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Using protein-DNA chimeras to detect and count small numbers of molecules

Nature Methods volume 2, pages 31–37 (2005)Cite this article

Abstract

We describe general methods to detect and quantify small numbers of specific molecules. We redirected self-splicing protein inteins to create 'tadpoles', chimeric molecules comprised of a protein head covalently coupled to an oligonucleotide tail. We made different classes of tadpoles that bind specific targets, including Bacillus anthracis protective antigen and the enzyme cofactor biotin. We measured the amount of bound target by quantifying DNA tails by T7 RNA polymerase runoff transcription and real-time polymerase chain reaction (PCR) evaluated by rigorous statistical methods. These assays had a dynamic range of detection of more than 11 orders of magnitude and distinguished numbers of molecules that differed by as little as 10%. At their low limit, these assays were used to detect as few as 6,400 protective antigen molecules, 600 biotin molecules and 150 biotinylated protein molecules. In crudely fractionated human serum, the assays were used to detect as few as 32,000 protective antigen molecules. Tadpoles thus enable sensitive detection and precise quantification of molecules other than DNA and RNA.

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Figure 1: Design and synthesis of chimeric detector molecules.
Figure 2: Characterization of anti-biotin tadpole.
Figure 3: Quantification of biotinylated BSA diluted in an excess of nonspecific organic molecules.
Figure 4: Statistical analysis of biotin quantification.
Figure 5: Quantification of recombinant PA diluted in BSA or human serum.
Figure 6: Statistical analysis of PA quantification.

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Acknowledgements

The authors thank R. Fu for technical assistance with early real-time PCR experiments, L. Lok for invaluable advice on statistical analysis, O. Resnekov for suggesting T-gel for fractionating serum, C. Cantor for the gene encoding the streptavidin core subunit, J. Maynard for plasmids encoding anti-PA scFvs, purified scFvs and antibodies, and A. Coleman-Lerner, K. Benjamin, A. Gordon, L. Lok, A. Mendelsohn, O. Resnekov, E. Serra and R. Yu for useful discussions and comments on the manuscript. Some support for this work came from grants from the National Science Foundation and Defense Advanced Research Projects Agency, but main support came from the Center for Genomic Experimentation and Computation, a National Institutes of Health Center of Excellence in Genomic Science (P50 HG02370).

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  1. Robert Carlson

    Present address: Department of Electrical Engineering, University of Washington, Box 352500, Seattle, Washington, 98195, USA

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  1. The Molecular Sciences Institute, 2168 Shattuck Avenue, Berkeley, 94704, California, USA

    Ian Burbulis, Kumiko Yamaguchi, Andrew Gordon & Roger Brent

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Correspondence to Ian Burbulis.

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Supplementary information

Supplementary Fig. 1

Verification of gaussian distribution. (PDF 176 kb)

Supplementary Fig. 2

Biotin enyzyme linked immuno-adsorption assay. (PDF 57 kb)

Supplementary Fig. 3

PA enyzyme linked immuno-adsorption assay. (PDF 61 kb)

Supplementary Table 1

Affinity protein heads. (PDF 66 kb)

Supplementary Table 2

DNA tails used to construct tadpoles. (PDF 51 kb)

Supplementary Table 3

Forward and reverse PCR primer sequences used to amplify specific tadpole tail DNAs. (PDF 50 kb)

Supplementary Table 4

Evanescent wave measurements of tadpole binding. (PDF 136 kb)

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Burbulis, I., Yamaguchi, K., Gordon, A. et al. Using protein-DNA chimeras to detect and count small numbers of molecules. Nat Methods 2, 31–37 (2005). https://doi.org/10.1038/nmeth729

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