The replaced GU base pair in the main chain trajectory P2 (cyan), the pseudoknot (PK) in P1, and the bound SAM molecule are all shown in cyan (O: red; N: dark blue).

[0027] Fig. 9 Different crystal stacking situations of 2'-dG-III-AU (PDB: 8Z8Q), 2'-dG-III-GU (PDB: 8KEB), and HmKt7-GU and U1A protein (PDB: 5FJ4) are shown. (A) Crystal stacking situation of 2'-dG-III-AU, whose space group is P1. (B) Crystal stacking situation of 2'-dG-III-GU, whose space group is C121. (C) Crystal stacking situation of HmKt7-GU and U1A protein, whose space group is C2221. (D) In ​​the 2'-dG-III unit cell, intramolecular hydrogen bonds are formed between the 5'-phosphate group and the O2' oxygen atom between the GU base pairs.

[0028] Fig.10 Demonstration of novel GAAA interactions between asymmetric units of MTR1. (A) Four MTR1 molecules related by crystallographic symmetry. On the left, the molecules (red) are associated with each other via a symmetry-related GAAA four-base ring with novel interactions (annotated within the dashed rectangle). On the right, symmetry-related molecules (green) are in contact with each other via backbone contacts proximal to a displaced GU base pair. On the far right, a zoomed-in view of the dashed box shows the precise interactions between the twofold symmetry-related molecules. Next to the illustration are the sequences of the interactions and a schematic diagram showing the non-Watson-Crick base pair types according to the Leontis-Westhof nomenclature (2). (B) (left) Non-Watson-Crick base pairs of GAAA interactions in MTR1; (right) two views showing the symmetry relationship between the GAAA four-base ring.

[0029] Fig.11The structures of G·U base pairs are shown. (A) One standard wobble G·U base pair (from PDB 1HQ1) and one non-standard anionic G·U base pair (from PDB 7K00). (B) Eight standard wobble G·U base pairs were observed in the structure of this proposal.

[0030] Fig.12 Metal ion binding in high-resolution RNA structures is shown. (A) Electron density maps for Broccoli-GU potassium bases G2 and U48 are shown. The electron density shows that electron density maps are missing from simulated annealing at 1.3σ. (B) Electron density maps for 2'-dG-III-GU sodium bases G2 and U70 are shown. The electron density shows that electron density maps are missing from simulated annealing outlined with 0.9σ contour lines. (C) Electron density maps for OR4K15-GC sodium bases G29 and C2 are shown. The electron density shows that electron density maps are missing from simulated annealing at 1.0σ. (D) Electron density maps for SAM-I-GU barium bases G30 and U22 are shown. The electron density shows that electron density maps are missing from simulated annealing at 1.5σ.

[0031] Fig.13 The positions of the introduced GU base pairs in LINE-1 ribozyme (A), 2'-dG-III riboswitch (B), Broccoli aptamer (C), and MTR1 ribozyme (D) and their corresponding resolutions are shown.

[0032] Fig.14 The position and energy changes of the GU base pairs on the double helix stem in the RNA structure analysis results are shown. The G of the six GUs is located on the 5' strand and the four Gs are located on the 3' strand. ΔE represents the difference in thermodynamic free energy caused by sequence changes (ΔE of HmKT7-19ntX3 is calculated by introducing the GAAA loop). The calculation of thermodynamic free energy uses the secondary structure prediction tool provided by MathewsGroup (https: / / rna.urmc.rochester.edu / RNAstructureWeb).

[0033] Fig.15Examples of solving the phase problem by artificially introducing GU base pairs that bind to heavy metals are shown. (AG) Schematic diagrams of the secondary structures of cricket paralysis virus (CrPV) IRES RNA (A)(4), dystrophin kinase type 1 (DM1) RNA (B)(5), SAM-II riboswitch (C)(6), bis-(3′-5′)-cyclic dimeric guanylate monophosphate (c-di-GMP)-II riboswitch (D)(7), guanidine-I riboswitch (E)(8), S-adenosyl-L-homocysteine ​​(SAH) riboswitch (F)(9), and potato leafroll virus (PLRV) RNA (G)(10) are shown. Pink spheres represent ligands. DETAILED DESCRIPTION

[0034] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application, rather than limiting the present application. Unless otherwise specified, all technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art.

[0035] It should be noted that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the exemplary embodiments of the present application. As used herein, unless otherwise expressly stated, terms in the singular form shall be deemed to include their plural forms. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates the presence of specific features, steps, operations, devices, components and / or combinations thereof, but does not exclude the presence of other features or elements not explicitly listed.

[0036] The present invention is now further described in conjunction with specific examples. The following examples are only intended to illustrate the present invention, but do not constitute a limitation on its scope. The test samples and test processes used in the following examples include the following (if the specific experimental conditions are not indicated in the examples, they are usually carried out according to conventional conditions or according to the conditions recommended by the reagent supplier; the reagents, consumables, etc. used in the following examples, if not otherwise specified, can all be obtained from commercial sources).

[0037] 1. Materials and Methods

[0038] (1) Chemical synthesis of RNA

[0039] Oligonucleotides were synthesized using tert-butyldimethylsilyl (t-BDMS) phosphoramidite chemistry following the procedure outlined by Wilson et al. The synthesis protocol was performed using an Applied Biosystems 394 DNA / RNA synthesizer and involved the use of t-BDMS (Link Technologies) to protect ribonucleic acid phosphoramidites. (Wilson, TJ, Zhao, ZY, Maxwell, K., Kontogiannis, L. and Lilley, DM (2001) Importance of specific nucleotides in the folding of the natural form of the hairpin ribozyme. Biochemistry, 40, 2291–2302.)

[0040] All oligonucleotides were dissolved in 100 μL of anhydrous DMSO and 125 μL of trifluorotriethylamine (Sigma-Aldrich) to facilitate the removal of the t-BDMS group. The solution was incubated at 65 °C in the dark for 2.5 h. After cooling on ice for 10 min, RNA precipitation was achieved using 1 mL of butanol, 70% ethanol, and then suspended in double distilled water. RNA lacking bromine modification was purchased from Precision Biotechnology Co., Ltd. (Hunan, China). LINE-1 ribozymes, HmKt-7, HmKt7-19ntX3, and OR4K15 ribozymes were chemically synthesized.

[0041] (2) RNA transcription

[0042] To promote efficient transcription initiation, the initial nucleotides of the native sequence were replaced by GCG / GGC / GGA / GGG. DNA templates containing T7 RNA polymerase promoter for in vitro transcription were prepared by polymerase chain reaction (PCR). After transcription, RNA molecules were purified by electrophoresis in polyacrylamide gels containing 7 M urea. The intact RNA transcripts were recovered by UV visualization, excision, and electroelution (using an electroelution system) after electrophoresis at 200 V in 0.5× TBE buffer for 12 h. Subsequently, RNA was precipitated with isopropanol, washed with 75% ethanol, and finally dissolved in water. SAM-I riboswitch, 2'-dG-III riboswitch, Broccoli RNA aptamer, and MTR1 ribozyme were synthesized by in vitro transcription.

[0043] (3) Crystallization, structure determination and purification

[0044] Solutions containing 10 μg / μL RNA in 5 mM HEPES (pH 7.5), 100 mM KCl, and 5 mM MgCl2 (HKM buffer) were refolded as described in Table 2. The RNA sequences and crystallization conditions are also summarized in Table 2. Crystallization was performed by mixing 0.2 μL of RNA or RNA-ligand complex with 0.2 μL of the stock solution using the sitting drop vapor diffusion method at 18 °C. Crystals were transferred to mother solutions containing additional 30% glycerol or MPD and then flash frozen by mounting in nylon loops and plunging into liquid nitrogen. X-ray diffraction data were collected on beamlines BL02U1, BL10U2, BL18U1, and BL19U1 at the Shanghai Synchrotron Radiation Facility (SSRF) and the National Facility for Protein Science (NFPSS) in Shanghai and processed with XIA2 or XDS. The resolution cutoff of the data was determined by inspecting CC1 / 2 and density maps. PDB 8Z9K (LINE-1-GC), 8Z8Q (2'-dG-III-AU), 8ZAU (MTR1-GU), and 8ZA4 (OR4K15-GU) were solved by PHASER molecular replacement, using 8Z8U (LINE-1-GU), 8KEB (2'-dG-III-GU), 7V9E (MTR1-GC / AU), and 8ZA0 (OR4K15-GC) as models. The resulting models were manually adjusted using Coot and refined through several iterations of Coot, phenix.refine, and PDB REDO. The geometry of the models and the fit to the electron density map were monitored using MOLPROBITY as well as validation tools in Coot. To effectively avoid overfitting, especially for structures with lower resolutions determined by molecular replacement, optimization weights and simulated annealing were introduced in phenix.refine. The experimental maps were calculated by Autosol, and the Fourier anomaly difference maps were calculated by phenix.refine. The simulated annealing omit maps were calculated by composite omit maps calculated using the annealing method in PHENIXsuite. The atomic coordinates and structure factor amplitudes have been deposited in the Protein Data Bank (PDB) and are listed in Table 3 together with the accession codes.

[0045] Among the crystal structures discussed in this article, four are new: LINE-1 ribozyme (8Z9K and 8Z8U), OR4K15 ribozyme (8ZA0 and 8ZA4), 2'-dG-III riboswitch (8Z8Q and 8KEB), and Broccoli RNA aptamer (8K7W). Four of the crystal structures have been reported: MTR1 ribozyme (7V9E), HmKt-7-GU (5FJ4), HmKt7-19ntX3 (5G4T), and SAM-I (4B5R and 5FJC) ( Figure 1and Table 1). These structures were obtained by screening 6 commercial crystallization kits, totaling 288 crystallization conditions, including Index1-96, Natrix I / II, and Crystal I / II, all purchased from Hampton Research. The specific crystallization details of the SAM-I riboswitch, HmKt-7, and HmKt7-19ntX3 RNA are recorded in their respective publications, outlining the process of structure resolution and refinement. A common nomenclature was used when designating the secondary structure: the first helix at 5' was named P1, followed by P2, the single-stranded helical link between P1 and P2 was called J12, and the head loop of the last helix had the same number as the helix, such as L3 covering P3.

[0046] 2. Results

[0047] (1) Innovation in crystallographic methods: GU base pair substitution

[0048] This study proposes an innovative strategy to promote RNA crystallization by replacing the existing helical Watson-Crick base pairs with wiggly GU pairs ( Figure 2 ) to promote RNA crystallization. Prior art X-ray diffraction crystallography relies on initial screening of the original sequence to obtain crystals. After the crystals are formed, the screening conditions are optimized and X-ray diffraction data are collected to resolve the structure. However, this process often fails to produce satisfactory results, such as low crystal quality or poor X-ray data. To address this problem, the strategy proposed in this scheme is to increase sequence diversity, thereby significantly increasing the probability of obtaining high-quality crystals. This strategy was not only successful in the initial screening stage, but also effectively optimized the crystal quality when the existing crystal resolution was low.

[0049] Figure 2 The flowchart on the left describes the standard process for producing RNA diffracting crystals and determining their structures. First, the original sequence for crystallization is designed and screened, as shown by the black arrows. If satisfactory crystals are not obtained or the X-ray data quality is poor, as shown by the gray arrows, the RNA sequence can be replaced or reversed with GU base pairs, as shown by the pink arrows, and then the crystals are rescreened or the crystallization conditions are optimized. The structures of LINE-1, Broccoli, OR4K15, 2'-dG-III, HmKt-7, and HmKt7-19ntX3 were obtained by the first method (crystal rescreening), while the structures of SAM-I and MTR1 were obtained by the second method (optimization of crystallization conditions).

[0050] Figure 2The right side shows the basic principle of replacing the position of GU base pairs, taking three-way junction RNA as an example. First, this protocol performs a preliminary screening of the original sequence to evaluate its potential for crystal formation. If the original sequence fails to form satisfactory crystals, or the resolution of the original sequence's X-ray diffraction is low, a pair of Watson-Crick base pairs will be replaced with GU base pairs at the center of the P1, P2, and P3 stems (the stems are numbered from the 5' end); if necessary, we will test the situation where multiple combinations of GU pairs are replaced simultaneously in these three regions. Double helical stems that support pseudoknots or hairpin loops for binding to protein auxiliary factors are also effective targets for replacement of GU pairs. It is well known that more than 60% of GU pairs in RNAs are within the helix (rather than at the end of the helix), and wobble base pairs and non-Watson-Crick base pairs preferentially appear in the middle of the helix. In addition, unlike Watson-Crick base pairs, GU pairs do not overlap with UG pairs in space, so either G or U can be tried to replace or be replaced, that is, forward replacements include AU-GU replacements, UA-UG replacements, CG-UG replacements, and / or GC-GU replacements, and reverse replacements include GU-AU replacements, UG-UA replacements, GU-GC replacements, and / or UG-CG replacements. The replaced GU base pairs should avoid being located near the active site or ligand binding region. If the crystal data does not meet expectations after initial modifications, this protocol explores different combinations of GU base pair replacements on other stems, including strategies to change multiple positions simultaneously.

[0051] (2) Resolution change results

[0052] Among the eight unique RNA crystals in this study, GC-GU substitutions significantly improved the resolution of four of them, including LINE-1 ribozyme, SAM-I riboswitch, Haloboxobacterium maritima Kt-7 (HmKt-7) kink turn, and a variant containing three tandem kink-turn (HmKt7-19ntX3) RNAs; AU-GU substitutions significantly improved the resolution of two other RNA crystals, including 2'-dG-III ribosomal switch and Broccoli RNA aptamer; reverse substitutions such as GU-GC substitutions improved the resolution of OR4K15 ribozyme. In contrast, for the MTR1 ribozyme, replacing GC and AU base pairs with GU base pairs did not significantly improve the crystal resolution ( Figure 1 and Table 1).

[0053] (3) Substitution of base pairs from GC to GU improves resolution

[0054] The GC-GU substitution significantly improved the resolution of four of the eight unique RNA crystals studied, including the LINE-1 ribozyme, the SAM-I riboswitch, and two different forms of HmKt-7. The first resolution of the LINE-1 ribozyme was achieved by introducing a GU base pair at the second base pair of its P1 region. Increase to ( Figure 3 A). Comparison of the structures of the two forms before and after GU substitution shows that their folding is similar. The root mean square deviation (RMSD) between the structures is ( Figure 3 C).

[0055] The SAM-I ribosomal switch is another very strong evidence for the remarkable ability of GU base pairs to improve resolution. Adding a GU to the penultimate base pair of the P2 region of the ribosomal switch (SAM-I-GU) increased the resolution from Increase to This is the highest resolution ever achieved for a SAM-I ribosomal switch. Figure 3 D) The P2 stem is located near the pseudoknot in the SAM-I ribosomal switch fold ( Figure 8 The overall structure of the SAM-I-GU ribosomal switch is realized by two sets of coaxially stacked helices ( Figure 3 E) The RMSD obtained by structural superposition before and after the introduction of the GU base pair is: The overall structural changes are minimal. The GU base pair and the adjacent base pairs C29-G21 and C31-G23 were examined in detail and found to have negligible changes ( Figure 3 F) In fact, for RNA molecules longer than 50 nucleotides, the crystal structure resolution is better than Relatively few. The wavelength collected The proposed method can easily solve the structure of SAM-I-GU riboswitch by using the anomalous scattering signal of barium. In addition, the crystals of SAM-I-GU riboswitch have high reproducibility, and the barium ions that promote crystal growth can be replaced by other metal ions without affecting the resolution, which is still better than This will be an excellent system to study the interaction of RNA with different metal ions. The high-resolution structure allowed the researchers to precisely locate the barium ion at position G74 and the water molecule associated with it ( Figure 3 G).

[0056] The third case is HmKt-7 RNA, which was edited into an artificial RNA molecule capable of binding to the U1A protein. In its original sequence, the GC base pairs were at positions 9 and 24, and this RNA molecule failed to crystallize. The GC-GU substitution at the above positions not only promoted crystallization, but also produced a resolution of The crystal ( Figure 4 A-4C). This observation further demonstrates the value of GU base pairs in RNA crystal formation and their ability to improve crystal resolution. Fig. 9 C shows that the replaced GU pair is located within the double helical stem, which supports the binding of the hairpin loop to the U1A protein used for cocrystallization.

[0057] Figure 4 D shows a molecular structure with six kink-turns, inspired by HmKt7-19nt, which highlights the effect of replacing different numbers of GC base pairs with GU base pairs. The overall shape of the kink-turn structure (HmKt7-19ntX3 6U) effectively cyclized by crystal symmetry is a triangle, with the edges defined by canonical helices (C) and the vertices formed by atypical helices (NC). Figure 4 D) The resolution of the HmKt7-19ntX3 variant without the GU base pair is Combining 6 GU base pairs (HmKt7-19ntX3 3U), the resolution was improved to In addition, the variant with 12 GU base pairs (HmKt7-19ntX3 6U) showed the most obvious improvement, with diffraction to ( Figure 4 E) Note that in this case, tandem GU pairs are introduced, which are the most common and stable in RNA structures, and the tandem GUs appear in the middle of each helical segment.

[0058] (4) Substitution of base pairs AU to GU improves resolution

[0059] We also observed that the resolution of the 2'-dG-III riboswitch and Broccoli RNA aptamer was significantly improved by replacing the AU base pair with GU. The substitution of AU to GU facilitated the structural elucidation of the 2'-dG-III riboswitch. In this structure, the adenine at the P3 position was replaced with a guanine, which increased the resolution from Increase to ( Figure 5 A). Compared with the dataset, The dataset not only provides better resolution, but also shows better statistical parameters. Notably, the overall Rmerge (a statistical indicator used to measure the consistency of repeated measurement data) decreased from 0.175 to 0.031 while maintaining similar redundancy (Table 3). dataset, whose structure was successfully determined by molecular replacement in this protocol.

[0060] The overall structure of 2'-dG-III RNA consists of a three-way stem-loop scaffold accompanied by loop-loop interactions between L2 and L3 ( Figure 5 B) The RMSD between the structures before and after the introduction of the GU base pair is Further analysis showed that there was only a small displacement between the GU base pair and its neighboring base pair, indicating that the GU base pair had little effect on the overall structure ( Figure 5 C). This is the only structure in which the space group changes after the insertion of the GU pair. Interestingly, two new intermolecular hydrogen bonds can be formed between the 5'-phosphate and O2' oxygen of the G of the GU pair and the two hydroxyl groups of the adjacent molecule ( Fig. 9 D).

[0061] This protocol also explored Broccoli RNA aptamers that can bind to the fluorescent small molecule DFHBI-1T. Initially, the presence of AU base pairs at positions 2 and 48 in the P1 region did not promote crystal formation. However, AU-GU substitutions at these positions promoted crystal formation with a diffraction resolution of ( Figure 5 D) Broccoli RNA aptamers are based on an elongated helical conformation, consisting of two stem regions P1 and P2, and a fluorophore binding site located between them. The fluorophore binding site consists of a G-quadruplex structure and a three-base interaction plane ( Figure 5 d and 5E). Detailed examination of the G2·U48 base pair and its neighboring base pairs revealed typical features of the RNA double helix structure ( Figure 5 F) These examples demonstrate that the introduction of a GU base pair can transform a crystal-incapable structure into a crystal-capable structure.

[0062] (5) Conversion of the original GU base pairs to GC can produce higher resolution structures

[0063] Introducing GU base pairs is not the only way to improve crystal resolution. Another approach is to convert naturally occurring GU base pairs into classic GC base pairs, an approach demonstrated in the OR4K15 ribozyme studied in this project, which enabled this project to determine the crystal structure of this ribozyme for the first time.

[0064] In this case, the crystal resolution of the P1 region was improved from 1.1 to 1.1 after the GU base pairs were converted to GC base pairs. Significantly increased to ( Figure 6 A) The OR4K15 ribozyme consists of two RNA strands that adopt a double helical configuration ( Figure 6 B). The RMSD value between the two structures before and after the GU base pair conversion is 0.382A, indicating that there is almost no significant difference between the two structures ( Figure 6 C). Further analysis of the two structures revealed only minor differences in the base pairs ( Figure 6 D) This finding suggests that strategies to improve RNA crystal resolution are not limited to introducing GU base pairs. When GU base pairs exist in natural RNA sequences and the crystal resolution is low, converting the GU base pairs of the inactive central stem to GC base pairs can increase sequence diversity and thus improve crystal resolution.

[0065] (6) GU base pair: potential site for binding barium ions in RNA crystals

[0066] Replacing GC pairs with GU wobble pairs does not always improve resolution. In our study of the MTR1 ribozyme, we replaced two Watson-Crick base pairs with GU pairs ( Figure 6 E), but the crystal resolution did not improve significantly. The resolution before and after the GU base pair substitution was and ( Figure 6 E). The overall structural features of the MTR1 ribozyme include a three-way junction consisting of the P1, P2, and P3 stems ( Figure 6 F). The RMSD values ​​between the structures before and after the introduction of the GU base pair are This indicates that the overall structure is highly consistent and has no significant changes ( Figure 6 G). Interestingly, this protocol observed a barium atom bound to the G59 U50 base pair ( Figure 6 H). Our findings in the case of the MTR1 ribozyme illustrate the multifaceted role of GU base pairs in RNA crystallography. Although they may not always improve resolution, they can facilitate the incorporation of heavy atoms, such as barium ions, which provides a new approach to solving phase problems. At the same time, our study observed a new GAAA interaction between the asymmetric units ( Fig.10 The above findings complement previous studies on the introduction of GU base pairs to promote barium ion binding to help solve the phase problem. In the experiment, this protocol designed two different pairs of GU base pairs in the P2 and P3 stems of MTR1, and only one pair was observed to bind barium ions.

[0067] 3. Analysis

[0068] The core of this method is to replace or reverse replace GU base pairs to increase sequence diversity when designing RNA sequences for crystallization. The results show that this method increases the possibility of obtaining high-quality crystals and accurate structures. In this study, all introduced GU base pairs in the 8 RNA crystals were observed in the standard wobble GU pair, and none of them existed in tautomeric form or anionic form ( Fig.11 This observation is consistent with the findings that the introduction of the GU base pair has little effect on the overall RNA structure. We compared the structures before and after the GU base pair replacement (Table 1). We found only one case, the 2'-dG-III ribosomal switch, in which the space group and crystal stacking were altered ( Fig. 9 A and S3B). When exploring the position of GU base pair substitutions, our approach observed metal ion binding in high-resolution structures in four RNA crystals, including brocoli-GU, 2'-dG-GU, SAM-I-GU, and OR4K15-GC ( Fig.12 ). This protocol assumes that this may be a factor leading to the improved resolution. However, it is important to note that some ions have poor electron density. Due to the poor resolution of the pre-modification structure, these ions may be difficult to observe in the unmodified crystal.

[0069] By analyzing the GU transformation site ( Fig.13 and 14 ), this approach found that only a few sites were effective. These effective modification sites appeared in the P1 stem. Specifically, in the 7 cases analyzed, 5 ( Fig.15 AE) in P1 stem, 2 cases ( Fig.15 FG) in other stems. This is consistent with previous observations on GU-assisted phase resolution. Therefore, this protocol recommends prioritizing modifications within the first 5' double helical stem (P1 stem). If modification of the P1 stem is unsuccessful, one can consider extending GU base pair modification to other stem regions.

[0070] Interestingly, the OR4K15 ribozyme provides a counterexample; RNA molecules have natural GU base pairs, and converting them to GC bases can also lead to improved resolution. This observation emphasizes the complex, environment-specific nature of RNA crystallography and highlights the importance of sequence diversity in RNA crystals. To better understand this phenomenon, this protocol examined large-scale deep mutation data for LINE-1 and OR4K15 ribozymes. This protocol found that in the LINE-1 ribozyme, the conversion of GC to GU has higher catalytic activity (relative activity changes from 1 to 1.43, an increase of 43%). The same is true for the opposite conversion of GU to GC in the OR4K15 ribozyme (relative activity changes from 1 to 1.82, an increase of 82%). Since the catalytic activity of RNA is closely related to its structural stability, the above results indicate that the improvement in RNA folding ability is the main reason for the improvement in crystal resolution. In addition, deep mutation scanning helps to find the best variants to improve RNA crystallization.

[0071] This protocol proposes a strategy to obtain RNA crystals with higher resolution. The current observations on the role of GU base pairs also help to select "crystallizable" sequences from a collection of homologous sequences. Therefore, this method is expected to be further applied and explored in the field of crystallographic research, with the potential to improve the ability of this field to elucidate the complex structure of RNA molecules and aid RNA sequence editing, especially in improving crystal quality and resolution.

[0072] Table 1. Summary of crystal properties of RNA structures

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[0075] Table 2. Sequences and conditions used in crystallization experiments

[0076] SAD-Ba: Single Wavelength Anomalous Diffraction using Barium. MR: Molecular Replacement. The nucleotides used for substitution are marked in bold and italic.

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[0080] Table 3. Crystallographic data submitted to the PDB. Values ​​in brackets are for the highest resolution shell.

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[0084] Obviously, the above embodiments of the present invention are only examples for clearly illustrating the technical solutions of the present invention, and are not intended to limit the specific implementation methods of the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the claims of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. A method for promoting RNA crystal growth and improving diffraction resolution, characterized in that: This includes forward and / or reverse substitution of GU base pairs with Watson-Crick base pairs in RNA.

2. A method for promoting RNA crystal growth and improving diffraction resolution according to claim 1, characterized in that: The forward substitution includes AU-GU substitution, UA-UG substitution, GC-GU substitution and / or CG-UG substitution.

3. A method for promoting RNA crystal growth and improving diffraction resolution according to claim 2, characterized in that: The position of the forward substitution is located in the middle of the paired region of the RNA stem.

4. A method for promoting RNA crystal growth and improving diffraction resolution according to claim 3, characterized in that: The RNA stem includes the first 5' duplex stem of the RNA and / or any inactive core stem.

5. A method for promoting RNA crystal growth and improving diffraction resolution according to claim 1, characterized in that: The reverse substitution includes GU-AU substitution, UG-UA substitution, GU-GC substitution and / or UG-CG substitution.

6. Use of the method according to any one of claims 1 to 5 in RNA crystal structure analysis.

7. A method for solving RNA crystal phase problems, characterized in that: The method comprises the forward replacement and combination of barium ions as described in claim 1.

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