CN119688749B - A method for promoting RNA crystal growth and improving diffraction resolution and its application - Google Patents

Abstract

The invention belongs to the field of nucleic acid crystallography structures, and particularly discloses a method for promoting RNA crystal growth and improving diffraction resolution and application thereof. The resolution limit of RNA crystals in X-ray diffraction is significantly increased by selectively using GU base pairs to replace watson-crick base pairs or vice versa in the RNA sequence. The method successfully obtained a high resolution structure of 8 unique RNA crystals and concluded that the introduction of GU base substitutions was placed in the middle of P1 (the first 5' double helical stem) of the RNA, or in the middle of the peripheral stem of any inactive center, sufficient to achieve the above effect. The scheme provides a simple and effective method from the aspect of RNA sequence editing, and the variety of base pairs is changed to further increase sequence diversity, so that the X-ray diffraction resolution of RNA crystals is remarkably improved. The method has important significance for analyzing the complex structure of RNA molecules and revealing functions, catalytic mechanisms, ligand recognition and intermolecular interaction.

Description

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

Technical Field

The invention belongs to the field of nucleic acid crystallography structures, and particularly relates to a method for promoting RNA crystal growth and improving diffraction resolution and application thereof.

Background

RNA molecules are a central component of many cellular processes. In addition to being a carrier of genetic information during protein synthesis, RNA molecules are known to exhibit a variety of catalytic and regulatory functions. The versatility of intracellular RNA molecules is conferred by their three-dimensional structure, whose precise folding plays a vital role in the organism.

X-ray crystallography, nuclear Magnetic Resonance (NMR) spectroscopy, and low temperature electron microscopy (cryo-EM) are the main tools for RNA structure biology. By day 7 of 2024, 3 records of the Protein Database (PDB) indicated that of 1861 pure RNA structures, about 62.4% were determined by X-ray crystallography, 31.6% by nmr and 6% by low temperature electron microscopy. Nuclear magnetic resonance spectroscopy has particular value in exploring the dynamic nature of RNA structures in solution, while cryogenic electron microscopy has unique advantages in addressing the structure of large RNA molecules and protein complexes, and does not require crystallization.

Until now, X-ray crystallography was the most successful method for determining RNA structure, and the core is to reveal the three-dimensional conformation of RNA with high accuracy by analyzing X-ray diffraction images. The high resolution of RNA structure by X-ray crystallography helps elucidate RNA function, catalytic mechanism, ligand recognition and RNA molecule interactions. In addition, these structural information is of indispensable significance in promoting the development of RNA-targeted therapies, promoting the advancement of gene editing techniques, and resolving the molecular mechanisms of RNA-related diseases. The resolution of the crystal data obtained by crystal X-ray diffraction is a key indicator for deriving the overall quality and accuracy of the structural model. The data resolution is directly related to the number of independent measurements that can be used to determine the molecular atomic parameters (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 generally usedIs measured, the smaller this number represents the higher the resolution (less than or equal toDefined as "atomic resolution"). 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. Second, the phase problem remains a core problem in this field, and accurate acquisition of phase information is critical to deriving accurate atomic structures from diffraction patterns, but the acquisition process is still fraught with technical difficulties.

A variety of techniques are known in the art for obtaining well-diffracting RNA crystals. For example, RNA binding proteins such as U1A proteins, fab antibodies, or tRNA scaffolds are used to facilitate the crystallization process. These molecular scaffolds may 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. Despite their potential, these methods have not been fully utilized in RNA crystallography due to the complexity of their manipulations. Among the methods for obtaining high resolution RNA crystal structures, there has been widely used a method for increasing RNA sequence diversity, which focuses on changing the length of the P1 stem (first 5' double helical stem) or modifying the unstable loop to form a stable tetranucleotide loop (Tetraloop) such as GAAA or UUCG. However, the search for well-diffracting RNA crystals requires constant exploration of new strategies. Unlike conventional methods, the present invention proposes a new strategy for manipulating the presence of GU base pairs in RNA sequences.

GU (guanine-uracil) base pairs are often observed in RNA structures, which have unique chemical, thermodynamic and structural properties. Among the various RNA molecules, GU base pairs play an important role in folding, ribozyme catalysis and interaction with proteins. However, the current academy uses fewer substitutions and inversions of GU and Watson-Crick base pairs, and deserves further exploration.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a method for promoting the growth of RNA crystals and improving diffraction resolution and application thereof. By changing the base types, the sequence diversity is increased, so that the X-ray diffraction resolution of the RNA crystal is obviously improved, and an effective means is provided for analyzing the complex structure of RNA molecules.

The technical scheme adopted by the invention is to provide a method for promoting the growth of RNA crystals and improving diffraction resolution, which comprises the forward substitution and/or the reverse substitution of GU base pairs and Watson-Crick base pairs of RNA.

Further, the forward substitution includes AU-GU substitution, UA-UG substitution, GC-GU substitution, and/or CG-UG substitution. In contrast to Watson-Crick base pairs, GU pairs and UG pairs do not have the property of spatially overlapping, and therefore either G or U can be replaced in an attempt to replace or be replaced, resulting in the several above-described alternatives, hereinafter referred to as GU base pair positive substitutions.

Further, the position of the forward substitution is located in the middle of the paired region of the RNA stem.

Further, the RNA stem comprises the first 5' duplex stem of RNA and/or the stem of any inactive center.

Further, the reverse substitutions include GU-AU substitutions, UG-UA substitutions, GU-GC substitutions, and/or UG-CG substitutions.

Existing X-ray diffraction crystallography relies on an initial screening by the original sequence, followed by optimization of screening conditions after crystal formation, and collection of diffraction data to resolve RNA structures. However, this process often has difficulties in obtaining satisfactory results, such as low crystal quality or poor X-ray data.

To solve this problem, the strategy proposed by the present scheme is to increase the sequence diversity significantly increasing the probability of obtaining high quality crystals. In one or more embodiments of the invention, the RNA sequence is engineered to replace a pair of AU or GC base pairs with GU base pairs at intermediate positions of the P1 (first 5 'duplex), P2 and P3 (non-active center stems numbered from the 5' end), or duplex stems with a pseudoknot or hairpin loop bound to a protein cofactor, respectively, to increase sequence diversity, thereby promoting RNA crystal growth and significantly increasing diffraction resolution of the crystal. In another embodiment of the invention, the sequence diversity is similarly increased after substitution of naturally occurring GU base pairs in the RNA sequence with GC base pairs, thereby significantly increasing the diffraction resolution of the crystal.

It is another object of the present invention to provide the use of a method for promoting RNA crystal growth and improving diffraction resolution in RNA crystal structure resolution. The scheme improves the X-ray diffraction resolution of the RNA crystal, and researchers can reveal the three-dimensional conformation of RNA with high precision by analyzing the X-ray diffraction image, thereby being helpful for elucidating the functions of RNA, the catalytic mechanism, ligand recognition and the interaction of RNA molecules. In addition, these structural information is of indispensable significance in promoting the development of RNA-targeted therapies, promoting the advancement of gene editing techniques, and resolving the molecular mechanisms of RNA-related diseases.

It is another object of the present invention to provide a method for solving the phase problem of RNA crystals, comprising forward substitution and binding of barium ions. In embodiments of the invention, a portion of the GU base pairs incorporate barium ions as heavy atoms that provide phase information for the RNA crystal while improving the X-ray diffraction results. This example demonstrates that GU base pairs have a versatile role in RNA crystallography, and although not always able to improve resolution, can promote the incorporation of heavy atoms, providing a new idea for solving the phase problem.

Compared with the prior art, the invention has the beneficial effects that:

The invention increases sequence diversity, improves crystal quality and further remarkably improves X-ray diffraction resolution by implementing forward and reverse substitution of GU base pairs and Watson-Crick base pairs, particularly base pair substitution at the middle position of the pairing part of RNA stems. RNA crystal phase information is also provided by binding barium ions through the forward substitution GU base pairs. The scheme provides a simple and efficient tool from the perspective of RNA sequence editing, and increases sequence diversity by changing base types, thereby improving the X-ray diffraction resolution of RNA crystals, being beneficial to analyzing the complex structure of RNA molecules and having important significance for elucidating the functions of RNA, the catalytic mechanism, ligand recognition and the interaction of RNA molecules.

Drawings

FIG. 1 is a scatter plot of 8 RNA crystal structure resolutions before and after base pair substitution. In the figure, the change in resolution is represented by using different colors, a dot indicating that the resolution is improved after the other base pairs are replaced with GU pairs, a square indicating that the change in resolution is not greatly affected by GU pairs, and a diamond indicating that the resolution is improved after GU is restored to GC.

FIG. 2 includes a flow chart of RNA crystals and determining their structure (left) and a schematic diagram of GU base pair editing (right).

FIG. 3 shows that the LINE-1 ribozyme and SAM-I riboswitch achieved higher resolution after substitution by the GU base pair by the GC base pair.

FIG. 4 shows that dead sea salt box bacteria king-turn-7 (HmKt-7) and HmKt7-19ntX3 significantly improved resolution upon GC base pair substitution to GU base pair.

FIG. 5 shows that the 2' -dG-III ribosomal switch and Broccoli RNA aptamer achieved higher resolution by substitution of AU base pairs to GU base pairs.

FIG. 6 shows that the OR4K15 ribozyme achieved higher resolution by GU to GC substitution.

FIG. 7 shows the major structural changes of a conventional type A RNA helix in the presence of a standard wobble U.G pair. Wherein U moves into the main groove between two Watson-Crick base pairs. The usual twist angle in RNA helices is 33℃and in U.G pairs the twist angle in the 5 'direction of U increases and the twist angle in the 3' direction decreases. These changes in torsion angle resulted in significant interchain stacking (with black boxes connecting) between the G and the 3' end residues of U in the u.g pair. Stacking occurs in the chains on both sides in the 5' direction of U.

FIG. 8 shows superimposed SAM-I-GC riboswitch (Red, PDB number: 4B 5R) and GU (cyan, PDB number: 5 FJC) models. The replacement GU base pairs in backbone trace P2 (cyan), the pseudo-knot structure Pseudoknot (PK) in P1, and the bound SAM molecules are all shown in cyan (O: red; N: dark blue).

FIG. 9 shows different crystal stacks of 2'-dG-III-AU (PDB: 8Z 8Q), 2' -dG-III-GU (PDB: 8 KEB), and HmKt-GU with U1A protein (PDB: 5FJ 4). (A) The stacking of crystals of 2' -dG-III-AU is carried out with a space group of P1. (B) The stacking of crystals of 2' -dG-III-GU is carried out with a space group of C121. (C) HmKt7 the crystal stacking of the 7-GU and U1A proteins, the spatial group was C222 ₁. (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.

Figure 10 shows novel GAAA interactions between asymmetric units of MTR 1. (a) four MTR1 molecules related by crystal symmetry. To the left, the molecules (red) are related to each other by symmetrically related GAAA four base rings with novel interactions (noted within the dashed rectangle). On the right, symmetrically related molecules (green) that contact each other by backbone contacts near the replacement GU base pairs. To the far right, an enlarged view of the dashed box shows the exact interaction between the two symmetrically related molecules. Alongside the illustration is the sequence of the interactions and a schematic showing the non-Watson-Crick base pair type according to nomenclature (2) Leontis-Westhof. (B) The GAAA-interacting non-Watson-Crick base pairs in MTR1 (left) and the symmetry between the GAAA four-base loops from two perspectives are shown.

FIG. 11 shows the structure of G.U base pairs. (A) One standard wobble G.U base pair (from PDB 1HQ 1) and one non-standard anion G.U base pair (from PDB 7K 00). (B) Eight standard wobble G.U base pairs were observed in the structure of this scheme.

Fig. 12 shows metal ion binding in RNA high resolution structures. (A) Electron density maps of Broccoli-GU potassium bases G2 and U48 are shown. Electron density shows a plot of simulated annealing missing electron density at 1.3σ. (B) Electron density maps of the 2' -dG-III-GU sodium bases G2 and U70 are shown. Electron density shows a simulated annealing missing electron density plot outlined with a 0.9σ contour. (C) Electron density maps of OR4K15-GC sodium ion bases G29 and C2 are shown. Electron density shows a plot of simulated annealing missing electron density at 1.0σ. (D) Electron density maps of SAM-I-GU barium ion bases G30 and U22 are shown. Electron density shows a plot of simulated annealing missing electron density at 1.5σ.

FIG. 13 shows the positions of the introduction of GU base pairs in LINE-1 ribozyme (A), 2' -dG-III riboswitch (B), broccoli aptamer (C), MTR1 ribozyme (D) and their corresponding resolutions.

FIG. 14 shows the position and energy change of GU base pairs on double helical stems in the results of RNA structural analysis. The G's in the six GUs are located on the 5' strand and the four G's are located on the 3' strand. ΔE represents the difference in thermodynamic free energy due to sequence changes (ΔE of HmKT, 7-19, ntX3 calculated by introducing the GAAA ring). The thermodynamic free energy was calculated using the two-level structure prediction tool provided by Mathews Group (https:// rna. Urmc. Rochester. Edu/RNAstructureWeb).

FIG. 15 shows an example of solving the phase problem by artificially introducing GU base pairs binding to heavy metals. (A-G) shows the secondary structure schematic of Gryllus paralysis virus (CrPV) IRES RNA (A) (4), dystrophin kinase type 1 (DM 1) RNA (B) (5), SAM-II riboswitch (C) (6), bis- (3 '-5') -cyclodimeric guanylate monophosphate (C-di-GMP) -II riboswitch (D) (7), guanidine-I riboswitch (E) (8), S-adenosyl-L-homocysteine (SAH) riboswitch (F) (9) and potato leaf curl virus (PLRV) RNA (G) (10). Pink spheres represent ligands.

Detailed Description

It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the application and is not intended to be limiting of the application. Unless otherwise indicated, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments of the application. As used herein, singular terms shall be deemed to include the plural thereof unless otherwise explicitly stated. Furthermore, it will be further understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of stated features, steps, operations, components, and/or groups thereof, but do not preclude the presence of other features or elements that are not explicitly listed.

The invention will now be further illustrated with reference to specific examples, which are intended to illustrate the invention only, but are not to be construed as limiting the scope thereof. The test specimens and test procedures used in the following examples include those (if the specific conditions of the experiment are not specified in the examples, generally according to conventional conditions or according to the recommended conditions of the reagent supplier; the reagents, consumables, etc. used in the examples described below are commercially available unless otherwise specified).

1. Materials and methods

(1) Chemical synthesis of RNA

The oligonucleotides were synthesized using t-butyldimethylsilyl (t-BDMS) phosphoramide chemistry following the procedure outlined by Wilson et al. The synthesis scheme is performed using applied biosystems 394DNA/RNA synthesizer involving the use of t-BDMS (Link Technologies) to protect ribonucleic acid phosphoramides .(Wilson,T.J.,Zhao,Z.Y.,Maxwell,K.,Kontogiannis,L.and Lilley,D.M.(2001)Importance of specific nucleotides in the folding of the natural form of the hairpin ribozyme.Biochemistry,40,2291–2302.)

All oligonucleotides were dissolved in 100. Mu.L of anhydrous DMSO and 125. Mu.L of trifluoroethylamine (Sigma-Aldrich) to facilitate removal of the t-BDMS group. The solution was incubated at 65℃for 2.5 hours in the dark. After cooling on ice for 10 minutes, RNA precipitation was achieved using 1mL butanol, one wash with 70% ethanol, and then suspended in double distilled water. RNA lacking the bromine modification was purchased from precision biotechnology (Hunan, china). LINE-1 ribozymes, hmKt-7, hmKt7-19ntX3 and OR4K15 ribozymes were chemically synthesized.

(2) RNA transcription

To promote efficient transcription initiation, the initial nucleotides of the native sequence are substituted with GCG/GGC/GGA/GGG. DNA templates containing the T7 RNA polymerase promoter for in vitro transcription were prepared by Polymerase Chain Reaction (PCR). After transcription, the RNA molecules were purified by electrophoresis in a polyacrylamide gel containing 7M urea. Intact RNA transcripts were recovered by UV development, excision and electroelution (using an electroelution system) after electrophoresis in 0.5 XTBE buffer for 12 hours at 200V electric field. 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.

(3) Crystallization, structure determination and purification

The solution containing 10. Mu.g/. Mu.L RNA, 5mM HEPES (pH 7.5), 100mM potassium chloride and 5mM MgCl2 (HKM buffer) was refolded as described in Table 2. The RNA sequences and crystallization conditions are also summarized in table 2. Crystallization was by mixing 0.2 μl of RNA or RNA ligand complex with 0.2 μl of stock solution, using a sitting-drop vapor diffusion method at 18 ℃. The crystals were transferred to a mother liquor containing an additional 30% glycerol or MPD and then flash frozen by mounting in nylon rings and injecting liquid nitrogen. The X-ray diffraction data were collected on the beam lines BL02U1, BL10U2, BL18U1 and BL19U1 of the Shanghai Synchrotron Radiation Facility (SSRF) and Shanghai national protein science facility (NFPSS) and processed with XIA2 or XDS. The resolution cut-off points of the data were determined by examining CC1/2 and density maps, and PDB 8Z9K (LINE-1-GC), 8Z8Q (2 '-dG-III-AU), 8ZAU (MTR 1-GU) and 8ZA4 (OR 4K 15-GU) were analyzed using PHASER molecular substitution, modeled as 8Z8U (LINE-1-GU), 8KEB (2' -dG-III-GU), 7V9E (MTR 1-GC/AU) and 8ZA0 (OR 4K 15-GC). The resulting model was manually adjusted using Coot and optimized through several iterations of Coot, phenyline and PDB REDO. The geometry of the model and the fit to the electron density map were monitored by verification tools in MOLPROBITY and Coot. In order to effectively avoid overfitting, in particular to determine lower resolution structures using molecular substitution, optimized weights and simulated annealing were introduced in the phix. Refine. The experimental plot was calculated by Autosol and the fourier outlier plot was calculated by phix. Simulated annealing thumbnail images are calculated by using the composite thumbnail image calculated by the annealing method in PHENIXsuite. The atomic coordinates and the structural factor amplitudes have been stored in a Protein Database (PDB) and are listed in table 3 together with the accession codes.

Among the crystal structures discussed herein, four new structures are included, LINE-1 ribozyme (8Z 9K and 8Z 8U), OR4K15 ribozyme (8 ZA0 and 8ZA 4), 2' -dG-III riboswitch (8Z 8Q and 8 KEB), and Broccoli RNA aptamer (8K 7W). Four of these crystalline structures have been reported for MTR1 ribozyme (7V 9E), hmKt-7-GU (5 FJ 4), hmKt7-19ntX (5G 4T) and SAM-I (4B 5R and 5 FJC) (FIG. 1 and Table 1). These structures were all obtained by screening 6 commercial crystallization kits for a total of 288 crystallization conditions, including Index1-96, natrix I/II and Crystal I/II, all purchased from Hampton Research. Specific crystallization details of SAM-I riboswitches, hmKt-7 and HmKt7-19ntX3 RNA are recorded in their respective published literature, outlining the process of structural resolution and refinement. The two-stage structure is designed by adopting a general naming method that the first spiral of the 5' is named as P1, the subsequent spiral is named as P2, the single-chain spiral link between the P1 and the P2 is named as J12, and the head circulation of the last spiral is the same as the marking number of the spiral, and the L3 covers the P3.

2. Results

(1) Crystallographic method innovation-GU base pair substitution

This study proposes an innovative strategy to promote RNA crystallization by replacing the existing helical Watson-Crick base pairs with the wobbled GU pairs (FIG. 2). Prior art X-ray diffraction crystallography relies on a preliminary screening of the original sequence to obtain crystals. After crystal formation, screening conditions are optimized and X-ray diffraction data is collected to resolve the structure. However, this process often has difficulties in obtaining satisfactory results, such as low crystal quality or poor X-ray data. In order to solve this problem, the strategy proposed by the present scheme is to increase the sequence diversity, thereby significantly increasing the probability of obtaining high quality crystals. This strategy not only succeeds in the initial screening stage, but also effectively optimizes crystal quality with lower resolution of the existing crystal.

The left-hand flow chart of fig. 2 depicts the standard procedure for producing RNA diffraction crystals and determining their structure. The original sequence of crystallization was first designed and screened as indicated by the black arrow. If satisfactory crystals are not obtained or the X-ray data is of poor quality, as indicated by grey arrows, the RNA sequence can be replaced or reversed using GU base pairs, as indicated by pink arrows, and then the crystals are rescreened or crystallization conditions optimized. The structures of LINE-1, broccoli, OR4K15, 2' -dG-III, hmKt-7 and HmKt-19 ntX were obtained by the ① method (crystal re-screening), while the structures of SAM-I and MTR1 were obtained by the ② method (optimizing crystallization conditions).

The basic principle of the substitution of GU base pair positions is shown on the right side of FIG. 2, taking three-way-linked RNA as an example. First, the protocol performs a preliminary screening of the original sequence to assess its potential for crystal formation. If the original sequence fails to form satisfactory crystals, or if the resolution of X-ray diffraction of the original sequence is low, then a pair of Watson-Crick base pairs will be substituted for GU base pairs at the center of the P1, P2 and P3 stems (stems numbered from the 5' end), and if necessary, we will test the case where multiple GU pairs are combined for simultaneous substitution in these three regions. Double helical stems supporting binding of pseudoknots or hairpin loops to protein cofactors are also effective alternatives to 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 that wobble base pairs and non-Watson-Crick base pairs occur preferentially in the middle of the helix. Furthermore, unlike Watson-Crick base pairs, the GU pair does not spatially overlap with the UG pair, so either G or U can be replaced in an attempt or replaced, i.e., forward replacement includes AU-GU replacement, UA-UG replacement, CG-UG replacement, and/or GC-GU replacement, and reverse replacement includes GU-AU replacement, UG-UA replacement, GU-GC replacement, and/or UG-CG replacement. The substituted GU base correspondence is avoided in the vicinity of the active site or ligand binding region. If the crystal data did not reach the expectations after the initial modification, the present protocol explored different combinations of GU base pair substitutions on other stems, including strategies to alter multiple positions simultaneously.

(2) Resolution change results

Among the 8 unique RNA crystals of this study, GC-GU substitutions significantly improved the resolution of 4 of the RNA crystals, including LINE-1 ribozyme, SAM-I riboswitch, dead sea salt box Kt-7 (HmKt-7) kink angle and one variant comprising three tandem king-turn (HmKt-19 ntX 3) RNAs, AU-GU substitutions significantly improved the resolution of 2 other RNA crystals, including 2' -dG-III riboswitch and Broccoli RNA aptamer, and reverse substitutions such as GU-GC substitutions improved the resolution of OR4K15 ribozyme. In contrast, substitution of GC and AU base pairs with GU base pairs did not significantly improve crystal resolution for MTR1 ribozymes (fig. 1 and table 1).

(3) Substitution of base pair GC to GU improves resolution

Among the 8 unique RNA crystals studied, GC-GU substitution significantly improved the resolution of 4 of the RNA crystals, including LINE-1 ribozyme, SAM-I riboswitch, and two different forms of HmKt-7 crystals. The first analysis of the LINE-1 ribozyme was accomplished by introducing a GU base pair at the second base pair of its P1 region. Crystal resolution fromTo be increased to(FIG. 3A). Structural comparison of the two forms before and after GU substitution shows that their folding is similar. Root Mean Square Deviation (RMSD) between structures is(FIG. 3C).

SAM-I ribosomal switch is another very strong evidence demonstrating the remarkable ability of GU base pairs to improve resolution. Adding a GU to the penultimate base pair of the P2 region (SAM-I-GU) of the ribosome switch to allow resolution fromTo be increased toThis is the highest resolution achieved by SAM-I ribosomal switches to date (fig. 3D). The P2 stem is located near the pseudoknot (pseudoknot) in the folding of the SAM-I ribosome switch (FIG. 8). The overall structure of the SAM-I-GU ribosomal switch is achieved by two sets of coaxially stacked helices (fig. 3E). Before and after GU base pair introduction, the structural superposition resulted in RMSD asThe overall structure changes little. A detailed examination of GU base pairs and adjacent base pairs C29-G21 and C31-G23 revealed negligible changes (FIG. 3F). In fact, in RNA molecules longer than 50 nucleotides, the resolution of the crystal structure is better than that of RNA moleculesRelatively small. Is utilized inCollected at a wavelength ofData, the structure of the device can be easily analyzed by using the abnormal scattering signals of the barium. In addition, the crystal of the SAM-I-GU riboswitch has high repeatability, and the barium ions which promote the crystal growth can be replaced by other metal ions without affecting the resolution, and is still superior to the crystalThis will be an excellent system to study the interaction of RNA with different metal ions. For example, SAM-I-GU ribosome switchingThe resolution structure enables researchers to pinpoint the barium ions and their associated water molecules at the G74 position (fig. 3G).

Third case HmKt-7 RNA, the study compiled it into an artificial RNA molecule capable of binding to U1A protein. In its original sequence, the GC base pairs are at positions 9 and 24, and the sequence RNA molecule fails to crystallize. GC-GU substitution of the above positions not only promotes crystallization, but also yields resolution ofIs shown (FIGS. 4A-4C). This observation further demonstrates the value of GU base pairs in RNA crystal formation and their ability to increase crystal resolution. As shown in FIG. 9C, the alternate GU pair was located within the double helical stem, which supported the binding of the hairpin loop to the U1A protein for co-crystallization.

FIG. 4D shows a molecular structure with 6 king-turn corners, the inspiration being from HmKt-19 nt, which underscores the impact of replacing a different number of GC base pairs with GU base pairs. The overall shape of the kine-turn corner structure (HmKt-19 ntx 36 u) effectively cyclized by crystal symmetry is a triangle, the edges being defined by a typical spiral (C), and the vertices being formed by an atypical spiral (NC) (fig. 4D). The HmKt7-19ntX variant without GU base pairs had a resolution ofCombining 6 GU base pairs (HmKt-19 ntX 3U) to increase resolutionIn addition, the improvement was most pronounced with the variant with 12 GU base pairs (HmKt.about.7-19 ntX3.6U), diffracting into(FIG. 4E). It should be noted that in this case, the introduction of tandem GU pairs is the most common and stable in RNA constructs, with tandem GUs occurring in the middle of each helical segment.

(4) Substitution of base pair AU to GU improves resolution

The scheme also observed that by substituting AU base pairs with GU, the resolution of the 2' -dG-III ribosomal switch and Broccoli RNA aptamer was significantly improved. Substitution of AU to GU facilitates structural resolution of the 2' -dG-III ribosomal switch. In this structure, adenine in the P3 position is replaced with guanine to provide resolutionTo be increased to(FIG. 5A). And (3) withIn contrast to the data set,The dataset not only provides better resolution, but also shows better statistical parameters. Notably, overall Rmerge (a statistical indicator of the consistency of duplicate measured data) falls from 0.175 to 0.031 (table 3) while maintaining similar redundancy. By means ofData sets, the present protocol successfully determines its structure by molecular replacement.

The overall structure of the 2' -dG-III RNA includes a three-way linked stem-loop scaffold, accompanied by loop-loop interactions between L2 and L3 (fig. 5B). RMSD between constructs before and after GU base pair introductionShowing the consistency of the overall structure. Further analysis showed only a small shift between the GU base pair and its adjacent base pair, indicating that the GU base pair had little effect on the overall structure (FIG. 5C). This is the only structure that changes the spatial group after insertion of a GU. Interestingly, two new intermolecular hydrogen bonds can be formed between the 5 '-phosphate and O2' oxygen of G of GU pair and the two hydroxyl groups of adjacent molecules (fig. 9D).

The present protocol also explores Broccoli RNA ligands that can bind to the fluorescent small molecule DFHBI-1T. Initially, the presence of AU base pairs at positions 2 and 48 of the P1 region did not promote crystal formation. However, AU-GU substitution at the above position promotes crystal formation with diffraction resolution of(FIG. 5D). Broccoli RNA the aptamer is based on an elongated helical conformation comprising two stem regions P1 and P2, and a fluorophore binding site located between them. The fluorophore-binding site comprises a G-quadruplex structure and a plane of three base interaction (FIGS. 5d and 5E). A detailed examination of the G2U 48 base pairs and their adjacent base pairs reveals typical features of RNA duplex structures (FIG. 5F). These examples demonstrate that the introduction of GU base pairs can transform structures that cannot form crystals into structures that can produce crystals.

(5) Conversion of original GU base pairs to GC can result in higher resolution structures

The introduction of GU base pairs is not the only way to increase the resolution of crystals. Another approach is to convert naturally occurring GU base pairs to classical GC base pairs, which was demonstrated in the case of the protocol study case OR4K15 ribozyme, which enabled the protocol to determine the crystal structure of this ribozyme for the first time.

In this case, after converting GU base pairs of the P1 region into GC base pairs, the crystal resolution thereof is changed fromSignificantly improve to(FIG. 6A). The OR4K15 ribozyme consists of two RNA strands, which adopt a double helix configuration (fig. 6B). RMSD value between the two structures before and after GU base pair conversion was 0.382A, indicating little significant difference in identity between the two structures (fig. 6C). Further analysis of these two structures showed only a slight difference between base pairs (fig. 6D). This finding suggests that strategies to increase the resolution of RNA crystals are not limited to the introduction of GU base pairs. When GU base pairs are present in the natural RNA sequence and the crystal resolution is low, converting GU base pairs of inactive central stems to GC base pairs can increase the sequence diversity and thus the crystal resolution.

(6) GU base pairs potential sites for barium ion binding in RNA crystals

Replacing GC pairs with GU wobble pairs does not always improve resolution. In the study of MTR1 ribozyme, 2 Watson Crick base pairs are replaced by GU pair (FIG. 6E), but the crystal resolution is not improved obviously, and the resolution before and after GU base pair replacement is respectivelyAnd(FIG. 6E). The overall structural features of the MTR1 ribozyme include a three-way junction consisting of P1, P2 and P3 stems (FIG. 6F). RMSD values between structures before and after GU base pair introduction were given asThe overall structure is illustrated to be highly consistent with no significant change (fig. 6G). Interestingly, this protocol observed a binding of one barium atom at G59U 50 base pairs (fig. 6H). The findings of this protocol in the case of MTR1 ribozymes demonstrate the versatile role of GU base pairs in RNA crystallography. While they may not always improve resolution, they may promote the incorporation of heavy atoms, such as barium ions, which provides a new idea for solving the phase problem. At the same time, this protocol observes a new GAAA interaction between asymmetric units (fig. 10). The above findings have complemented 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, only one of which was observed to bind barium ions.

3. Analysis

The core of the method of this embodiment 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 likelihood of obtaining high quality crystals and accurate structures. In this study, all introduced GU base pairs in 8 RNA crystals were observed in standard wobble GU pairs, none of which were in tautomeric form or in anionic form (fig. 11). This observation is consistent with the results of the study, i.e., the introduction of GU base pairs has little effect on the overall RNA structure. This protocol compares the structure before and after GU base pair substitution (Table 1). This scheme only found one case, the 2' -dG-III ribosome switch, where the space group and crystal stack were changed (fig. 9A and S3B). When the position of GU base pair substitution was explored, the present protocol observed metal ion binding in high resolution structures including brocoli-GU, 2' -dG-GU, SAM-I-GU, and OR4K15-GC in 4 RNA crystals (FIG. 12). This scheme assumes that this may be one factor leading to increased resolution. However, it is noted that some ions have poor electron densities. Due to the lower resolution of these pre-change structures, these ions may be difficult to observe in unmodified crystals.

By analyzing GU engineering sites (FIGS. 13 and 14), this protocol found that only a few sites were effective. These effective remodelling sites occur within the P1 stem. Specifically, of the 7 cases analyzed, 5 (FIGS. 15A-E) were in the P1 stem and 2 (FIGS. 15F-G) were in the other stems. This is consistent with previous observations of GU-aided phase resolution. Thus, the present solution suggests prioritizing modifications within the first 5' double helical stem (P1 stem). If the modification of the P1 stem is unsuccessful, it is contemplated that the GU base pair modification may be extended to other stem regions.

Interestingly, the OR4K15 ribozyme provides a counter example, and the conversion of RNA molecules with native GU base pairs to GC base pairs also results in improved resolution. This observation underscores the complex, environment-specific nature of RNA crystallography and underscores the importance of sequence diversity in RNA crystals. To better understand this phenomenon, the present protocol examined large-scale deep mutation data for LINE-1 and OR4K15 ribozymes. This protocol found that in the LINE-1 ribozyme, the GC conversion to GU had higher catalytic activity (relative activity was changed from 1 to 1.43, 43% increase). The same is true for the reverse conversion of GU to GC in OR4K15 ribozymes (relative activity changed 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 of RNA folding ability is a major cause of the improvement of crystal resolution. Furthermore, deep mutation scanning helps to find the best variant to improve RNA crystallization.

The present solution proposes a strategy to obtain RNA crystals with higher resolution. Current observations of GU base pairing effect also aid in the selection of "crystallizable" sequences from the collection of homologous sequences. Therefore, the method is expected to be further applied and explored in the field of crystallography research, and is possible to improve the capability of elucidating the complex structure of RNA molecules in the field and help RNA sequence editing, in particular to improve the quality and resolution of crystals.

TABLE 1 summary of crystal Properties of RNA Structure

TABLE 2 sequences and conditions used in crystallization experiments

SAD-Ba Single wavelength anomalous scattering using barium (SINGLE WAVELENGTH Anomalous Diffraction using Barium). MR: molecular replacement (Molecular Replacement). Nucleotides for substitution have been marked with bolded inclinations.

Table 3. Crystallographic data submitted to PDB. The values in brackets are for the highest resolution shell.

It should be understood that the foregoing examples of the present invention are provided for the purpose of clearly illustrating the technical aspects of the present invention and are not intended to limit the specific embodiments of the present invention. Any modification, equivalent replacement, improvement, etc. that comes within the spirit and principle of the claims of the present invention should be included in the protection scope of the claims of the present invention.

Claims (6)

1. A method of promoting RNA crystal growth and improving diffraction resolution comprising a forward substitution or a reverse substitution of a GU base pair with a watson-crick base pair by RNA, the reverse substitution comprising a GU-GC substitution and/or a UG-CG substitution.

2. The method of claim 1, wherein the forward substitution comprises AU-GU substitution, UA-UG substitution, GC-GU substitution, and/or CG-UG substitution.

3. A method of promoting RNA crystal growth and improving diffraction resolution as claimed in claim 2, wherein the position of the forward substitution is located in the middle of the mating region of the RNA stem.

4. A method of promoting RNA crystal growth and improving diffraction resolution according to claim 3, wherein the RNA stem comprises a first 5' duplex stem of RNA and/or a stem of any inactive center.

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

6. A method of solving the problem of RNA crystal phase comprising the forward substitution and binding of barium ions of claim 1.