Purification, characterization and cytotoxic activities of individual tRNAs from Escherichia coli

Journal Pre-proofs Purification, characterization and cytotoxic activities of individual tRNAs from Escherichia coli Kai-Yue Cao, Yu Pan, Tong-Meng Yan, Zhi-Hong Jiang PII: DOI: Reference:

S0141-8130(19)35869-6 https://doi.org/10.1016/j.ijbiomac.2019.09.106 BIOMAC 13351

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International Journal of Biological Macromolecules

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27 July 2019 12 September 2019 13 September 2019

Please cite this article as: K-Y. Cao, Y. Pan, T-M. Yan, Z-H. Jiang, Purification, characterization and cytotoxic activities of individual tRNAs from Escherichia coli, International Journal of Biological Macromolecules (2019), doi: https://doi.org/10.1016/j.ijbiomac.2019.09.106

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Purification, characterization and cytotoxic activities of individual tRNAs from Escherichia coli Kai-Yue Cao1,2, Yu Pan1,2, Tong-Meng Yan1,2, Zhi-Hong Jiang1,2*

1State

Key Laboratory of Quality Research in Chinese Medicine, Macau University of

Science and Technology, Avenida Wai Long, Taipa, Macau SAR, China. 2Macau

Institute for Applied Research in Medicine and Health, Macau University of

Science and Technology, Avenida Wai Long, Taipa, Macau SAR, China. *Corresponding author: Dr. Zhi-Hong Jiang, State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Avenida Wai Long, Taipa, Macau SAR, China. E-mail: [email protected] (Zhi-Hong Jiang)

1

ABSTRACT Transfer RNAs (tRNAs) are the most abundant class in small non-coding RNAs which have been proved to be pharmacologically active. In the present study, we evaluated the potential anticancer activities of tRNAs from Escherichia coli MRE 600 to investigate the relationship between non-pathogenic Escherichia coli strain and colorectal cancer. To purify individual tRNAs, we firstly developed a two-dimensional liquid chromatography (2D-LC) and successfully obtained two pure tRNAs. Nuclease mediated base-specific digestions coupled with UHPLC-MS/MS techniques led to an identification of these two tRNAs as tRNA-Val(UAC) and tRNA-Leu(CAG) with typical cloverleaf-like secondary structure. MTT assay demonstrated that both tRNA1 and tRNA-2 exhibit strong cytotoxicity with IC50 of 113.0 nM and 124.8 nM on HCT8 cells in a dose-dependent manner. Further clonogenic assay revealed that the purified tRNAs exhibit significant inhibition in colony formation with survival percentage of 79.0 ± 1.6 and 71.2 ± 2.2 at the concentration of 100 nM. These findings provided evidences of anticancer activities of tRNAs from non-pathogenic Escherichia coli strain,

indicating

that

the

pharmacological

effects

of

these

neglected

2

biomacromolecules from microorganisms should be emphasized. This study put new insights into the therapeutic effects of intestinal microorganism on human diseases, therefore broadened our knowledge of the biological functions of gut microbiota. Keywords: non-pathogenic Escherichia coli; tRNAs; two-dimensional liquid chromatography; purification; characterization; anticancer activities

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1. Introduction RNA therapeutics like siRNAs and miRNAs have been facilitated as a promising market due to their great therapeutic promise to regulate gene expression, which are non-toxic and highly effective [1]. Recently, small RNA fraction from corn has been reported with anticancer activity via inducing apoptosis in Hela cells, this finding raised the potential therapy of small non-coding RNAs [2]. As the most abundant class in small RNA, transfer RNAs (tRNAs) with functions of RNA splicing, RNA translation and DNA replication have been discovered as fundamental composition of the translational machinery in life entities [3]. For instances, mutations or chemical modifications in tRNA would alter the structures and expression levels of RNA [4, 5], which have been proven to be closely linked to physiological activities in animals and plants [6-9]. Furthermore, tRNA-derived RNA fragments have been discovered as potential biomarkers of multiple diseases, leading more studies on biological functions of natural tRNA [10-12]. Therefore, separation and purification of highly purified tRNA are crucial for further understanding their bioactive functions.

4

Mammals are initiated into the colonization of exogenous microorganisms after birth [13]. The human intestinal tract is colonized by gut microbiota that contains 100 trillion microorganisms [14]. Previous studies revealed that gut microbiota is related to multiple diseases like obesity, type 2 diabetes, inflammatory bowel, non-alcoholic fatty liver, colorectal cancer, etc. [15]. As one of the most distributed Gram-negative anaerobes in colon, non-pathogenic strain of Escherichia coli has been proven to benefit hosts by producing vitamin K2 [16], although its pathogenic strain was found to induce the development of colorectal cancer [17]. This promotes us to investigate the possible relationship between non-pathogenic E. coli and colorectal cancer. In this study, non-pathogenic E. coli MRE600 was selected for tRNA purification [18]. Nowadays, tRNA purification is manly depended on two commonly used methods. The first one is based on molecular biology techniques. As a matured technique for purification of biomacromolecules, affinity method has been employed for the capture of tRNA with optimized DNA probe with strong sequence-specificity and selectivity [19]. Although this approach is fast, simple and reproducible, its denaturing process would open the spatial structure of tRNA molecule which is important to the biological

5

function of tRNAs [20]. Chemical approach is another technique for tRNA purification. A specific aminoacyl-tRNA tagged by a hydrophobic group is separated from untagged RNA by reversed-phase chromatography followed by deacylation to produce free tRNA [21]. However, tRNA isoacceptors are difficult to be separated by using this method. Therefore, these two methods are not appropriate for our investigation. Chromatographic separation drives much development in life sciences. Lots of efforts have been made to separate tRNAs by using liquid chromatography. In the past decades, one-dimensional liquid chromatography (1D-LC) was the most frequently used approach to purify tRNA [22]. To simplify the preparative-scale purification of tRNA, Pearson et al further investigated a polychlorotrifluoroethylene resin in a reversed-phase chromatography to decrease the overlap of adjacent tRNA chromatographic peaks and yield many sharp peaks from crude tRNA of E. coli [23]. Holmes et al used high concentrations of ammonium sulfate at pH 4.5 to make E. coli tRNAs to bind to Sepharose 4B, and a gradient elution of ammonium sulfate from high to slow concentrations led the fractionation of tRNA-Leu into five isoacceptors [24]. With the development of chromatographic techniques, two-dimensional liquid

6

chromatography (2D-LC) is being gradually used to separate tRNA because 2D-LC based on different separation principles is more efficient than 1D-LC [25]. However, reported 2D-LC methods failed to separate tRNA isoacceptors, which resulted in insufficient purity of prepared tRNA [26, 27]. To our best knowledge, no study on 2DLC separation of individual tRNAs was reported. In the present study, a 2D-LC method integrating weak anion-exchange and ion-pair chromatographies was developed for the separation of individual tRNAs from E. coli MRE600, which was further identified and characterized by UHPLC-MS techniques. Furthermore, cytotoxic activities of the purified tRNAs on colorectal cancer cells were evaluated.

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2. Materials and Methods 2.1 Chemicals and reagents Escherichia coli MRE 600 total transfer ribonucleic acid as a source of total RNA for chromatographic purification was purchased from Roche (Basel, Switzerland). MicroRNA marker and low range ssRNA ladder as marker of urea-polyacrylamide gel electrophoresis analysis were purchased from New England BioLabs (Massachusetts, U.S.A.). Diethylpyrocarbonate (DEPC)-treated water, S1 nuclease, RNase T1 and polyacrylamide containing a ratio of Acrylamide/Bis (19:1, w/w) were purchased from Thermo (Massachusetts, U.S.A.). Guanidinium thiocyanate, triethylammonium acetate, hexafluoro-2-propanol and fluorouracil (5-FU) were purchased from Sigma (Missouri, U.S.A.). Deionized water was prepared by a Millipore Milli-Q Plus system (Millipore, U.S.A.). All the other reagents were of analytical grade.

2.2 High-performance liquid size-exclusive chromatographic (SEC) analysis of E. coli total tRNAs SEC was performed on an Agilent 1100 HPLC system (Agilent Technologies, Santa Clara, CA, USA) using a Bio-Sil SEC-125 column (7.8×300 mm i.d., 5 μm, Bio-

8

Rad, California, U.S.A.) maintained at 30°C, coupled with a diode array detector to record chromatogram under 260 nm. 1 μg of total tRNA in 10 μL mobile phase was directly injected and isocratic elution was carried out by 10 mM phosphate buffer (Na2HPO4 and KH2PO4, pH=7.4) and 130 mM NaCl at flow rate of 0.1 mL/min. 2.3 High-performance liquid weak-anion chromatographic (WAC) fractionation of E. coli total tRNAs

Fractionation was performed on an Agilent 1100 HPLC system using a TSKgel DNA-STAT column (4.6×100 i.d., 5 μm, Tosoh, Tokyo, Japan) maintained at 25°C, coupled with a diode array detector to record chromatogram under 260 nm. The flow rate was set as 0.2 mL/min. The column was equilibrated with 20 mM Tris buffer (pH=8.5). Gradient elution with (A) 20 mM Tris buffer (pH=8.5) and (B) 20 mM Tris buffer + 1 M NaCl (pH=8.5) was 0-120 min, 53%-63% B. 30 μg of total tRNA in 10 μL mobile phase was directly injected and repeated 5 times (100 μg of total tRNA for each), the same fractions were combined and freeze-dried using a Speed-Vac system RVC 2-18 (Marin Christ, Germany). Powders of RNA and inorganic salts were then dissolved with DEPC-treated water. Equal volume of 6 M guanidinium thiocyanate

9

solution was added to 3 M concentration, followed by adding ethanol to a final concentration of 55%. Then the mixed solutions were added to a silicon-membrane column (mirVana™ miRNA Isolation Kit, Invitrogen) for desalting.

2.4 High-performance liquid ion-pair chromatographic (IPC) purification All separations were performed on an Agilent 1100 HPLC system using a DNAPac RP column (3.0×100 i.d., 4 μm, Thermo) maintained at 40°C, coupled with a diode array detector to record chromatogram under 260 nm. The flow rate was set as 0.2 mL/min. The column was equilibrated with 100 mM triethylammonium acetate (pH=7.0). Gradient elution with (A) 100 mM triethylammonium acetate (pH=7.0) and (B) 25% acetonitrile in A was 0-5 min, 30%-37% B; 5-25 min, 37%-45% B; 25-35 min, 45%-100% B; 35-45 min, 100% B. Individual tRNA peak was collected and freezedried to remove organic solvents and essential salts. 2.5 Ribonuclease hydrolysis of purified tRNAs 2.5.1 RNase T1 hydrolysis For RNase T1 hydrolysis, 1 μg of purified individual tRNA dissolved in DEPCtreated water was mixed with RNase T1 (the final amount was 50 U, respectively) and

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ammonium acetate (220 mM in final), followed by digestion at 37°C for 1.5 h. Then the mixtures were incubated at 70°C for 10 min to stop the reaction. The digested products of RNase T1 were featured by a terminal 3' monophosphate guanosine that is commonly used for RNA characterization [28]. Finally, after centrifugation (10,000 xg for 1 min), the supernatants contained hydrolysates were collected for UHPLC-MS/MS analysis. 2.5.2 S1 nuclease hydrolysis For S1 nuclease hydrolysis, each 500 ng of purified individual tRNA dissolved in DEPC-treated water was mixed with S1 nuclease (the final amount of S1 nuclease was 8 U, respectively), 2 μL 5X reaction buffer, and Milli Q-Plus water to a final volume of 20 μL, followed by digestion at 25°C for 40 min. Then 0.5 μL of EDTA solutions (0.5 M) was added to the mixtures to stop the reaction. Finally, after centrifugation (10,000 xg for 1 min), the supernatants contained hydrolysates were collected for UHPLC-MS/MS analysis. 2.6 Urea-polyacrylamide gel electrophoresis (urea-PAGE) analysis

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Urea-PAGE was carried out as following procedures. Briefly, all samples were separated using a vertical slab gel electrophoresis apparatus, Mini-Protean Tetra System (Bio-Rad). 15% urea-polyacrylamide gel was applied for separation. Electrophoresis was performed at room temperature. Samples were electrophoresed at 250 V for 1 h. After electrophoresis, gels were stained with 1 X SYBR Gold nucleic acid gel stain (Thermo) in Milli Q-Plus water for 30 min, followed by imaging using a Bio-Rad imaging system under UV light. 2.7 UHPLC-MS/MS analysis UHPLC-MS/MS was performed on an Agilent UHPLC 1290 system (Agilent Technologies, Santa Clara, CA, USA), equipped with a vacuum degasser, a quaternary pump, an autosampler, a diode array detector and an Agilent ultrahigh definition 6545 Q-TOF mass spectrometer. Separation was carried out on an ACQUITY UPLC OST C18 Column (2.1×100 mm i.d., 1.7 μm, Waters, Massachusetts, U.S.A.) at 60°C. The flow rate was set at 0.2 mL/min and sample injection volume was 20 μL. Gradient elution with (A) 100 mM hexafluoro-2-propanol (HFIP)+15 mM trimethylamine (TEA) and (B) 50% MeOH in A was 0-1.5 min, 2% B, 1.5-8.3 min, 2%-28% B, 8.3-16.5 min,

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28%-34% B, followed by washing with 80% B and equilibration with 2% B. ESI conditions were as follows: gas temperature 320°C, spray voltage 3.5 kV, sheath gas flow and temperature were set as 12 L/min and 350°C, respectively. For MS experiment, samples were analyzed in negative mode over an m/z range of 500 to 3200. For MS/MS experiment, samples were analyzed in negative mode over an m/z range of 300 to 2000. The information of sequence and modification were obtained from the collisioninduced dissociation analysis of target oligonucleotides. 2.8 Sequence mapping of RNase T1 enzymatic digestions of purified RNAs RNAModMapper was used for automated MS/MS data analysis and sequence annotation.

The

program

and

user

manual

were

downloaded

from

http://bearcatms.uc.edu/. E. coli tRNA sequences with modifications were obtained from Modomics (http://modomics.genesilico.pl/). 2.9 Evaluation of anticancer activities of purified tRNAs from E. coli 2.9.1

Cell culture HCT-8 human ileocecal colorectal adenocarcinoma cells purchased from

American Type Culture Collection (ATCC) was cultured in RPMI 1640 medium

13

(Gibco,

New

Zealand)

containing

10%

fetal

bovine

serum

(FBS),

1%

penicillin/streptomycin (P/S) in a humidified 5% CO2 atmosphere at 37°C. All tested RNA samples were dissolved in nuclease free water and stored in -80°C before use. 5FU was dissolved in dimethyl sulfoxide (DMSO) as positive control. 2.9.2

MTT assay of purified tRNAs on HCT-8 cells Briefly, 5×103 cells in 100 μL RPMI 1640 medium (Thermo) were seeded in 96-

well plates to adhere for 20 h before treatment. Serial concentrations of RNA sample solutions with LipofectamineTM RNAiMAX Transfection Reagent in Opti-MEM medium (Thermo) were then added according to manufacturer’s instructions. Cells without any treatment were used as control, cells with only liposomes treatment were used to assay cytotoxicity of transfection reagents. After treated for 48 h, MTT [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thermo] solution (50 μL per well, 1 mg/mL solution) were added to each well and incubated for 4 h at 37°C. Subsequently, 200 μL DMSO were added and the optical densities of the resulting solutions were calorimetrically determined by A570 nm using a SpectraMax 190 microplate reader (Molecular Devices, Sunnyvale, CA, U.S.A). Dose-response curves

14

were obtained, and the IC50 values were calculated by GraphPad Prism 5.0 (GraphPad, U.S.A.). Each experiment was carried out for three times. IC50 results were expressed as means ± standard deviation. 2.9.3

Clonogenic assay The clonogenic assay was performed based on minor modification of the

published method [29]. Briefly, HCT-8 cells were plated at a density of 1000 cells/well with 1640 medium in 6-well plates. The medium was changed to appropriate solutions of purified tRNAs, 5-FU or blank Opti-MEM after 20 h when the cells adhered. After 48 h, all solutions were changed to normal culture medium and the cells were incubated for 14 days. After fixation for 20 min with a 4% paraformaldehyde fix solution (Beyotime, China), the cells were stained with a crystal violet staining solution (Beyotime, China) for 10 min. Finally, the plates were imaged by a camera and colonies with more than 50 individual cells were counted using ImageJ software. 2.10

Statistical and data analysis All experiments results were expressed as mean ± SD. Statistical significance was

analyzed using a two-tailed Student t test (GraphPad Prism 5.0). **P<0.01,

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***P<0.001, ****P<0.0001. Moreover, the digital plot image of urea-PAGE analysis were generated and analyzed by ImageJ software (NIH, Bethesda, U.S.A.). 3. Results and discussion 3.1 Development of two-dimensional liquid chromatographic method 3.1.1 HPLC-SEC As a class of small RNA, tRNAs have a narrow length range from 76 to 90 nucleotides [30], resulting in big challenge of their chromatographic separation. To date, WAC, IPC and SEC are widely used for qualitative analysis or preparation of oligonucleotides [31, 32]. In order to optimize appropriate chromatographic method, the separation ability of the three commonly used chromatographies for RNA separation were compared on a same instrument. SEC has the ability to separate RNAs of wide range of molecular weights, which could be used to evaluate the impurities of aggregates and degrades [33]. Thus, this method has been developed to separate free small-interfering RNA (siRNA) and liposome encapsulated siRNA for quality control [34]. We firstly examined the ability of SEC to fractionate total tRNA of E. coli (Figure

16

1A). Although low concentration phosphate buffer together with low elution rate were used, satisfactory separation of total tRNA was not achieved. 3.1.2 HPLC-IPC IPC offers good separation to fractionate total RNA into ribosomal RNA (28S/18S RNA) and small RNA species (total tRNA, 5S and 5.8S rRNA) in short time. Meanwhile, it has been applied to purify aimed RNA product by removing ribonuclease [35, 36]. The second method based on IPC was further examined. The results demonstrated that multiple RNA peaks were observed with good resolution under slow elution by using wide-pore hydrophobic poly(styrene-divinylbenzene) resin. 3.1.3 HPLC-WAC WAC is a powerful method to efficiently separate homogeneous RNA with overall difference in charge, especially for shorter oligonucleotides [37-40]. The third method, a non-porous polymethacrylate resin with trimethyl-amino group of WAC was found to generate more chromatographic peaks than both SEC and IPC (Figure 1B), as well as better resolution and narrower peak width were obtained. However, most of

17

chromatographic peaks were not baseline separated by the WAC methods, which is not suitable for direct purification of individual tRNA. Since none of single chromatography could directly purify tRNA, as well as WAC and IPC generate significantly higher resolution than SEC, we developed a new strategy of 2D-LC integrating WAC and IPC for the purification of individual tRNAs of E. coli, which performed better resolution than the reported methods. Our developed method takes only a few hours, which is significant faster than the methods reported in the literatures. Furthermore, the ion-pair reagents used in IPC is more compatible with ESI mass spectrometry, thus could be further applied to tRNA analysis by online LC-MS. 3.2 Purification of individual tRNA Since WAC enables good resolution for separation of total tRNA, it was selected as first dimension in our study. Considering that time-consuming is a big shortage for WAC application, we fastened the gradient elution to save half of the separation time (160 min to 80 min). 100 μg of E. coli total tRNA was directly injected into WAC for 5 times and six fractions were separately collected (Figure 2A). The results showed that the prepared RNA fractions with good quality have over 80% recovery rate (Table 1).

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Urea-PAGE analysis further indicated that tRNAs with different charge have been successfully fractionated by WAC (Figure 2B). Subsequently, pure tRNA-1 and tRNA2 with good quality were isolated from Fraction 2 and Fraction 3 with yield of 0.484% and 1.096% (Table 1), respectively by IPC purification. 3.3 Chemical characterization of purified tRNAs 3.3.1 Purity analysis of purified tRNAs UHPLC-MS technique was employed in this study to analyze the purity of purified tRNAs. The results showed that both tRNA-1 and tRNA-2 displayed a single peak in total ion chromatogram, and the deconvolution mass spectrum indicated that their molecular weight is 24681.31 and 28157.52 Da, respectively (Figure 3A, B). Meanwhile, their purity was confirmed by analysis of urea-polyacrylamide gel electrophoresis. As shown in Figure 3C, both tRNA-1 and tRNA-2 have a clear single gel band, each of which displayed a single peak in plot image. 3.3.2 Sequence information of purified tRNAs To verify their sequence information, RNase T1, an endoribonuclease specifically hydrolyzed the 3'-OH phosphodiester bond of a guanosine nucleotide, was applied to

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degrade tRNA-1 and tRNA-2. MS/MS data from the UHPLC-MS analysis of the digested products were exported and mapped by RNAModMapper software [41] based on c-, y-, w-, a-B types ions (Figure 4A, B). The results indicated that identified RNA fragments (Figure 5A, B) of tRNA-1 and tRNA-2 were in accordance to RNase T1 signature digestion products (Table 2) of tRNA-Val(UAC) and tRNA-Leu(CAG) [42], and their molecular weights are exactly the same with the theoretical values [43]. Moreover, the sequence coverages of target RNAs are both above 70%. Thus, tRNA-1 and tRNA-2 were determined as tRNA-Val(UAC) and tRNA-Leu(CAG) of E. coli. 3.3.3 Elucidation of secondary structure of purified tRNAs For tRNA, not only nucleotide mutations and chemical modifications, but also cloverleaf secondary folding and L-shaped tertiary architectures could influence their biological functions [20], such as recognition by aminoacyl-tRNA synthetases [44] or post-transcriptional modification enzymes [45]. Therefore, specific cloverleaf-like secondary structure of tRNAs is important to their downstream studies. Unfortunately, secondary structure of purified tRNA in some previous reports were misfolded due to the complicated separation procedures or long-time denaturing [46, 47]. Here, S1

20

nuclease was used to degrade purified tRNAs for obtaining tRNAs half fragments, which is a specific method for elucidation of cloverleaf-like secondary structure of tRNA [48]. For tRNA-1, its digested products displayed two peaks whose deconvoluted mass are 10698.49 Da and 12656.82 Da, which is in accordance with the molecular weights of 5' and 3' tRNA-half of tRNA-Val(UAC) (Figure 6A). S1 digestion of tRNA2 provided two products whose deconvoluted mass are 11784.28 Da and 16391.01 Da, which is in accordance to the molecular weights of 5' and 3' tRNA-half of tRNALeu(CAG) (Figure 6B). Thus, the individual tRNAs purified by our method hold a cloverleaf-like secondary structure, which could satisfy the requirement of their downstream investigations. 3.4 Biological assay 3.4.1 MTT assay MTT method was employed to evaluate the cytotoxicity of tRNA-1 and tRNA-2 from E. coli toward cancer cells. With liposomal transfection, almost 50% population of HCT-8 cells dead at the concentration of 100 nM of both tRNAs. The results showed that tRNA-1 and tRNA-2 exhibited strong cytotoxicity on HCT-8 cells in a dose-

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dependent manner. Their IC50 are 113.0 nM and 124.8 nM, which are almost 400 times less than that of the positive control, 5-FU, with an IC50 of 41.4 μM (Figure 7A). Meanwhile, no significant cytotoxicity of the negative control double-stranded RNA (Forward: 5'-UUCUCCGAACGUGUCACGUTT-3', Reverse: 5'-ACGUGACACGUU CGGAGAATT-3') was observed by dose-dependent investigation (data not shown), suggesting that the cytotoxicity of tRNA-1 and tRNA-2 has sequence-specificity. 3.4.2 Clonogenic assay To further evaluate the cytotoxic effectiveness of two tRNAs on colorectal cancer, clonogenic assay was carried out on HCT-8 cells. The results indicated that tRNA-1 and tRNA-2 markedly reduced the clonogenic ability of HCT-8 cells with survival percentage of 79.0 ± 1.6 and 71.2 ± 2.2 at the concentration of 100 nM (Figure 7B). While the survival percentage of positive control 5-FU at a much higher concentration of 50 μM is 53.8 ± 4.7 in colony formation. Together with the MTT results, we found that tRNAs from non-pathogenic E. coli exhibited cytotoxicity activities against cancer cells. The present study is the first report that tRNA is pharmacologically active. Moreover, the present study provided sequence information for further development of

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nucleic acid therapeutics for colorectal cancer treatment. Further purification, in vivo antitumor activities and chemical modifications characterization of different tRNA species from E. coli MRE600 are meaningful to investigate their structure-activity relationships. 4. Conclusion For the first time, a 2D-LC method integrating WAC and IPC was successfully applied to purify two tRNAs from non-pathogenic E. coli MRE600 strain in this study. This new methodology is universal, simple and fast for purifying tRNAs from biological sources without need of information of sequence, which is helpful for their biochemical and structural studies. Bioassay showed that both purified tRNAs, i.e. tRNA-1 and tRNA-2, exhibit stronger cytotoxic activities than 5-FU, a clinical drug for the treatment of colorectal cancer, on HCT-8 human ileocecal colorectal adenocarcinoma cells. These findings firstly provided evidences of anticancer activities of individual tRNAs from non-pathogenic Escherichia coli strain, indicating that the pharmacological effects of these neglected biomacromolecules from microorganisms should be emphasized. This study put new insights into the therapeutic effects of

23

intestinal microorganisms on human diseases, therefore broadened our knowledge of the biological functions of gut microbiota.

Declaration of Competing Interest The authors declare that there are no conflicts of interest.

Acknowledgements This work was financially funded by The Science and Technology Development Fund, Macau SAR (File no. 015/2017/AFJ).

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Figure Legends

Figure 1. Comparison of three chromatographies for tRNA separation. (A) Typical chromatograms of SEC, IPC and WAC under UV 260 nm. (B) Total numbers of chromatographic peaks using different chromatography.

Figure 2. Fractionation of total tRNA using WAC. (A) Preparative WAC chromatogram of total tRNA under UV 260 nm. (B) Denaturing urea-polyacrylamide gel electrophoresis of WAC prepared fractions. T, total tRNA; 1-6, Fraction 1 to Fraction 6.

Figure 3. Purification and identification of individual tRNA-1 from Fr. 2 (A) and tRNA-2 from Fr. 3 (B) of WAC prepared fractions. (C) Urea-polyacrylamide gel electrophoresis analysis and plot image of prepared tRNAs analyzed by ImageJ software.

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Figure 4. Detailed MS/MS spectra of RNase T1 signature digestion of tRNA-1 (A) and tRNA-2 (B).

Figure 5. Annotation of RNase T1 digestions and sequence mapping of purified tRNA1 (A) and tRNA-2 (B).

Figure 6. Elucidation of cloverleaf-like secondary structure by nuclease S1 hydrolysis. (A) Typical structure of tRNA-1, TIC chromatograms and deconvolution MS results of its 5' and 3' tRNA half. (B) Typical structure of tRNA-2, TIC chromatograms and deconvolution MS results of its 5' and 3' tRNA half.

Figure 7. MTT assay (A) and clonogenic assay (B) of purified tRNAs from E. coli and 5-FU on HCT-8 cells.

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Table Legends

Table 1. Quality and yield of prepared tRNA fractions and purified tRNAs.

Table 2. MS/MS data of RNase T1 signature digestion products of purified tRNAs.

36

Figure 1.

37

Figure 2.

38

Figure 3.

39

Figure 4.

40

41

Figure 4 (continued).

42

Figure 5.

43

Figure 6.

44

Figure 7.

45

Table 1. RNA

Concentration (ng/μL)

A260/A280

A260/A230

Weight (μg)

Yield (%)

Fraction 1

1725.8

1.96

1.86

86.29

17.258

Fraction 2

951.1

2.03

1.72

47.55

9.510

Fraction 3

958.4

2.01

1.78

47.92

9.584

Fraction 4

543.0

2.02

1.46

27.15

5.430

Fraction 5

779.5

1.97

1.39

38.98

7.796

Fraction 6

3330.1

1.99

1.98

166.51

33.302

tRNA-1

121.2

1.99

1.89

2.42

0.484

tRNA-2

274.1

2.01

1.82

5.48

1.096

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Table 2. tRNA

Signature T1 digestion sequence

Calculated massa (Da)

Deconvoluted mass (Da)

Differenceb (Da)

m/z

Measured mass (Da)

AU[s4U]AGp

1649.189

1649.189

0

[M-2H]2-

823.587

CUCAGp

1608.216

1608.216

0

[M-H]-

1607.206

CDGp

976.138

976.139

0.001

[M-H]-

975.132

CACCUCCCU[cmo5U]AC[m6A]AGp

4820.644

4820.652

0.008

[M-3H]3-

1605.876

[m7G]UCGp

1333.186

1333.185

-0.001

[M-H]-

1332.177

TΨCGp

1294.163

1294.164

0.001

[M-H]-

1293.155

AUCCCGp

1913.257

1913.262

0.005

[M-2H]2-

955.623

UCAUCACCCACCA

4001.590

4001.593

0.003

[M-3H]3-

1333.191

AAGp

1021.161

1021.164

0.003

[M-H]-

1020.156

AADD[Gm]Gp

1996.305

1996.315

0.01

[M-2H]2-

997.150

DAGp

1000.150

1000.154

0.004

[M-H]-

999.146

ACGp

997.150

997.154

0.004

[M-H]-

996.149

CUAGp

1303.175

1303.182

0.007

[M-H]-

1302.171

CUUCAGp

1914.241

1914.251

0.010

[M-2H]2-

956.118

ΨUAGp

1304.159

1304.161

0.002

[M-H]-

1303.154

tRNA-1

tRNA-2

47

UCCUUACGp

2525.307

2525.320

0.013

[M-2H]2-

1262.159

TΨCAAGp

1952.268

1952.278

0.01

[M-2H]2-

975.131

UCCCCCCCCUCGp

3720.476

3720.492

0.016

[M-3H]3-

1239.492

CACCA

1511.271

1511.279

0.008

[M-2H]2-

754.633

a, theoretical monoisotopic mass b, Difference=(Deconvoluted mass)-(Calculated mass)

48

Highlights 1. A two-dimensional liquid chromatographic method integrating weak-anion exchange chromatography and ion-pair chromatography was firstly developed for purification of individual tRNAs from non-pathogenic E. coli MRE600. 2. Two pure tRNAs were obtained from E. coli MRE600. 3. Purified tRNAs were characterized as tRNA-Val(UAC) and tRNA-Leu(CAG) of E. coli MRE600. 4. Purified tRNAs showed potent cytotoxicity activities toward colorectal cancer cells.

49