Using protein-DNA chimeras to detect and count small numbers of molecules.
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.
Burbulis
Ian
I
The Molecular Sciences Institute, 2168 Shattuck Avenue, Berkeley, California 94704, USA. burbulis@molsci.org
Yamaguchi
Kumiko
K
Gordon
Andrew
A
Carlson
Robert
R
Brent
Roger
R
D000942:Antigens, Bacterial / Q000378:metabolism; D001408:Bacillus anthracis / Q000378:metabolism*; D001427:Bacterial Toxins / Q000378:metabolism; D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D019921:Biotinylation; D004247:DNA / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D005821:Genetic Techniques; D047668:Inteins; D016014:Linear Models; D008956:Models, Chemical; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D012313:RNA / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C030325:anthrax toxin; D012313:RNA; D001710:Biotin; D004247:DNA