CN115418392A - Method for detecting miRNA based on click chemistry terminal transferase and CRISPRCs 12a - Google Patents

Abstract

The invention discloses a miRNA detection method based on click chemistry terminal transferase and CRISPRCs 12 a. First, the target miRNA-21 is used as a template, two nucleic acid probes are connected by click chemistry, and the product can be combined with Magnetic Beads (MBs). Then, the connected nucleic acid probe and the complementary strand miRNA-21 are extended by TdT. The extended poly-T tail activates the trans-cleavage capability of CRISPR/Cas12a, thereby cleaving the reporter gene to generate a fluorescent signal. The specificity and sensitivity of microRNAs (miRNAs) detection play a crucial role in the early diagnosis of cancer and the treatment of various diseases.

Description

Method for detecting miRNA based on click chemistry terminal transferase and CRISPRCs 12a

Technical Field

The invention belongs to the technical field of biological detection, and relates to a method for detecting miRNA based on click chemistry terminal deoxynucleotidyl transferase combined with CRISPR/Cas12 a.

Background

MiRNA is a non-coding endogenous single-stranded RNA with high conservation and small molecular weight (18-25 nt), and is widely present in a human genetic genome. Some miRNAs are closely related to a variety of diseases, such as psychiatric disorders, cardiovascular disorders and tumorigenesis. MiRNAs can bind to specific target genes, regulate transcription and translation of downstream target genes, and play different roles in various diseases. For example, miRNA-21, as an oncogene, may be a biomarker and therapeutic target for the diagnosis of various diseases and cancers. Many traditional miRNAs detection strategies (northern blotting, microarray, real-time quantitative PCR) have been widely used. Northern blotting is a commonly used method for detecting miRNAs, but the method has the disadvantages of low throughput, low sensitivity, long service life and large sample consumption. Microarray technology is particularly effective for high throughput analysis of a variety of miRNAs, but has low sensitivity and selectivity. Compared with the two methods, the real-time fluorescent quantitative PCR (qRT-PCR) has higher sensitivity, accuracy and practicability and is the gold standard for the quantitative expression of genes at present. The method has the problems of complicated analysis, high cost and low flux.

The aggregated regular spacer short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) establish new approaches for molecular diagnostics. After CRISPR/Cas12 arbitrarily cleaves non-target nucleic acids (referred to as trans-nucleic acid activity), it has shown great potential as a biosensor. Due to the inherent high resolution, simplicity and self-signaling capability of the CRISPR/Cas12a system, a new opportunity is provided for miRNA detection methods. Many CRISPR/Cas12 a-based biosensors combine nucleic acid isothermal amplification techniques such as cross-linking reaction (HCR), catalytic Hairpin Assembly (CHA), rolling Circle Transcription (RCT), and strand displacement amplification to improve the sensitivity of miRNAs detection. The HCR circuit acts as a signal sensor, converting each miRNA into multiple programmable DNA duplexes, activating the trans-cleavage activity of CRISPR/Cas12 a. A simple and universal miRNA detection platform is developed based on CRISPR/Cas12a binding HCR. Modular CHA is used to convert and amplify the target miRNA into multiple programmable DNA duplexes, initiating the trans-cleavage ability of CRISPR/Cas12 a. By combining the CRISPR/Cas12a and the CHA circuit, a universal sensing system for the amplification detection of miRNAs is established. The target miRNA specifically initiates CHA, which then activates the blocked crRNA to a precursor crRNA for CRISPR/Cas12a processing, thereby generating mature crRNA-guided CRISPR/Cas12a recognition activators and unlocking the trans-cleavage ability of CRISPR/Cas12 a. Chen et al established a one-step miRNA detection platform by integrating RNA-based CHA circuits with CRISPR/Cas12 a. Chen et al established a one-step miRNA detection platform by integrating RNA-based CHA circuits with CRISPR/Cas12 a. RCT is specifically initiated by miRNA to generate long single-stranded RNA, resulting in the generation of large amounts of CRISPR/Cas12a precursor crRNA repeats for self-crRNA trimming and construction, further activating its trans-cleavage activity. Based on RCT combined with CRISPR/Cas12a, a biosensor for miRNA detection is established. The "invader-stacked primer" amplification reaction serves as a strand displacement amplification reaction, converting each miRNA into a large amount of DNA, triggering the trans-lytic nuclease activity of CRISPR/Cas12 a. Plum et al made a platform for the instant detection of miRNA by "invader-stacking primer" amplification reaction and CRISPR/Cas12 a. Plum et al introduced a cascade toe-mediated strand displacement reaction (CTSDR) and used in miRNA detection in conjunction with CRISPR/Cas12a trans-cleavage. In this study, dynamic CTSDR is initiated after hybridization of a target miRNA with a rationally designed probe, resulting in enzyme-free target cycling and generation of multiple programmable DNA duplexes, triggering CRISPR/Cas12a trans-cleavage. By combining ligase chain reaction with CRISPR/Cas12a, the inventors developed an ultrasensitive and uniform miRNA detection strategy. In addition, double-strand specific nucleases (DSNs) exhibit a specific substrate specificity, hydrolyzing only DNA in DNA/RNA hybrids, without regard to nucleotide fragments. Through a dual specificity nuclease assisted CRISPR/Cas12a strategy, a biosensing platform for detecting miRNA is established.

Click chemistry was first proposed in 2001 by Sharpless, kolb and Finn to be a novel compound useful for rapid synthesis via carbon heteroatom linkage. In recent years, copper-free click chemistry has attracted considerable attention as a signal amplification strategy. Unlike DNA or RNA ligases, click chemistry relies on a chemical reaction between two groups, azide (N3) and dibenzocyclooctene (Aza-DBCO), with higher ligation efficiency and better compatibility. In the presence of a target sequence, aza-DBCO and N3 can be hybridized with each other closely under the conditions of constant temperature and no metal ion catalysis, and a click reaction is completed spontaneously. In addition, aza-DBCO and N3 are not affected by carboxylic acid, amine and thiol, and interference caused by coexistence of protein and other molecules is avoided, so that click chemistry has the potential of analyzing complex samples. Therefore, click chemistry reactions have been widely used in the fields of nucleic acid analysis, biology, DNA nanotechnology, and the like.

TdT is a template-independent DNA polymerase that catalyzes the repetitive addition of deoxyribonucleotides to the 3' -OH ends of single-and double-stranded DNA/RNA. The TdT reaction requires a short sequence comprising at least 3 bases as a primer. When RNA is used as a template, tdT may be strictly dependent on the 3' terminal tertiary structure of the acceptor RNA and the type of nucleotides. The polymerization activity of TdT is independent of the template strand, and the polymerization reaction can be completed under isothermal conditions. TdT is used as a multifunctional tool to quantify metal ions, target RNA and exosomes. The publication "a fluorescent biosensor for simple and sensitive detection of miRNAs based on DSN/TdT cycling digestion strategy", is detailed in: he, J.L., et al, DSN/TdT recycling diagnostic based amplification protocol for microRNA assay. Talanta, 2020.219. Historically, et al developed an electrochemical assay for label-free and highly sensitive detection of thrombin in human serum by catalysis of hairpin assembly and TdT-catalyzed DNA extension. Based on the advantages of click chemistry and TdT, the invention establishes a click chemistry-TdT (ccTdT) nucleic acid isothermal amplification technology.

Disclosure of Invention

The invention provides a method for detecting miRNA based on click chemistry terminal deoxynucleotidyl transferase combined CRISPR/Cas12 a. The method comprises the step of attaching DNA chains connected with miRNA complementary fragments to the surfaces of Magnetic Beads (MBs) through streptavidin-biotin interaction, wherein the MBs are retained through magnetic separation. In the presence of dTTP and TdT, the exposed free 3' -OH ends of the ligated DNA strand and the complementary strand miRNA can generate a continuous poly-T. The recognition region of crRNA contains a21 bp poly-A tail. The expanded poly-T tail can be used as an activator to supplement crRNA, activate trans-cleavage of CRISPR/Cas12a, cleave the reporter gene and generate a fluorescent signal.

Portions of Probe A (PA) and Probe B (PB) are complementary to the miRNA-21 sequence. When miRNA-21 is present, PA and PB hybridize to miRNA-21. Since hybridization brings the two groups close to each other, it allows an efficient click chemistry reaction between DBCO and N3. When TdT and dTTP are present, the free 3' -OH ends of miRNA-21 and PB may participate in TdT-induced polymerization processes, forming a continuous poly-T product. To activate Cas12a trans-cleavage activity using poly-T product, we designed a21 bp crRNA recognition sequence. Formation of a hybrid between crRNA and poly-T tail initiates trans-cleavage of several CRISPR/Cas12a units, initiating catalytic cleavage of the ssDNA reporter (reporter labeled with HEX and BHQ1 at the 5 'and 3' ends, respectively). The fluorescein group is then released and a significant fluorescent signal is generated. However, in the absence of miRNA-21, there was no reaction between the two groups. In this case, since DBCO modifies the 3' end of PA to block TdT-induced non-specific amplification. Thus, the 3' end of the PA is not only unable to generate a poly-T tail to activate Cas12a, but also does not cause significant changes in the fluorescence signal.

The technical scheme adopted by the invention is as follows:

a method for detecting miRNA based on click chemistry terminal transferase and CRISPRCs 12a comprises the following steps:

step 1: preparation of crRNA

(1) mu.L of crRNA template and 2. Mu.L of T7 promoter (100. Mu.M) were mixed in 14. Mu.L of enzyme-free water. The mixture was incubated at 95 ℃ for 5min, then gradually cooled to 25 ℃ and left for 2h.

(2) mu.L of 100mM dNTP buffer and 2. Mu.L of T7 RNA polymerase were mixed. 16h at 37 ℃ and thus a large amount of target crRNA was transcribed.

(3) The crRNA template was degraded with DNaseI at 37 ℃ for 15min. The crRNA obtained was then purified using an RNA clearing kit and redissolved in DNase/rNase-free water.

(4) The concentration of crRNA was measured using a NanoDrop 1000 (Waltham, MA, USA) and stored at-80 ℃ prior to use.

And 2, step: detection of miRNA

(1) miRNA-21 at different concentrations were mixed into 30. Mu.L of reaction buffer containing 2. Mu.L of probe A and 2. Mu.L of probe B (0.5. Mu.M each, table S1). After incubation at 37 ℃ for 30min, 3. Mu.L of MBs was added, followed by incubation at 37 ℃ for 30min, and repeated magnetic separation washing 3 times with 50. Mu.L of PBS solution (pH 7.6) through a magnet, to retain MBs.

(2) To the reaction were added 2.5. Mu.L of 2.5mM cobalt chloride, 2.5. Mu.L of 10 XTdT buffer, 2. Mu.L of 10mM dTTP, 2U of TdT and 10. Mu.L of enzyme-free water, and incubated at 37 ℃ for 40min. The reaction system was heated at 80 ℃ for 10min to inactivate the catalytic activity of TdT. mu.L of the reaction solution containing 2. Mu.L of 0.8. Mu.M Cas12a, 2. Mu.L of 5 Xbuffer solution and 1. Mu.L of 1.5. Mu.M crRNA, preincubated at 25 ℃ for 10min, 2. Mu.L of the reaction product and 1. Mu.L of 10. Mu.M HEX were added. The HEX cleavage reaction catalyzed by cas12a was maintained at 37 ℃ for 1h. And measuring the fluorescence signal by using an ND-3300 fluorescence spectrometer under the conditions that the excitation wavelength is 495nm and the emission wavelength is 556nm, and further detecting miRNA-21.

And step 3: gel electrophoresis analysis

mu.L of the reaction product was premixed with 6 Xloading buffer (2. Mu.L). Each mixture (10. Mu.L) was injected into a polyacrylamide gel electrophoresis (PAGE) system, and the gel was run in 1 XTBE buffer (89 mM boric acid, 89mM Tris and 2.0mM EDTA, pH 8.2). Electrophoresis was carried out at 180V for 45min and stained with 10 XSSYBR GreenII for 20min. The gel was visualized using Azure biosystems C600.

In the presence of miRNA-21, the connected DNA and the exposed 3' -OH end of miRNA-21 form a continuous poly-T tail (activator), and the trans-cleavage activity of CRISPR/Cas12a can be activated. In addition, the activated CRISPR/Cas12a has a signal enhancing effect itself, and can easily cleave a beacon molecule modified with a fluorophore and a fluorescence quenching group nearby. The proposed fluorescent biosensor containing the multiple amplification strategy can realize the ultra-sensitivity detection of miRNA, and has application prospects in molecular diagnosis and biomedical research.

Drawings

Fig. 1 is a workflow diagram of ccTdT in combination with Cas12a to detect miRNA.

FIG. 2 is a diagram of a feasibility analysis of the biosensor.

FIG. 3 is a feasibility analysis diagram; (A) is polyacrylamide gel electrophoresis (PAGE); (B) In the middle, 1 is click product, and 2 is click product without dTTP; 3, clicking products without TdT; lane 4 is click product + dTTP + TdT;5, experimental groups; 6 channels are PA + dTTP + TdT;7 lanes are control group; lane 8 crRNA.

FIG. 4 optimization of the proposed biosensor; wherein (a) click chemistry reaction time (B) TdT extension time (C) TdT concentration (D) Cas12a cleavage time (E) Cas12a concentration (F) crRNA concentration.

FIG. 5 is a graph of the performance of the miRNA-21 detection system; wherein (A) fluorescence spectra in the presence of different concentrations (100-105 pM) of miRNA (B) the linear relationship between fluorescence intensity and logarithm of miRNA concentration

FIG. 6 is a schematic diagram showing the specificity of the method for detecting miRNA21 of the invention.

Detailed Description

The oligonucleotides used in the examples of the present invention were all synthesized by Biotechnology engineering (Shanghai) Inc., and the sequences of the oligonucleotides are listed in Table 2-1. All primer purification methods adopt a High Performance Liquid Chromatography (HPLC) purification method.

TABLE 2-1 oligonucleotide sequences

Table 2-1Oligonucleotides sequence

The reagents and apparatus referred to in the examples are shown in tables 2-2 and 2-3

TABLE 2-2 reagents

Table 2-2reagents

TABLE 2-3 Instrument Equipment

Table 2-3instruments and equipment

As shown in FIG. 2, the blank control group without miRNA-21 showed no significant change in fluorescence. In contrast, when miRNA-21 was added, the observed fluorescence signal was significantly enhanced, indicating that the trans-cleavage activity of CRISPR/Cas12a was unlocked and the reporter gene was triggered. The significant difference in fluorescence between these two systems indicates the feasibility of our miRNA-21 detection strategy. In the absence of TdT, dTTP and Cas12a in the reaction system, no fluorescence change occurred, indicating that TdT, cas12a and dTTP play an essential role in the sensor system.

To further demonstrate the feasibility of the miRNA-21 detection mechanism proposed by the present invention, we performed PAGE analysis. First, it was demonstrated that click chemistry reactions can occur in the presence of miRNA-21. As shown in FIG. 3A, lanes 1-3 correspond to miRNA-21, PB, and PA, respectively. Lane 4 is the click product where miRNA-21, PA and PB are present. Lane 5 shows PA and PB, without miRNA-21. It is clear that click chemistry reactions can only occur in the presence of miRNA-21 and are effective. Next, after the click chemistry reaction was completed, the amplification process of TdT and the activation process of Cas12a were verified. As shown in fig. 3B, the bands in lane 1 correspond to the click products described above. Lane 2 shows the click product with only TdT and no dTTP. Lane 3 shows the click product with only dTTP and no TdT. Lane 4 shows the product when both dTTP and TdT are present. Lane 5 is the experimental group. Lane 6 represents PA (DBCO) with both TdT and dTTP present. Lane 7 represents the control group. The bright band in lane 8 corresponds to crRNA. Some large amount of DNA fragments appeared in the upper part of the gel (lane 4), mainly due to poly-T sequence amplified from free 3' -OH of DNA/RNA fragments in the presence of dTTP and TdT. The extended poly-T tail can unlock the trans-cleavage ability of Cas12a (lane 5), but the 3' end of PA is modified with DBCO to prevent tdt-induced non-specific amplification (lane 6), and thus cannot activate Cas12a activity (lane 7).

Using F/F ₀ Expressed in the presence or absence of miRNA-21The ratio of the fluorescence signals is used to estimate the current fluorescence signal. Since click chemistry is the first step in a reaction, which determines whether subsequent reactions will proceed, we first optimize the click time. As shown in FIG. 4A, F/F ₀ The fluorescence value of (A) is from 10min to 30min ₀ The fluorescence value of (a) gradually increases and then tends to decrease. Thus, 30 minutes was selected as the optimized click time to complete the click reaction. This time was chosen because when the click time was too short, the limited ligation between the probes affected the polymerization of TdT, resulting in a lower fluorescence signal from the system. However, when the click time is long, coupling between the probes occurs, resulting in an increase in the fluorescence intensity of the control group. The extension of TdT determines the length of the poly-T tail, and how much specific sequence can activate the trans-cleavage activity of Cas12a, which is closely related to the enhanced efficiency of the biosensor. The reaction time and concentration of TdT were optimized. In FIG. 4B, F/F increases with time of reaction with TdT ₀ Gradually increases to a maximum value at 40min, after which F/F ₀ And begins to fall. The optimal reaction time for TdT is 40min. FIG. 4C shows that as the TdT concentration is increased to 0.3U/. Mu.L, F/F ₀ Continuously increase, then F/F ₀ And begins to fall. The optimum concentration is 0.3U/. Mu.L, and when the extension time of the system is too long and the TdT concentration is too high, non-specific amplification may occur, resulting in non-specific recognition.

Different reaction conditions comprise the concentration of CRISPR/Cas12a, the enzyme cutting time of CRISPR/Cas12a and the dosage of crRNA. FIG. 4D shows the effect of enzyme digestion time on F/F0, from which it can be seen that F/F ₀ The value of (b) is increased when the enzyme digestion time is 20 to 60min, and then gradually decreased, so that an optimal enzyme digestion time of 60min is selected. At the same time, as the concentration of CRISPR/Cas12a increased from 0.2. Mu.M to 0.8. Mu.M, F/F ₀ Continues to increase and tends to stabilize (fig. 4E). By studying the effect of the crRNA concentration on the method, it was found that F/F was found when the crRNA concentration was 0.5. Mu.M to 1.5. Mu.M ₀ The values increased with increasing crRNA concentration and then were relatively stable (fig. 4F). 1.5. Mu.M was selected as the optimal crRNA concentration.

As shown in fig. 5A, with the targetStandard miRNA-21 concentration (100-10) ⁵ pM), the fluorescence intensity of the sensing system gradually increases. F/F0 is obviously linear with the logarithm of miRNA-21 concentration. The standard equation is Y = 0.7293X +1.572 (R) ² = 0.932), where X is the logarithm of the miRNA-21 concentration and Y represents the current fluorescence signal (fig. 5B). According to the 3 σ/k rule, the lower limit of detection (LOD) is estimated to be 88fM. Therefore, the proposed biosensor has excellent performance and sensitivity in the detection of miRNA-21. The comparison of this method with other reported similar methods for detecting miRNA is shown in Table 3-1. In contrast, the detection method of the invention has more excellent sensitivity and shorter detection time.

TABLE 3-1 comparison of different miRNA assay methods

Table 3-1Comparison of different miRNA determination methods

See the following documents:

37.Xu,H.,et al.,RCA-enhanced multifunctional molecule beacon-based strand-displacement amplification for sensitive microRNA detection.Sensors and Actuators B:Chemical,2018.258:p.470-477.

38.Zhang,Y.,et al.,A simple electrochemical biosensor for highly sensitive and specific detection of microRNA based on mismatched catalytic hairpin assembly.Biosens Bioelectron,2015.68:p.343-349.

39.Li,C.,et al.,Oriented Tetrahedron-Mediated Protection of Catalytic DNA Molecular-Scale Detector against in Vivo Degradation for Intracellular miRNA Detection.Anal Chem,2019.91(18):p.11529-11536.

40.Hosseinzadeh,E.,et al.,Colorimetric detection of miRNA-21by DNAzyme-coupled branched DNA constructs.Talanta,2020.216:p.120913.

in order to prove the specificity and selectivity of the ccTdT-Cas12a amplification system for miRNA-21 detection, the fluorescence signals of mirnas (miRNA 21-1, miRNA21-2, miRNA21-3, miRNA 21-4) and miRNA-141 with different base mutation numbers were measured using this method and compared with the fluorescence signal of miRNA-21. As shown in fig. 6, the addition of miRNA-21 alone resulted in a significant change in fluorescence intensity compared to other miRNAs sequences tested. Even a single base mutation in miRNA can be recognized by the biosensor. Apparently, other mirnas are not able to cleave activity in trans of Cas12a together. These results indicate that the proposed biosensor has superior specificity. Research on the practical application value and reliability of the combined ccTdT-Cas12a amplification system in a biological sample, and three miRNA-21 (10) with different concentrations ² 、10 ³ And 10 ⁴ pM) was added to 10% biological serum. The performance index of the reporter system was then assessed by the recovery rate, which was defined as the ratio of the average detected concentration of the biosensor to the added concentration of miRNA-21. As shown in Table 3-2, the average recoveries obtained for the serum samples ranged from 100.3% to 114.1% with a Relative Standard Deviation (RSD) of less than 9%.

Table 3-2Recovery of miRNA in real serum sample

Claims (2)

1. A method for detecting miRNA based on click chemistry terminal transferase and CRISPRCs 12a is characterized in that: attaching DNA chains connected with miRNA complementary fragments to the surfaces of Magnetic Beads (MBs) through streptavidin-biotin interaction, and keeping the MBs through magnetic separation; in the presence of dTTP and TdT, the exposed free 3' -OH ends of the ligated DNA strand and complementary strand mirnas may generate a continuous poly-T; the recognition region of crRNA contains a21 bp poly-A tail; the expanded poly-T tail can be used as an activator to supplement crRNA, activate trans-cleavage of CRISPR/Cas12a, cleave the reporter gene and generate a fluorescent signal.

2. The method for detecting miRNA based on click chemoterminal transferase and CRISPRCs 12a as claimed in claim 1, comprising the steps of:

step 1: preparation of crRNA

(1) mu.L of crRNA template and 2. Mu.L of T7 promoter (100. Mu.M) were mixed in 14. Mu.L of enzyme-free water; incubating the mixture at 95 ℃ for 5min, then gradually cooling to 25 ℃, and standing for 2h;

(2) Mix 10. Mu.L of 100mM dNTP buffer and 2. Mu.L of T7 RNA polymerase; transcribing for 16h at 37 ℃ to transcribe a large amount of target crRNA;

(3) Degrading the crRNA template with DNaseI at 37 ℃ for 15min; the crRNA obtained was then purified using an RNA-clearing kit and redissolved in DNase/rNase-free water;

(4) The concentration of crRNA was measured using NanoDrop 1000 (Waltham, MA, USA) and stored at-80 ℃ prior to use;

step 2: detection of miRNA

(1) Mixing miRNA-21 with different concentrations into 30 μ L of reaction buffer containing 2 μ L of probe A and 2 μ L of probe B (0.5 μ M each, table S1); incubating at 37 deg.C for 30min, adding 3 μ L of MBs, incubating at 37 deg.C for 30min, washing with 50 μ L of PBS solution (pH 7.6) by magnetic separation repeatedly for 3 times, and retaining MBs;

(2) To the reaction were added 2.5. Mu.L of 2.5mM cobalt chloride, 2.5. Mu.L of 10 XTdT buffer, 2. Mu.L of 10mM dTTP, 2U TdT and 10. Mu.L of enzyme-free water and incubated at 37 ℃ for 40min; heating the reaction system at 80 ℃ for 10min to inactivate the catalytic activity of TdT; 10 μ L of reaction solution containing 2 μ L of 0.8 μ M Cas12a,2 μ L of 5 × buffer solution and 1 μ L of 1.5 μ M crRNA, preincubated at 25 ℃ for 10min, 2 μ L of reaction product and 1 μ L of 10 μ M HEX were added; the HEX cleavage reaction catalyzed by cas12a was maintained at 37 ℃ for 1h; measuring the fluorescence signal by an ND-3300 fluorescence spectrometer under the conditions that the excitation wavelength is 495nm and the emission wavelength is 556nm, and further detecting miRNA-21;

and 3, step 3: gel electrophoresis analysis

10. Mu.L of the above reaction product was premixed with 6 Xloading buffer (2. Mu.L); each mixture (10. Mu.L) was injected into a polyacrylamide gel electrophoresis (PAGE) system, the gel was run in 1 XTBE buffer (89 mM boric acid, 89mM Tris and 2.0mM EDTA, pH 8.2); electrophoresis was carried out at 180V for 45min, stained with 10 XSSYBR GreenII for 20min; the gel was visualized using Azure biosystems C600.

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