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Nuclease p1

Manufactured by Merck Group
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Nuclease P1 is a lab equipment product manufactured by Merck Group. It is an enzyme that catalyzes the hydrolytic cleavage of single-stranded and double-stranded DNA and RNA into 5'-mononucleotides. The core function of Nuclease P1 is to facilitate the breakdown of nucleic acids in a controlled and precise manner for various research and analytical applications.

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Lab products found in correlation

Alkaline phosphatase, by Merck Group (64 mentions) Phosphodiesterase 1, by Merck Group (14 mentions) Antarctic phosphatase, by New England Biolabs (13 mentions) Pei cellulose plate, by Merck Group (12 mentions) Molecular dynamics storm 860 phosphorimager, by Cytiva (10 mentions) Dnase 1, by New England Biolabs (9 mentions) Trizol, by Thermo Fisher Scientific (9 mentions) Dynabeads mrna purification kit, by Thermo Fisher Scientific (8 mentions) Calf intestinal phosphatase, by New England Biolabs (8 mentions) Rnase a, by Merck Group (7 mentions) Alkaline phosphatase, by New England Biolabs (7 mentions) Trizol reagent, by Thermo Fisher Scientific (7 mentions) Pyrophosphatase, by Roche (6 mentions) 6410 qqq triple quadrupole lc mass spectrometer, by Agilent Technologies (6 mentions) Fastap thermosensitive alkaline phosphatase, by Thermo Fisher Scientific (6 mentions) Rnase t1, by Merck Group (6 mentions) Phosphodiesterase 1, by Worthington (6 mentions) γ 32p atp, by PerkinElmer (6 mentions) Alkaline phosphatase, by Thermo Fisher Scientific (5 mentions) 8 oxo detection elisa kit, by Cayman Chemical (5 mentions)

264 protocols using nuclease p1

1

Quantitative Aminoacylation Assay for tRNA

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A 20 μl aminoacylation reaction contained the following components: 50 mM HEPES-KOH (pH 7.2), 25 mM KCl, 10 mM MgCl2, 5 mM DTT, 10 mM ATP, 25 μg/ml pyrophosphatase (Roche Applied Science). All tRNA aminoacylation levels were determined at 37°C with according to the reactions conditions descried above with 100 nM tRNA synthetases, 10 nM 32P-labeled tRNA. Time points were taken at 5 min, 10 min and 20 min by removing 2 μl aliquots from the reaction and immediately quenching the reaction into an ice-cold 3 μl quench solution (0.66 μg/μl nuclease P1 (Sigma) in 100 mM sodium citrate (pH 5.0)). For each reaction, 2 μl of blank reaction mixture (containing no enzyme) was added to the quench solution as the start time point. The nuclease P1 mixture was then incubated a room temperature for 30 min, and 1 μl aliquots were spotted on PEI-cellulose plates (Merck) and developed in running buffer containing 5% acetic acid and 100 mM ammonium acetate. Radioactive spots for AMP and AA-AMP (representing free tRNA and aminoacyl-tRNA, respectively) were separated and then visualized and quantified by phosphorimaging by a Molecular Dynamics Storm 860 phosphorimager (Amersham Biosciences). The ratio of aminoacylated tRNA to total tRNA was determined to monitor reaction progress.
Fan C., Xiong H., Reynolds N.M, & Söll D. (2015). Rationally evolving tRNAPyl for efficient incorporation of noncanonical amino acids. Nucleic Acids Research, 43(22), e156.
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2

Quantifying tRNA Aminoacylation Kinetics

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The assay was modified from the original method [13 (link)]. A 20 µl reaction contained the following components: 50 mM HEPES-KOH (pH 7.2), 25 mM KCl, 10 mM MgCl2, 5 mM DTT, 10 mM ATP, 25 µg/ml pyrophosphatase (Roche Applied Science), 1 µM tRNA. All tRNA aminoacylation levels were determined at 37°C with synthetase, 10 nM 32P-labeled tRNA. Time points were taken at 5 min, 10 min and 30 min by removing 2 µl aliquots from the reaction and immediately quenching the reaction into an ice-cold 3 µl quench solution (0.66 µg/µl nuclease P1 (Sigma) in 100 mM sodium citrate (pH 5.0)). For each reaction, 2 µl of blank reaction mixture (containing no enzyme) was added to the quench solution as the start time point. The nuclease P1 mixture was then incubated a room temperature for 30 min and 1 µl aliquots were spotted on PEI-cellulose plates (Merck) and developed in running buffer containing 5% acetic acid and 100 mM ammonium acetate. Radioactive spots of AMP and AA-AMP (representing free tRNA and aminoacyl-tRNA, respectively) were separated and then visualized and quantified by phosphorimaging in a Molecular Dynamics Storm 860 phosphorimager (Amersham Biosciences). The ratio of amino-acyl-tRNA to total tRNA was determined to monitor reaction progress.
Fan C., Ip K, & Söll D. (2016). Expanding the Genetic Code of Escherichia coli with Phosphotyrosine. FEBS letters, 590(17), 3040-3047.
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3

Quantitative Aminoacylation Assay

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A 20 μl aminoacylation reaction contained the following components: 50 mM HEPES-KOH (pH 7.2), 25 mM KCl, 10 mM MgCl2, 5 mM DTT, 10 mM ATP, 25 μg/ml pyrophosphatase (Roche Applied Science), 2 mM amino acids. All plateau tRNA aminoacylation levels were determined at 37 °C according to the reaction conditions described above with 500 nM enzyme, 5 μM unlabeled tRNA plus 100 nM 32P-labeled tRNA. Time points were taken at 5 min, 20 min and 60 min by removing 2 μl aliquots from the reaction and immediately quenching the reaction into an ice-cold 3 μl quench solution (0.66 μg/μl nuclease P1 (Sigma) in 100 mM sodium citrate (pH 5.0)). For each reaction, 2 μl of blank reaction mixture containing no enzymes was added to the quench solution as the start time point. The nuclease P1 mixture was then incubated at room temperature for 30 min and 1 μl aliquots were spotted on PEI-cellulose plates (Merck) and developed in running buffer containing 5% acetic acid and 100 mM ammonium acetate. Radioactive spots for AMP and AA-AMP (representing free tRNA and aminoacyl-tRNA, respectively) were separated and then visualized and quantified by phosphorimaging by a Molecular Dynamics Storm 860 phosphorimager (Amersham Biosciences). The ratio of aminoacylated tRNA to total tRNA was determined to monitor reaction progress.
Amiram M., Haimovich A.D., Fan C., Wang Y.S., Aerni H.R., Ntai I., Moonan D.W., Ma N.J., Rovner A.J., Hong S.H., Kelleher N.L., Goodman A.L., Jewett M.C., Söll D., Rinehart J, & Isaacs F.J. (2015). Evolution of translation machinery in recoded bacteria enables multi-site incorporation of nonstandard amino acids. Nature biotechnology, 33(12), 1272-1279.
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4

Aminoacylation of Pyrrolysyl-tRNA Variants

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The aminoacylation of tRNAPyl variants was carried out at 37°C in the buffer containing 50 mM HEPES-KOH (pH 7.2), 25 mM KCl, 10 mM MgCl2, 5 mM DTT, 10 mM ATP, 10 mM amino acids, 100 nM PylRS variants, 24 μM unlabeled tRNAPyl, and 3.6 μM 32P-labeled tRNAPyl with a total volume of 25 μL. Various concentrations of BocK (0.1–12.8 mM), Pyl44 (link) (5–500 μM for chPylRS and 0.1–10mM for variant 32A), and tRNA (0.5–16 μM) were used to determine KM values for corresponding substrates. A 2 μL aliquot was taken out from each of the reaction mixtures at the time points of 5 min, 20 min and 30 min, and the reactions were immediately quenched by adding 3 μL quenching solution [0.66 μg/μL nuclease P1 (Sigma) in 100 mM sodium citrate (pH 5.0)]. The nuclease P1 mixtures were then incubated at room temperature for 30 min and 1 μL aliquots were spotted on PEI-cellulose plates (Merck) and developed in the running buffer containing 5% acetic acid and 100 mM ammonium acetate. Radioactive spots for AMP and AA-AMP (representing free tRNA and aminoacyl-tRNA, respectively) were separated and visualized and quantified by phosphorimaging using a Molecular Dynamics Storm 860 phosphorimager (Amersham Biosciences). The ratio of aminoacylated tRNA to total tRNA was determined to monitor reaction progress.
Suzuki T., Miller C., Guo L.T., Ho J.M., Bryson D.I., Wang Y.S., Liu D.R, & Söll D. (2017). Crystal structures reveal an elusive functional domain of pyrrolysyl-tRNA synthetase. Nature chemical biology, 13(12), 1261-1266.
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5

Aminoacylation of Pyrrolysyl-tRNA Variants

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The aminoacylation of tRNAPyl variants was carried out at 37°C in the buffer containing 50 mM HEPES-KOH (pH 7.2), 25 mM KCl, 10 mM MgCl2, 5 mM DTT, 10 mM ATP, 10 mM amino acids, 100 nM PylRS variants, 24 μM unlabeled tRNAPyl, and 3.6 μM 32P-labeled tRNAPyl with a total volume of 25 μL. Various concentrations of BocK (0.1–12.8 mM), Pyl44 (link) (5–500 μM for chPylRS and 0.1–10mM for variant 32A), and tRNA (0.5–16 μM) were used to determine KM values for corresponding substrates. A 2 μL aliquot was taken out from each of the reaction mixtures at the time points of 5 min, 20 min and 30 min, and the reactions were immediately quenched by adding 3 μL quenching solution [0.66 μg/μL nuclease P1 (Sigma) in 100 mM sodium citrate (pH 5.0)]. The nuclease P1 mixtures were then incubated at room temperature for 30 min and 1 μL aliquots were spotted on PEI-cellulose plates (Merck) and developed in the running buffer containing 5% acetic acid and 100 mM ammonium acetate. Radioactive spots for AMP and AA-AMP (representing free tRNA and aminoacyl-tRNA, respectively) were separated and visualized and quantified by phosphorimaging using a Molecular Dynamics Storm 860 phosphorimager (Amersham Biosciences). The ratio of aminoacylated tRNA to total tRNA was determined to monitor reaction progress.
Suzuki T., Miller C., Guo L.T., Ho J.M., Bryson D.I., Wang Y.S., Liu D.R, & Söll D. (2017). Crystal structures reveal an elusive functional domain of pyrrolysyl-tRNA synthetase. Nature chemical biology, 13(12), 1261-1266.
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6

Decapping and deNADding Assay Protocol

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32P-NAD-cap labeled or 32P-m7G-capped RNAs were incubated with the indicated amount of recombinant proteins in decapping buffer containing 10 mM Tris-HCl pH 7.5, 100 mM KCl, 2 mM DTT, 2 mM MgCl2 and 2 mM MnCl2 as previously described39 (link) and incubated at 37 °C for 30 min. Reactions were stopped with 30 mM EDTA. For reactions involving Nuclease P1 treatment, reactions were first extracted with phenol followed by chloroform and 1U of Nuclease P1 (Sigma-Aldrich) was added. The reactions were incubate at 37 °C for 30 min. Decapping products were resolved by PEI-cellulose TLC plates (Sigma-Aldrich) and developed in 0.5 M LiCl or 0.45 M (NH4)2SO4 in a TLC chamber at room temperature40 (link). deNADding assays in Fig. 4S were similarly carried out except, following termination of the reaction with 30mM EDTA, the RNA were resolved by 15% denaturing polyacrylamide gel electrophoresis and dried. Reaction products were visualized with a Molecular Dynamics PhosphorImager (Storm860).
Grudzien-Nogalska E., Wu Y., Jiao X., Cui H., Mateyak M.K., Hart R.P., Tong L, & Kiledjian M. (2019). Structural and mechanistic basis of the mammalian Nudt12 RNA deNADding. Nature chemical biology, 15(6), 575-582.
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7

Quantification of Cellular NAD Levels

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Yeast strains were grown in YPD media at 30 °C according to standard protocols. All strains were harvested for the experiments at exponential phase (OD600 ~1) and total RNA was isolated using the acidic hot phenol method48 (link). To remove residual free NAD, purified RNAs were dissolved in 10 mM Tris-HCl (pH 7.5) containing 2 M urea. Samples were incubated 2 min at 65 °C and immediately precipitated with isopropanol in the presence of 2 M ammonium acetate. NAD-capQ was carried out as previously described21 (link). Briefly, 50 μg of total RNA was digested with 2 U of Nuclease P1 (Sigma-Aldrich) in 20 μL of 10 mM Tris (pH 7.0), 20 μM ZnCl2 at 37 °C for 1 h to release 5′-end NAD. The control samples lacking Nuclease P1 were prepared by incubating 50 μg of RNA treated with the same reaction condition. The NADH standard curve was generated for each experiment in the same buffer condition as above for assays containing Nuclease P1. Following digestion with Nuclease P1, 30 μL of NAD/NADH Extraction Buffer (NAD/NADH Quantitation Kit, Sigma-Aldrich) was added to each sample. In the second step, 50 μL samples were used in a colorimetric assay according to the manufacturer’s protocol (NAD/NADH Quantitation Kit, Sigma-Aldrich) as described21 (link).
Sharma S., Yang J., Grudzien-Nogalska E., Shivas J., Kwan K.Y, & Kiledjian M. (2022). Xrn1 is a deNADding enzyme modulating mitochondrial NAD-capped RNA. Nature Communications, 13, 889.
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8

Enzymatic Digestion for HPLC Nucleoside Analysis

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Prior to HPLC analysis, 20 pmol of each mRNA sample were digested to the nucleosides level according to the following protocol (56 (link)): samples were incubated in presence of 1/10 volume of 10× nuclease P1 buffer (0.2 M NH4OAc pH 5.0, ZnCl2 0.2 mM), 0.3 U nuclease P1 (Sigma-Aldrich, Munich, Germany), and 0.1 U snake venom phosphodiesterase (Worthington, Lakewood, CO, USA) at 37°C for 2 h. Next, 1/10 volume of 10× fast alkaline phosphatase buffer (Fermentas, St. Leon-Roth, Germany) and 1 U fast alkaline phosphatase (Fermentas, St. Leon-Roth, Germany) were added, and samples were incubated for additional 60 min at 37°C. For the calibration series of EU, commercially available EU triphosphate was digested analogously.
Hellmuth I., Freund I., Schlöder J., Seidu-Larry S., Thüring K., Slama K., Langhanki J., Kaloyanova S., Eigenbrod T., Krumb M., Röhm S., Peneva K., Opatz T., Jonuleit H., Dalpke A.H, & Helm M. (2017). Bioconjugation of Small Molecules to RNA Impedes Its Recognition by Toll-Like Receptor 7. Frontiers in Immunology, 8, 312.
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9

Quantification of NAD and NADH in HEK293T Cells

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HEK293T cells (6 × 106) were seeded in 100 mm plates a day before the experiment and cells were collected for RNA extraction at ∼80% confluency. NAD-capQ was carried out as previously described (15 (link)) (Figure 2A). Briefly, 50 μg of total RNA was digested with 2 U of Nuclease P1 (Sigma-Aldrich) in 20 μl of 10 mM Tris (pH 7.0), 20 μM ZnCl2 at 37°C for 1 h to release 5′ end NAD. The control samples lacking Nuclease P1 were prepared by incubating 50 μg of RNA treated with the same reaction condition and supplemented with 5% glycerol lacking the enzyme. The NADH standard curve was generated for each experiment in the same buffer condition as above for assays containing Nuclease P1. Following digestion with Nuclease P1, 30 μl of NAD/NADH Extraction Buffer (NAD/NADH Quantitation Kit, Sigma-Aldrich) was added to each sample. Fifty microliters of each sample was used in a colorimetric assay according to the manufacturer's protocol (NAD/NADH Quantitation Kit, Sigma-Aldrich) as described (15 (link)). Values were corrected for background absorbance and concentrations of NAD and NADH were derived from the standard curves.
Sharma S., Grudzien-Nogalska E., Hamilton K., Jiao X., Yang J., Tong L, & Kiledjian M. (2020). Mammalian Nudix proteins cleave nucleotide metabolite caps on RNAs. Nucleic Acids Research, 48(12), 6788-6798.
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10

RNA Nucleoside Digestion for LC-MS/MS Analysis

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Prior to LC-MS/MS analysis, RNA samples were digested into nucleosides according to the following protocol: samples were incubated in presence of 1/10 volume of 10× nuclease P1 buffer (0.2 M ammonium acetate pH 5.0, ZnCl2 0.2 mM), 0.3 U nuclease P1 (Sigma Aldrich, Munich, Germany) and 0.1 U snake venom phosphodiesterase (Worthington, Lakewood, USA) at 37°C for 2 h. Next, 1/10 volume of 10× fast alkaline phosphatase buffer (Fermentas, St Leon-Roth, Germany) and 1 U fast alkaline phosphatase (Fermentas, St Leon-Roth, Germany) were added, and samples were incubated for additional 60 min at 37°C. After digestion, 1/10 volume of 13C-labeled total RNA (S. cerevisiae, 10 ng/μl), prepared as described in (23 ), was added as internal standard for m1A quantification.
Hauenschild R., Tserovski L., Schmid K., Thüring K., Winz M.L., Sharma S., Entian K.D., Wacheul L., Lafontaine D.L., Anderson J., Alfonzo J., Hildebrandt A., Jäschke A., Motorin Y, & Helm M. (2015). The reverse transcription signature of N-1-methyladenosine in RNA-Seq is sequence dependent. Nucleic Acids Research, 43(20), 9950-9964.
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