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

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.

Protein detection via direct enzymatic amplification of short DNA aptamers

“…This observation has been previously reported by Burbulis et al, who used 'confidence belt' statistical analyses to extract further information from the non-linear region at the transition between specific and non-specific interactions. (35) Enhanced LoD can also be accomplished by decreasing the amount of aptamer, as a 20-fold reduction in the initial aptamer concentration results in an approximate 20-fold enhancement in the LoD (to ~100 fM; see supplementary data section Figure S2). Conversely, increasing the concentration of aptamer above 2 nM resulted in a concomitant reduction in sensitivity (data not shown).…”

“…(31)(32)(33)(34) A drawback of these approaches is that the synthesis of the antibody-DNA hybrids can be problematic as controlling the location and number of DNA conjugates per protein is not always straightforward, often leading to heterogeneous ratios of DNA tags per antibody. Recent developments in site-specific conjugation of oligonucleotide tags to proteins using intein chemistry (or chemical ligation) have been very successful, (35) although conjugate preparation remains laborious. A main advantage of solely DNA-based reagents is the possibility of combining both the high affinity of aptamers and amplification techniques for sensitive detection in a single molecular platform, thus reducing synthetic complexity.…”

Method for generation of in vivo biotinylated recombinant antibodies by yeast mating

“…Finally, this method may also be used to generate reagents for emerging technologies such as antibody-based proximity ligation (Fredriksson et al, 2002), antibody-based competition assays using tadpoles (Burbulis et al, 2005), bead-based assays (Vignali, 2000;Gorelik et al, 2005;Scholler et al, 2006) and protein or antibody arrays (Chen et al, 2005;Gao et al, 2005;Utz, 2005;Boozer et al, 2006). In conclusion, the yeast expression system described here allows efficient generation of directly biotinylated high-affinity reagents that are needed for a large range of applications, including evaluation of the products of large-scale proteomics discovery projects.…”

Protein Splicing Mechanisms and Applications

“…They have been used to extend size limitations in NMR analysis by partial labeling, to introduce post-translational modifications, unnatural amino acids, transition state analogs and biosensors, to limit unwanted transgene transfer in plants by splitting the transgene between the nucleus and the chloroplast, and to express cytotoxic proteins (Table 1) (3 -5, 26). The 'tadpole' technology uses EPL to link a binding protein to a DNA sequence that contains a T7 RNA polymerase promoter and a PCR barcode sequence (25). Protein binding can be quantitated by real-time PCR or runoff transcription.…”