T7 RNA polymerase as a self-replicating label for antigen quantification.

Enzymes are used widely as labels in binding assays for protein analytes, because they provide signal amplification. Efforts at improving the assay sensitivity have been focused mainly on the synthesis of novel substrates, e.g. fluorogenic and chemiluminogenic ones. We report the investigation of T7 RNA polymerase (T7RP) as a label with unique characteristics for antigen quantification. In an in vitro, coupled (one-step) transcription/translation reaction, T7RP catalyzes the expression of an enzyme-coding DNA template to produce free enzyme (luciferase) in solution. We demonstrate that the generated luciferase is linearly related to the input T7RP in a range covering over four orders of magnitude. It is also shown that T7RP exhibits a significant level of self-replication (100-fold) in vitro by acting on a DNA template comprising the T7RP cDNA downstream of a T7 promoter. By combining the self-replication reaction with the expression of luciferase DNA, as low as 1400 T7RP molecules are detectable. Furthermore, the T7RP is biotinylated, complexed with streptavidin and used for antigen quantification in a microtiter well-based assay with high sensitivity and reproducibility.

[1]  J. Eberwine,et al.  Protein quantification from complex protein mixtures using a proteomics methodology with single-cell resolution , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[2]  Andreas Plückthun,et al.  Picomolar affinity antibodies from a fully synthetic naive library selected and evolved by ribosome display , 2000, Nature Biotechnology.

[3]  I. Tomlinson,et al.  Antibody arrays for high-throughput screening of antibody–antigen interactions , 2000, Nature Biotechnology.

[4]  S. Gygi,et al.  Evaluation of two-dimensional gel electrophoresis-based proteome analysis technology. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[5]  S. Kingsmore,et al.  Immunoassays with rolling circle DNA amplification: a versatile platform for ultrasensitive antigen detection. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[6]  T. Kodadek,et al.  Peptides Selected to Bind the Gal80 Repressor Are Potent Transcriptional Activation Domains in Yeast* , 2000, The Journal of Biological Chemistry.

[7]  A D Ellington,et al.  In vitro selection of nucleic acids for diagnostic applications. , 2000, Journal of biotechnology.

[8]  A. Skerra,et al.  Applications of a peptide ligand for streptavidin: the Strep-tag. , 1999, Biomolecular engineering.

[9]  T. K. Christopoulos,et al.  Two-site expression immunoassay using a firefly luciferase-coding DNA label. , 1999, Clinical chemistry.

[10]  T K Christopoulos,et al.  Signal amplification system for DNA hybridization assays based on in vitro expression of a DNA label encoding apoaequorin. , 1999, Nucleic acids research.

[11]  S. Gygi,et al.  Correlation between Protein and mRNA Abundance in Yeast , 1999, Molecular and Cellular Biology.

[12]  R. Tsien,et al.  green fluorescent protein , 2020, Catalysis from A to Z.

[13]  L. Wodicka,et al.  Genome-wide expression monitoring in Saccharomyces cerevisiae , 1997, Nature Biotechnology.

[14]  P. Hajduk,et al.  Discovering High-Affinity Ligands for Proteins: SAR by NMR , 1996, Science.

[15]  P. Brown,et al.  Parallel human genome analysis: microarray-based expression monitoring of 1000 genes. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[16]  K. Doyle.  Promega protocols and applications guide , 1996 .

[17]  H. Deng,et al.  Self-amplifying expression from the T7 promoter in 3T3 mouse fibroblasts. , 1994, Gene.

[18]  T. Hunt,et al.  Plasmid cDNA-directed protein synthesis in a coupled eukaryotic in vitro transcription-translation system. , 1992, Nucleic acids research.

[19]  C R Cantor,et al.  Immuno-PCR: very sensitive antigen detection by means of specific antibody-DNA conjugates. , 1992, Science.

[20]  V. Gurevich,et al.  Preparative in vitro mRNA synthesis using SP6 and T7 RNA polymerases. , 1991, Analytical biochemistry.

[21]  F. Studier,et al.  Creation of a T7 autogene. Cloning and expression of the gene for bacteriophage T7 RNA polymerase under control of its cognate promoter. , 1991, Journal of molecular biology.

[22]  E. Diamandis,et al.  The biotin-(strept)avidin system: principles and applications in biotechnology. , 1991, Clinical chemistry.

[23]  I. Weeks,et al.  Chemiluminescence immunoassay: an overview. , 1986, Clinical science.

[24]  I. Weeks,et al.  Chemiluminescence immunoassay , 1985 .

[25]  F. Studier,et al.  Cloning and expression of the gene for bacteriophage T7 RNA polymerase. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[26]  S C Suffin,et al.  Fluorescence immunoassays. , 1979, Analytical chemistry.