Pcp cy3

Manufactured by Jena Biosciences

Sourced in Germany

PCp-Cy3 is a fluorescently labeled nucleotide analog for incorporation into DNA or RNA. It can be used for various applications, such as labeling and detection of nucleic acids.

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8 protocols using pcp cy3

Fluorescent RNA Structure Probing

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In vitro transcribed RNA of ppiC was 3’ end-labeled with 10 μM pCp-Cy3 (Jena Bioscience) using 15 U T4 Ligase 1 (NEB). 2 μg of fluorescently-labeled RNA was structure probed with 0.05 U of RNase V1 (Ambion) or with a dilution 1:7000 of combined RNase A/T1 (Thermo Scientific), in conditions identical to the PARS experiment. The digestion was stopped with phenol chlorophorm extraction, precipitated overnight at 4°C and resuspended in 10 μl of 2x RNA Loading Dye (Thermo Scientific). In parallel, a ddNTP-Sanger sequencing PCR reaction was performed using 20 pmol of a 3’-fluorescently(Cy3)-labeled primer, in the presence of 400 ng of DNA template, 10 μM dNTPs, 1.25 U Pfu DNA Polymerase (Thermo Scientific), Pfu Polymerase Buffer and 1 mM of each ddNTP. PCR was performed according to the manufacturer instructions in a volume of 15 μl. After addition of 2x RNA Loading Dye, all samples were boiled for 3 min at 95°C and loaded on a 6% PA, 1x TBE, 7M UREA gel (50x40 cm), already pre-run for 30 min at 50W. The gel was then run for 3 h at 50W and the fluorescence was detected using a fluorescent gel imager.

Del Campo C., Bartholomäus A., Fedyunin I, & Ignatova Z. (2015). Secondary Structure across the Bacterial Transcriptome Reveals Versatile Roles in mRNA Regulation and Function. PLoS Genetics, 11(10), e1005613.

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RNA Oligonucleotide Labeling and Purification

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RNA oligonucleotides (Supplementary Table S1) were chemically synthesized (Biomers) and 3′ labeled with T4 RNA ligase (Thermo Fisher) and pCp-Cy3 (Jena Bioscience). Labeled RNA substrates were subsequently separated from excess pCp-Cy3 by ethanol precipitation. RNA was mixed in 1:1 ratio in 100 mM HEPES(NaOH) pH 7.0, 100 mM NaCl, 50 mM KCl, 5 mM MgCl₂, 0.5 U/μl RNasin (Promega), 2 mM dithiothreitol, and 0.1 μg/μl bovine serum albumin and incubated at 30°C for 30 min before an equal volume of RNA loading buffer (98% formamide, 18 mM EDTA, 0.025 mM SDS) was added. The samples were heated at 95°C for 3 min before separation on denaturing 7 M urea, 20% polyacrylamide Tris–borate–EDTA gels and 0.5-fold Tris–borate buffer.

Vogel D., Thorkelsson S.R., Quemin E.R., Meier K., Kouba T., Gogrefe N., Busch C., Reindl S., Günther S., Cusack S., Grünewald K, & Rosenthal M. (2020). Structural and functional characterization of the severe fever with thrombocytopenia syndrome virus L protein. Nucleic Acids Research, 48(10), 5749-5765.

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Probing p66 RT-tRNA Interaction by SEC

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All Size Exclusion Chromatography (SEC) experiments used a 24-ml analytical Superdex 200 Increase 10/300 GL column (GE Healthcare), mobile phase of 25 mM Bis-tris buffer, pH 7.0, containing 100 mM NaCl with 0.02% sodium azide at a flow rate of 0.5 ml/min. Injection volume was 50 μl, and protein elution was monitored by UV absorbance at 254 and 280 nm. Elution profiles of 40 μM p66 RT were evaluated in the absence of tRNA, or following preincubation with 5 μM tRNA containing tracer tRNA 3′-end labeled with pCp-Cy3 (Jena Bioscience, Jena, Germany). As a control, the labeled tRNA was injected without mixing with p66. With the SEC experiments that contain labeled tRNA, in addition to UV, the fluorescence emission at 560 nm (excitation 485 at nm) was also measured using an in-line Shimadzu RF-10AXL Fluorescence Detector.

Ilina T.V., Slack R.L., Elder J.H., Sarafianos S.G., Parniak M.A, & Ishima R. (2018). Effect of tRNA on the maturation of HIV-reverse transcriptase. Journal of molecular biology, 430(13), 1891-1900.

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Reconstitution of U1snRNP complex

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Cytoplasmic extracts from the differentiated neurons were prepared using NE-PER nuclear and cytoplasmic extraction kit (Thermo Scientific 78835) and the protein concentration were measured by Pierce^TM BCA protein assay kit. U1snRNA were transcribed from gel-eluted and linearized DNA template by in vitro transcription using T7 RNA polymerase and m7G cap analog. pCp-Cy3 (Cytidine-5′-phosphate-3′-(6-aminohexyl) phosphate) (Jena Bioscience) was transferred to the 3′-hydroxyl group on U1snRNA by T4 RNA ligase (Thermo Fisher). The snRNP assembly reaction was carried out by incubating 5 µg of pCp-Cy3-labeled U1snRNAs with 50 µg of cytoplasmic extract, 10 µM tRNA, and 2.5 mM ATP at 30 °C for one and half hours^{9 (link),45 (link)}. The reaction mix were loaded onto native 6% TBE polyacrylamide gel (Novex/Life Technologies). The gel was run at 150 V at 4 °C and was imaged using LI-COR imager.

Kour S., Rajan D.S., Fortuna T.R., Anderson E.N., Ward C., Lee Y., Lee S., Shin Y.B., Chae J.H., Choi M., Siquier K., Cantagrel V., Amiel J., Stolerman E.S., Barnett S.S., Cousin M.A., Castro D., McDonald K., Kirmse B., Nemeth A.H., Rajasundaram D., Innes A.M., Lynch D., Frosk P., Collins A., Gibbons M., Yang M., Desguerre I., Boddaert N., Gitiaux C., Rydning S.L., Selmer K.K., Urreizti R., Garcia-Oguiza A., Osorio A.N., Verdura E., Pujol A., McCurry H.R., Landers J.E., Agnihotri S., Andriescu E.C., Moody S.B., Phornphutkul C., Sacoto M.J., Begtrup A., Houlden H., Kirschner J., Schorling D., Rudnik-Schöneborn S., Strom T.M., Leiz S., Juliette K., Richardson R., Yang Y., Zhang Y., Wang M., Wang J., Wang X., Platzer K., Donkervoort S., Bönnemann C.G., Wagner M., Issa M.Y., Elbendary H.M., Stanley V., Maroofian R., Gleeson J.G., Zaki M.S., Senderek J, & Pandey U.B. (2021). Loss of function mutations in GEMIN5 cause a neurodevelopmental disorder. Nature Communications, 12, 2558.

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Size Exclusion Chromatography of p66 and tRNA

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Size Exclusion Chromatography (SEC) experiments were performed using
a 24-ml analytical Superdex 200 Increase 10/300 GL column (GE Healthcare),
at room temperature at a flow rate of 0.5 mL/min. Either p66 protein at 20
– 46 μM, with 0 – 25 μM tRNA^Lys3 or
the tRNA^Lys3 alone that contains tracer tRNA 3′-end
labeled with pCp-Cy3 (Jena Bioscience, Jena, Germany) was prepared in 25 mM
Bis-tris buffer, pH 7.0, containing 100 mM NaCl, 1% DMSO, and 0.02% sodium
azide. RPV was added at [p66:p66]:[RPV] = 1: 1.3 or 1.5 ratio. Each
injection volume was 50 μL. Elution profiles were monitored by UV
absorbance at 254 and 280 nm and, for the samples with tRNA^Lys3,
additionally by in-line Shimadzu RF-10AXL Fluorescence Detector with
fluorescence excitation at 485 nm and the emission at 560 nm.

Slack R.L., Ilina T.V., Xi Z., Giacobbi N.S., Kawai G., Parniak M.A., Sarafianos S.G., Sluis-Cremer N, & Ishima R. (2019). Conformational changes in HIV-1 reverse transcriptase that facilitate its maturation. Structure (London, England : 1993), 27(10), 1581-1593.e3.

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RNA Library Labeling Protocol

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All RNA probes in the RNA structure libraries were labeled with a fluorescent dye at the 3' end. Ten micromolar RNA structure library, 100 μM pCp-Cy5 or pCp-Cy3 (Jena Bioscience), and 0.5 U/μL T4 RNA Ligase (Thermo Fisher Scientific) were mixed in 100 μL of 1× T4 Ligase Buffer (Thermo Fisher Scientific). The mixture was incubated at 16 °C for 48 h on a ThermoMixer (Eppendorf) with ThermoTop (Eppendorf). After incubation, the labeled RNA was purified using Zymo RNA Clean and Concentrator (Zymo Research) and stored at -28 °C until use.

Nagasawa R., Onizuka K., Komatsu K.R., Miyashita E., Murase H., Ojima K., Ishikawa S., Ozawa M., Saito H, & Nagatsugi F. (2024). Large-scale analysis of small molecule-RNA interactions using multiplexed RNA structure libraries. Communications chemistry, 7(1).

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Fluorescent Labeling of mRNA

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Synthesized mRNAs were 3′ end labeled with T4 RNA ligase and pCp-Cy3 (Jena Bioscience) using the manufacturer’s proceedings. Unbound pCp-Cy3 was removed with the use of MicroSpin G-25 columns (GE Healthcare Life Sciences). For microscopy, 18 ng/μL of labeled mRNA was incubated with 1 μM Ded1-GFP in the presence or absence of 18 ng/μL tRNA.

Iserman C., Desroches Altamirano C., Jegers C., Friedrich U., Zarin T., Fritsch A.W., Mittasch M., Domingues A., Hersemann L., Jahnel M., Richter D., Guenther U.P., Hentze M.W., Moses A.M., Hyman A.A., Kramer G., Kreysing M., Franzmann T.M, & Alberti S. (2020). Condensation of Ded1p Promotes a Translational Switch from Housekeeping to Stress Protein Production. Cell, 181(4), 818-831.e19.

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Fluorescent RNA Labeling Protocol

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Cy3- and Cy5-labelled RNAs were prepared by ligation using T4 RNA ligase (Ambion), pCp-Cy3 (Jena Bioscience) and pCp-Cy5 (Jena Bioscience). Cy3- and Cy5-labelling was performed with 150 pmol RNA using 10 U T4 RNA ligase, 3 nmol pCp-Cy3 or pCp-Cy5 and 10% (v/v) dimethyl sulfoxide in 10 µl at 16 °C for 36–48 h. The Cy3- and Cy5-labelled RNAs were purified using an RNeasy MinElute Cleanup Kit (Qiagen). After the recovery of RNAs, the RNA concentration was measured in a NanoDrop (Thermo Scientific). The labelling efficiencies of Cy3 and Cy5 were calculated from the absorbance of Cy3 and Cy5, respectively.

Shibata T., Fujita Y., Ohno H., Suzuki Y., Hayashi K., Komatsu K.R., Kawasaki S., Hidaka K., Yonehara S., Sugiyama H., Endo M, & Saito H. (2017). Protein-driven RNA nanostructured devices that function in vitro and control mammalian cell fate. Nature Communications, 8, 540.

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