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RNA Applications

baseclick GmbH

Simple click labeling of RNA and transfection into cells

Do you need to prepare labeled RNA? Or are you looking for efficient transfection of mRNA into cells?

We have your solutions for simple and reliable labeling of RNA and transfection. We offer ready-to-use custom oligonucleotides, but also provide corresponding building blocks to prepare click-ready RNA by enzymatic synthesis. Actually, a click-ready nucleoside can be fed to cells to allow de novo RNA detection. In addition, kits for mRNA preparation, purification and transfection enable mRNA technology for everyone.

The key attributes of baseclick’s RNA application products are:

  • High-purity custom oligos for meaningful experiments help you save time
  • A reliable mRNA transfection agent enables transient gene expression experiments in cell culture
  • High quality nucleotides e.g. for in vitro transcription enzymatic preparation provide decent mRNA yields and quality.
  • Simple kits for RNA labeling and purification enable click reactions for everyone

Applications

Baseclick offers all components to prepare labeled short and long RNA oligonucleotides to allow full flexibility for your experiments. They should be considered whenever the standard nucleobases and functionalities are not enough or when detection in complex mixtures is required, e.g. for mRNA tracking inside of cells.

Which kit or reagent is the right for me?

Our nucleotides are ideal for researchers to prepare labeled oligonucleotides, applying chemo-enzymatic approaches, e.g. in vitro transcription of RNA. Even the modified nucleoside 5-ethynyluridine can be an option for metabolic labeling. In case you already have your mRNA of interest, baseclick’s transfection agent is an easy option for transient expression in cell culture. Our kit systems are available for easy preparation of labeled RNAs.

You are not sure what you are looking for or you need a quote? Just contact us.

Literature

RNA Applications

  • Click chemistry applications on RNA molecules[1][2]
  • (m)RNA as a therapeutic agent[3][4]

Selected references

[1] E. Paredes, S. R. Das, ChemBioChem 2011, 12, 125–131.

[2] S.Croce, S. Serdjukow, T. Carell, T. Frischmuth, ChemBioChem, 2020, 21, 1-7

[3] J. Lieberman, Nat. Struct. Mol. Biol. 2018, 25, 357–364.

[4] U. Sahin, K. Karikó, Ö. Türeci, Nat. Rev. Drug Discov. 2014, 13, 759–780.

FAQs

  • What is the best way to handle RNA to avoid degradation?

    Due to its chemical structure RNA is less stable than DNA. At high temperatures, under basic conditions (pH>9) and in the presence of high Mg2+ concentrations, chemical reactions that favour hydrolysis of the RNA molecule take place. Additionally, RNases are common contaminations in standard laboratory conditions compared to DNases.

    We recommend to clean the benches and equipment with RNase inhibitors or specific detergent mixtures and to wear fresh gloves when handling the tubes (gloves that have been worn or that e.g. touched door/refrigerator handles are considered contaminated). Moreover, RNase-free H2O and tubes should be used for buffer preparation and to dissolve the samples.

  • What is the best way to store RNA to avoid degradation?

    For storage we recommend to store RNA at –80 °C, which should conserve RNA for one year without significant degradation. Alternatively,
    in our experience, short synthetic RNAs (up to 50mer) are more stable than long single-stranded RNAs from biological samples and therefore can be stored at –20 °C in a dry format.

  • Are you offering RNA oligos?

    We are offering modified and unmodified synthetic DNA, RNA, LNA and PNA oligonucleotides with a focus on modified oligos. Our online request oligo formula gives you an overview of the most standard configurations and prices thereof.

  • Can I use 5-ethynyl uridine (EU) for labeling of nascent RNA inside cells?

    EU can be fed to cells and detected using a fluorescent dye via click labeling. Our own experiments indicate that it will be first integrated in de novo RNA, but we have also observed incorporation into DNA after some incubation time. This is most likely via conversion of the EU ribonucleoside into EdU aided by intracellular ribonucleotide reductases.

    Research from several groups demonstrates successful EU cellular labeling for divers applications. (1)(2)

    1. Jao,C.Y. and Salic,A. (2008) Exploring RNA transcription and turnover in vivo by using click chemistry. Proc. Natl. Acad. Sci. U. S. A., 105, 15779–84.

    2. Bao,X., Guo,X., Yin,M., Tariq,M., Lai,Y., Kanwal,S., Zhou,J., Li,N., Lv,Y., Pulido-quetglas,C., et al. (2018) Capturing the interactome of newly transcribed RNA. 15.

  • What kits are available from Baseclick for RNA modification?

    We offer several kits to prepare labelled RNAs that are based on enzymatic incorporation of alkyne- or azide-modified nucleotides and subsequent click labelling.

    The RNA Labeling Kit allows you to introduce several alkyne groups randomly in your RNA through the incorporation of the 5-ethynyl-UTP (EUTP) by the T7 RNA polymerase during in vitro transcription.

    The TTT mRNA prep Kit was developed and specifically designed for the production of alkyne decorated messenger RNA by the help of T7 RNA polymerase and EUTP.

    The 3’END Labeling Kit introduces site specifically at the 3’ end of an RNA a single azide group via the incorporation azide-ATP derivative.

  • How does the DNA template influence in vitro transcription performance?

    DNA template quality directly influences yield and quality of transcription reactions. Linearized plasmid DNA needs to be fully digested and should be free of contaminating RNase, protein and salts. We recommend selecting restriction enzymes that generate blunt ends or 5´-overhangs and purification after the enzymatic restriction (e.g. via silica-membrane based purification columns).

    Alternatively, a PCR mixture can be used directly, but better yields will usually be obtained with purified PCR products (e.g. via silica-membrane based purification columns).

  • What do I need to consider when planning my transcription experiments using your kits?

    Promotor choice: Different RNA polymerase promoters such as T7, T3 and SP6, are commonly used for the in vitro production of RNA. Our kits, TTT RNA prep and RNA labelling kit, are based on a T7 RNA polymerase promoter and will not function upon usage of a different one.

    mRNA production: For the production of functional mRNA please ensure that the DNA template encodes the required structural features e.g. 3’-UTR, 5’-UTR, correctly orientated target sequence and ideally a poly A-tail. Alternatively, polyA-tailing can be introduced post-transcriptionally using a Poly(A) polymerase.

  • What is the promoter sequence of T7 RNA Polymerase?

    5′ TAATACGACTCACTATAG 3′

    T7 RNA polymerase starts transcription at the underlined G in the promoter sequence. The polymerase then transcribes using the opposite strand as a template from 5’->3’. The first base in the transcript will be a G.

  • Can the 3’ END labelling kit be used with other RNAs?

    Indeed, it can be used for other kinds of RNA. Make sure that your RNA does not have secondary structures that can interfere with enzyme activity. If you are not sure about secondary structures, it can help to heat your samples shortly (5 minutes at 65 °C and then on ice for other 5 minutes) before applying the reagents.

    We recommend to try such a step first with a smaller test sample, as too harsh handling can also affect your RNA.

    Importantly, make sure that your RNA is longer than 100 nucleotides, since the purification method provided with the kit will not be able to retain shorter fragments.

  • Does my RNA need to end with an A nucleotide for efficient 3’ END labelling?

    No this is not necessary, the poly(A) polymerase inside the kit is able to add the modification whatever the last natural base is.

  • Can the TTT mRNA Prep kit be used for other RNA?

    No, this kit has been designed for preparation and labeling of mRNA from DNA templates, only. Due to the nucleotide mix present in the kit, the resulting mRNA will contain a CAP structure and pseudo-uridine for enhanced translation efficiency and intracellular stability, besides the clickable modification.

  • What kind of CAP is introduced with the TTT mRNA prep kit?

    The CAP structure is introduced co-transcriptionally and results in a CAP 1 structure.

  • How much RNA can I expect after in vitro transcription using the TTT mRNA prep Kit?

    Starting from 1 µg of DNA template, it is common to end up with a solution containing from 10 to 12 µg of mRNA.