A method for promoting RNA crystal growth and improving diffraction resolution and its application

Abstract

The present invention belongs to the field of nucleic acid crystallography structure, and specifically discloses a method and application thereof for promoting RNA crystal growth and improving diffraction resolution. By selectively using GU base pairs to replace Watson-Crick base pairs or reverse substitutions in RNA sequences, the resolution limit of RNA crystals in X-ray diffraction is significantly improved. The method successfully obtained high-resolution structures of 8 unique RNA crystals, and concluded that the introduction of GU base substitutions is placed in the middle of the P1 (first 5' double helix stem) of RNA, or in the middle of the peripheral stem of any inactive center, which is sufficient to achieve the above effects. This solution provides a simple and effective method from the perspective of RNA sequence editing, which significantly improves the X-ray diffraction resolution of RNA crystals by changing the type of base pairs and thereby increasing sequence diversity. This method is of great significance for parsing the complex structure of RNA molecules and revealing their functions, catalytic mechanisms, ligand recognition and intermolecular interactions.

Description

Technical Field

[0001] The present invention belongs to the field of nucleic acid crystallographic structure, and particularly 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. Besides their role as carriers of genetic information during protein synthesis, RNA also exhibits diverse catalytic and regulatory functions. The versatility of RNA molecules within cells is conferred by their three-dimensional structure, whose precise folding plays a crucial role in biology.

[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 particularly valuable in exploring the dynamic properties of RNA structures in solution, while cryo-EM has unique advantages in resolving 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, and 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. Resolution is usually expressed in To measure, the smaller the number, the higher the resolution (less than or equal to Researchers face numerous challenges in high-resolution RNA crystallography. The molecular dynamics and conformational fluctuations of RNA molecules make obtaining high-quality crystals extremely difficult. Furthermore, the phase problem remains a core challenge in the field. Accurately acquiring phase information is crucial for deriving precise atomic structure from diffraction patterns, but its acquisition remains fraught with technical difficulties.

[0005] There are various techniques available in the art for obtaining well-diffracting RNA crystals. For example, RNA-binding proteins such as U1A protein, Fab antibodies, or tRNA scaffolds are used to facilitate the crystallization process. These molecular scaffolds can stabilize the RNA structure, thereby increasing the likelihood 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 yet been fully utilized in RNA crystallography due to their operational complexity. Among the methods for obtaining high-resolution RNA crystal structures, one method has been widely used, namely increasing RNA sequence diversity. This method focuses on changing the length of the P1 stem (the first 5' double helix stem) or modifying unstable loops to generate stable tetranucleotide loops (tetraloops) such as GAAA or UUCG. However, the pursuit of well-diffracting RNA crystals requires continuous exploration of new strategies. Unlike traditional methods, the present invention proposes a new strategy, namely manipulating the presence of GU base pairs in the RNA sequence.

[0006] GU (guanine-uracil) base pairs are frequently observed in RNA structures and possess unique chemical, thermodynamic, and structural properties. In various RNA molecules, GU base pairs play important roles in folding, ribozyme catalysis, and protein interactions. However, the substitution and reverse substitution of GU with Watson-Crick base pairs are currently understudied and warrant further exploration. Summary of the Invention

[0007] The present invention aims to overcome the shortcomings of existing technologies by providing a method and application for promoting the growth of RNA crystals and improving diffraction resolution. By varying the base types and increasing 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 the growth of RNA crystals and improving the diffraction resolution, including forward substitution and / or reverse substitution of RNA GU base pairs and Watson-Crick base pairs.

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

[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 crystallography relies on initial screening using raw sequences, followed by optimizing the screening conditions after crystals form, and collecting diffraction data to solve RNA structures. However, this process often yields unsatisfactory results, such as low-quality crystals or poor X-ray data.

[0014] 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 to replace a pair of AU or GC base pairs with GU base pairs in the middle position of P1 (the first 5' double helix stem), P2 and P3 stems (the inactive central stems are numbered starting from the 5' end), or on the double helix stem where the pseudoknot or hairpin loop binds to the protein auxiliary factor, thereby increasing sequence diversity, thereby promoting RNA crystal growth 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 approach improves the X-ray diffraction resolution of RNA crystals, enabling researchers to precisely reveal the three-dimensional conformation of RNA by analyzing X-ray diffraction images. This helps elucidate RNA function, catalytic mechanisms, ligand recognition, and interactions between RNA molecules. Furthermore, this structural information is essential for promoting the development of RNA-targeted therapies, advancing gene editing technologies, and elucidating the molecular mechanisms of RNA-related diseases.

[0016] Another object of the present invention is to provide a method for resolving phase issues in RNA crystals, comprising forward substitution and incorporation of barium ions. In an embodiment of the present invention, some GU base pairs are bound to barium ions, which act as heavy atoms to provide phase information for RNA crystals and improve X-ray diffraction results. This embodiment demonstrates the multifaceted role of GU base pairs in RNA crystallography. While not always improving resolution, they can facilitate the incorporation of heavy atoms, providing a new approach to resolving phase issues.

[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 substitutions of GU base pairs with Watson-Crick base pairs, especially performing base pair substitutions in the middle position of the paired part of the RNA stem, thereby significantly improving X-ray diffraction resolution. In addition, RNA crystal phase information is provided by combining barium ions with the forward substituted GU base pairs. This solution provides a simple and efficient tool from the perspective of RNA sequence editing. By changing the base types, the sequence diversity is increased, thereby improving the X-ray diffraction resolution of RNA crystals, which is conducive to analyzing the complex structure of RNA molecules and is of great significance for clarifying the function of RNA, catalytic mechanism, ligand recognition and interaction of RNA molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a scatter plot showing the resolution changes of eight RNA crystal structures before and after base pair substitutions. Different colors are used to represent the change in resolution: circles indicate an improvement in resolution after replacing other base pairs with GU pairs; squares indicate a minimal effect of the GU pair substitution on resolution; and diamonds indicate an increase in resolution after reverting GU 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 ribozymes and SAM-I riboswitches achieve higher resolution by replacing GC base pairs with GU base pairs.

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

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

[0024] Figure 6 We show that OR4K15 ribozyme achieves higher resolution by substitution of GU to GC.

[0025] Figure 7This figure shows the main structural changes in a conventional A-form RNA helix in the presence of a standard wobble U·G pair. The U shifts into the major groove between the two Watson-Crick base pairs. The typical torsion angle in an RNA helix is ​​33°; in a U·G pair, the torsion angle in the 5' direction of the U increases, while the torsion angle in the 3' direction decreases. These changes in torsion angle lead to significant interstrand stacking between the G in the U·G pair and the 3'-terminal residue of the U (connected by a black box). In the 5' direction of the U, stacking occurs within both strands.

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

[0027] Figure 9 The crystal stacking patterns of 2'-dG-III-AU (PDB: 8Z8Q), 2'-dG-III-GU (PDB: 8KEB), and HmKt7-GU and U1A proteins (PDB: 5FJ4) are shown. (A) Crystal stacking pattern of 2'-dG-III-AU, with a space group of P1. (B) Crystal stacking pattern of 2'-dG-III-GU, with a space group of C121. (C) Crystal stacking pattern of HmKt7-GU and U1A proteins, with a space group of C2221. (D) In ​​the 2'-dG-III unit cell, intramolecular hydrogen bonds are formed between the 5'-phosphate group of the GU base pair and the O2' oxygen atom.

[0028] Figure 10 Demonstration of novel GAAA interactions between asymmetric units of MTR1. (A) Four MTR1 molecules related by crystal symmetry. Left, molecules (red) are associated with each other via a symmetry-related GAAA four-base ring with novel interactions (annotated within the dashed rectangle). Right, symmetry-related molecules (green) contacting each other via backbone contacts near replacement GU base pairs. On the far right, a magnified view of the dashed box shows the precise interactions between the twofold symmetry-related molecules. Next to the illustration are the interacting sequences 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] Figure 11The structures of G·U base pairs are shown. (A) A standard wobble G·U base pair (from PDB 1HQ1) and a non-standard anionic G·U base pair (from PDB 7K00). (B) Eight standard wobble G·U base pairs are observed in the structure of this scheme.

[0030] Figure 12 Figure 1. Metal ion binding in high-resolution RNA structures. (A) Electron density map for potassium-ion bases G2 and U48 in Broccoli-GU. Electron density shows missing electron density from simulated annealing at 1.3σ. (B) Electron density map for sodium-ion bases G2 and U70 in 2'-dG-III-GU. Electron density shows missing electron density from simulated annealing, outlined at 0.9σ. (C) Electron density map for sodium-ion bases G29 and C2 in OR4K15-GC. Electron density shows missing electron density from simulated annealing at 1.0σ. (D) Electron density map for barium-ion bases G30 and U22 in SAM-I-GU. Electron density shows missing electron density from simulated annealing at 1.5σ.

[0031] Figure 13 The positions of 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] Figure 14 The position and energy changes of GU base pairs in the double helix stem are shown in the RNA structure analysis results. The six GUs are located on the 5' strand, and four are located on the 3' strand. ΔE represents the difference in thermodynamic free energy resulting from the sequence change (ΔE for HmKT7-19ntX3 was calculated by introducing a GAAA loop). Thermodynamic free energy calculations were performed using the secondary structure prediction tool provided by the Mathews Group (https: / / rna.urmc.rochester.edu / RNAstructureWeb).

[0033] Figure 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 dipolyguanylate 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 description is illustrative and intended to provide further explanation of the present application, rather than a limitative definition of the present application. Unless otherwise specified, all technical and scientific terms used herein have the meanings commonly understood by those skilled 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 should 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, they indicate the presence of specific features, steps, operations, devices, components and / or combinations thereof, but do not exclude the presence of other features or elements not expressly listed.

[0036] The present invention will now be further described in conjunction with specific examples. The following examples are intended only to illustrate the present invention but are not intended to limit its scope. The test samples and test procedures used in the following examples include the following (if the specific experimental conditions are not specified in the examples, they are usually based on conventional conditions or the conditions recommended by the reagent supplier; the reagents, consumables, etc. used in the following examples, unless otherwise specified, can 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) phosphoamidite chemistry following the procedures outlined by Wilson et al. This synthesis protocol was performed using an Applied Biosystems 394 DNA / RNA synthesizer and involved the use of t-BDMS (Link Technologies) to protect the ribonucleic acid phosphoamidite. (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 triethylamine (Sigma-Aldrich) to facilitate the removal of the t-BDMS group. The solution was incubated at 65°C in the dark for 2.5 hours. After cooling on ice for 10 minutes, RNA was precipitated using 1 mL of butanol, washed once with 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 with GCG / GGC / GGA / GGG. A DNA template containing a T7 RNA polymerase promoter for in vitro transcription was prepared by polymerase chain reaction (PCR). After transcription, the RNA molecules were purified by electrophoresis on a polyacrylamide gel containing 7 M urea. The intact RNA transcripts were recovered by UV visualization, excision, and electroelution (using an electroelution system) in 0.5× TBE buffer at 200 V for 12 hours. Subsequently, the RNA was precipitated with isopropanol, washed with 75% ethanol, and finally dissolved in water. The 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] A solution containing 10 μg / μL RNA in 5 mM HEPES (pH 7.5), 100 mM KCl, and 5 mM MgCl2 (HKM buffer) was refolded according to the method 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 an 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 at beamlines BL02U1, BL10U2, BL18U1, and BL19U1 at the Shanghai Synchrotron Radiation Facility (SSRF) and the Shanghai National Facility for Protein Science (NFPSS) and processed with XIA2 or XDS. The resolution cutoff for the data was determined by examining the CC1 / 2 and density maps. PDB structures 8Z9K (LINE-1-GC), 8Z8Q (2'-dG-III-AU), 8ZAU (MTR1-GU), and 8ZA4 (OR4K15-GU) were solved using the PHASER molecular replacement method, 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 model geometry and fit to the electron density map were monitored using MOLPROBITY and validation tools within Coot. To effectively avoid overfitting, particularly for lower-resolution structures determined by molecular replacement, optimization weights and simulated annealing were implemented in phenix.refine. Experimental maps were calculated using Autosol, and Fourier anomaly difference maps were calculated using phenix.refine. Simulated annealing omit maps were calculated using composite omit maps calculated using the annealing method in PHENIX suite. Atomic coordinates and structure factor amplitudes have been deposited in the Protein Data Bank (PDB) and are listed in Table 3 along with the accession codes.

[0045] Among the crystal structures discussed in this article, four are new: LINE-1 ribozymes (8Z9K and 8Z8U), OR4K15 ribozymes (8ZA0 and 8ZA4), 2'-dG-III riboswitches (8Z8Q and 8KEB), and Broccoli RNA aptamers (8K7W). Four of these 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 six commercial crystallization kits, totaling 288 crystallization conditions. Index 1-96, Natrix I / II, and Crystal I / II were purchased from Hampton Research. Specific crystallization details for the SAM-I riboswitch, HmKt-7, and HmKt7-19ntX3 RNA are documented in their respective publications, outlining the structure resolution and refinement processes. A common nomenclature was used for the secondary structure designations: the first 5' helix was designated P1, followed by P2. The single-stranded helical link between P1 and P2 was designated J12. The head loop of the final helix has the same number as the helix itself, for example, L3 overlays 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 swingable GU pairs ( Figure 2 ) to promote RNA crystallization. Existing 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 solve 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 not only succeeded 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 indicated by the black arrows. If satisfactory crystals are not obtained or the X-ray data quality is poor, as indicated by the gray arrows, the RNA sequence can be replaced with GU base pairs or reversed, as indicated by the pink arrows, and then the crystals can be rescreened or the crystallization conditions can be 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 illustrates the rationale for replacing GU base pairs, using a three-way junction RNA as an example. First, the original sequence is screened to assess its crystal formation potential. If the original sequence fails to form satisfactory crystals or if X-ray diffraction of the original sequence yields low resolution, a Watson-Crick base pair is replaced with a GU base pair at the center of the P1, P2, and P3 stems (stems are numbered starting from the 5' end); if necessary, multiple combinations of GU pairs are tested for simultaneous replacement in these three regions. Double helical stems, which support pseudoknots or hairpin loops for protein cofactor binding, are also effective targets for GU pair replacement. It is well known that over 60% of GU pairs in RNAs are located within the helix (rather than at the ends), and that wobble base pairs and non-Watson-Crick base pairs are preferentially located in the middle of the helix. Furthermore, unlike Watson-Crick base pairs, GU and UG pairs do not spatially overlap, so either G or U can be attempted to replace or be replaced. Forward substitutions include AU-GU, UA-UG, CG-UG, and / or GC-GU, while reverse substitutions include GU-AU, UG-UA, GU-GC, and / or UG-CG. Replaced GU base pairs should be avoided near the active site or ligand-binding region. If the crystallographic data did not meet expectations after initial modifications, this protocol explored different combinations of GU base pair replacements on other stems, including strategies that simultaneously altered multiple positions.

[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) Substituting GC for 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 that GU base pairs can significantly improve the resolution. Increase to This is the highest resolution achieved to date for a SAM-I riboswitch. 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. A detailed examination of the GU base pair and the adjacent base pairs C29-G21 and C31-G23 revealed negligible changes ( Figure 3 F) In fact, for RNA molecules longer than 50 nucleotides, the crystal structure resolution is better than The use of The wavelength collected The proposed method can easily resolve the structure of the SAM-I-GU riboswitch by utilizing the anomalous scattering signal of barium. In addition, the crystals of the SAM-I-GU riboswitch are highly reproducible, 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 for studying the interaction of RNA with different metal ions. The high-resolution structure allowed the researchers to precisely locate the barium ion and its associated water molecule at position G74 ( 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 this study. In its original sequence, GC base pairs were at positions 9 and 24, and this RNA molecule failed to crystallize. GC-GU substitutions at these positions not only facilitated 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. Figure 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 emphasizes 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 circularized by crystal symmetry is a triangle, with the edges defined by canonical helices (C) and the vertices ( Figure 4 D) The resolution of the HmKt7-19ntX3 variant without GU base pairs is Combined with 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) Substituting the base pair AU for GU improves resolution

[0059] This protocol 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 AU to GU substitution facilitated the structural elucidation of the 2'-dG-III riboswitch. In this structure, the adenine at the P3 position was replaced by a guanine, which increased the resolution from Increase to ( Figure 5 A). With 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.

[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 adjacent 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 ( Figure 9 D).

[0061] This protocol also explored Broccoli RNA aptamers that could 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, with 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 reveals typical features of the RNA double helix structure ( Figure 5 F) These examples demonstrate that the introduction of GU base pairs can transform a non-crystallizable structure into a crystal-forming one.

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

[0063] Introducing GU base pairs isn’t 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, enabling the first crystal structure determination of this ribozyme.

[0064] In this case, after converting the GU base pairs in the P1 region to GC base pairs, the crystal resolution increased from 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 for improving RNA crystallographic resolution are not limited to introducing GU base pairs. When GU base pairs are present in natural RNA sequences and the crystallographic resolution is low, converting the GU base pairs in the inactive central stem to GC base pairs can increase sequence diversity and thus improve crystallographic resolution.

[0065] (6) GU base pairs: potential sites for barium ion binding 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 resolutions before and after the GU base pair substitution were 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 there is no significant change ( Figure 6 G). Interestingly, this scheme 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. While they may not always improve resolution, they can facilitate the incorporation of heavy atoms, such as barium ions, providing a new approach to resolving phase problems. Furthermore, our study observed a new GAAA interaction between asymmetric units ( Figure 10 These findings complement previous research that has shown that introducing GU base pairs can help resolve phase issues by promoting barium ion binding. In this study, two different GU base pairs were designed in the P2 and P3 stems of MTR1, and barium ion binding was observed in only one of these pairs.

[0067] 3. Analysis

[0068] The core of this approach is to replace or reverse-replace GU base pairs when designing RNA sequences for crystallization to increase sequence diversity. Results show that this approach increases the likelihood of obtaining high-quality crystals and accurate structures. In this study, all introduced GU base pairs in the eight RNA crystals were observed in the standard wobble GU pair, and none of them existed in tautomeric or anionic forms ( Figure 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 packing were altered ( Figure 9 A and S3B). When exploring the positions of GU base pair substitutions, our approach observed metal ion binding in high-resolution structures of four RNA crystals, including brocoli-GU, 2'-dG-GU, SAM-I-GU, and OR4K15-GC ( Figure 12 This protocol hypothesizes that this may be a factor contributing to the improved resolution. However, it should be noted that some ions have poor electron density. Due to the lower resolution of the pre-modified structure, these ions may be difficult to observe in the unmodified crystal.

[0069] By analyzing the GU transformation sites ( Figure 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 cases ( Figure 15 AE) in P1 stem, 2 cases ( Figure 15 FG) in other stems. This is consistent with previous observations of GU-assisted phase resolution. Therefore, this protocol recommends prioritizing modifications within the first 5' duplex stem (P1 stem). If modification of the P1 stem is unsuccessful, consideration can be given to extending GU base pair modification to other stem regions.

[0070] Interestingly, the OR4K15 ribozyme provides a counterexample; RNA molecules with naturally occurring GU base pairs can also be converted to GC base pairs, resulting in improved resolution. This observation highlights the complex, context-specific nature of RNA crystallography and underscores the importance of sequence diversity in RNA crystals. To better understand this phenomenon, we examined large-scale deep mutational data from the LINE-1 and OR4K15 ribozymes. We found that in the LINE-1 ribozyme, conversion of GC to GU resulted in higher catalytic activity (relative activity increased from 1 to 1.43, a 43% increase). The same was true for the reverse conversion of GU to GC in the OR4K15 ribozyme (relative activity increased from 1 to 1.82, an 82% increase). Because RNA catalytic activity is closely linked to its structural stability, these results suggest that enhanced RNA folding is the primary driver of the improved crystal resolution. Furthermore, deep mutational scanning facilitates the search for optimal variants that improve RNA crystallization.

[0071] This protocol proposes a strategy for obtaining RNA crystals with relatively high resolution. The current observations on the role of GU base pairs also facilitate the selection of "crystallizable" sequences from a collection of homologous sequences. Therefore, this method is expected to be further applied and explored in the field of crystallography, potentially improving the field's ability to elucidate the complex structures of RNA molecules and aiding RNA sequence editing, particularly 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 bolded and italicized.

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[0079]

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

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[0084] Obviously, the above embodiments of the present invention are merely examples for the purpose of 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 within the scope of protection 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 or reverse substitution of a GU base pair with a Watson-Crick base pair in RNA, wherein the reverse substitution includes a GU-GC substitution and / or a UG-CG substitution.

2. A method for promoting RNA crystal growth and improving diffraction resolution according to claim 1, characterized in that: The forward substitution includes an AU-GU substitution, a UA-UG substitution, a GC-GU substitution and / or a 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 the stem of any inactive center.

5. Use of the method according to any one of claims 1 to 4 in RNA crystal structure analysis.

6. 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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