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Synthesis of biotin–AMP conjugate for 5′ biotin labeling of RNA through one-step in vitro transcription

Nature Protocols volume 3, pages 1848–1861 (2008)Cite this article

Abstract

Biotin-labeled RNA has found broad applications in chemistry, biology and biomedicine. In this protocol, we describe a simple procedure for 5′ RNA biotin labeling by one-step in vitro transcription. A biotin–AMP (adenosine 5′-monophosphate) conjugate, biotin-HDAAMP (adenosine 5′-(6-aminohexyl) phosphoramide; where HDA is 1,6-hexanediamine), is chemically synthesized. Transcription initiation by biotin-HDAAMP under the T7 φ2.5 promoter produces 5′ biotin-labeled RNA with high labeling efficiency. The procedure is especially useful for biotin labeling of RNA that is larger than 60 nucleotides. In addition, the protocol provides an attractive alternative to chemical synthesis of biotin-labeled small RNA of less than 60 nucleotides, particularly when the desired quantity of RNA is low. The whole procedure, from chemical syntheses to isolated biotin-labeled RNA, can be completed within 2 weeks.

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Figure 1: Comparison of two T7 promoters φ2.5 and φ6.5.
Figure 2: Procedure for biotin labeling of RNA by in vitro transcription and analysis of RNA labeling efficiency by PAGE.
Figure 3: Scheme for the chemical synthesis of the transcription initiator biotin-HDAAMP.
Figure 4: HPLC analysis of HDAAMP synthesis from HDA and AMP in the presence of EDAC after 90 min reaction at room temperature.
Figure 5: HPLC analysis of biotin-HDAAMP synthesis from biotin-NHS and HDAAMP after 5 h reaction at room temperature.
Figure 6: Purification of biotin-HDAAMP by semipreparative HPLC: column—Alltech Econosphere C18, 10 mm × 250 mm, flow rate—5 ml min−1.
Figure 7: HPLC analysis of biotin-HDAAMP purity.
Figure 8
Figure 9: Gel image of optimization of DNA template preparation by PCR amplification.
Figure 10: PAGE analysis of RNA biotin-labeling efficiency under different transcription conditions.
Figure 11: Separation of biotin-labeled 100 nt RNA from unlabeled pppRNA by 40-cm-long denaturing PAGE (8%).

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Acknowledgements

This study was supported by a NASA grant NNX07AI98G. We thank Adam York for NMR characterization of biotin-HDAAMP. Previous members of our laboratory, Guocan Wang, Tricia Coleman and Na Li made contributions to the development of the current protocol.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of Southern Mississippi, Hattiesburg, 39406, Mississippi, USA

    Faqing Huang, Jun He & Yilin Zhang

  2. Department of Biological Sciences, University of Southern Mississippi,

    Yanlin Guo

  3. Hattiesburg, 39406, Mississippi, USA

    Yanlin Guo

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Correspondence to Faqing Huang.

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Huang, F., He, J., Zhang, Y. et al. Synthesis of biotin–AMP conjugate for 5′ biotin labeling of RNA through one-step in vitro transcription. Nat Protoc 3, 1848–1861 (2008). https://doi.org/10.1038/nprot.2008.185

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