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Analytical Chemistry

DNA Trio Assembles To Detect Proteins In New Assay

Medical Diagnostics: An easy-to-run assay senses picomolar levels of proteins in a single tube

by Laura Cassiday
July 1, 2014

Four-Piece Assay
Schematic of a DNA assembly method to detect proteins.
Credit: Anal. Chem.
Two different molecules (yellow square/circle) bind to a protein target (blue oval), causing complementary DNA sequences (green) attached to the molecules to hybridize. A third DNA strand (MB) then binds to the complex at two other complementary sequences (red and purple). To bind, the third strand must open up, separating a fluorophore (F) from its quencher (Q). A nicking endonuclease (purple “Pac Man”) cleaves a site (yellow) in the third strand, releasing the now fluorescent DNA from the other two strands. Other third strands can then bind to the complex, amplifying the fluorescent signal.

A new assay easily detects picomolar concentrations of specific proteins in a single step without the need for complex reagent mixtures (Anal. Chem. 2014, DOI: 10.1021/ac5011316). All researchers need is a trio of DNA strands and an enzyme. The method could simplify diagnostic testing, particularly in regions with limited resources, the study authors say.

Currently, the gold standard for diagnostic protein detection is the enzyme-linked immunosorbent assay, or ELISA. However, this method requires several reagents and steps. Hongquan Zhang, a bioanalytical chemist at the University of Alberta, developed the new, one-step process to make protein detection easier.

The assay adds a simple readout to a technique Zhang developed previously, called binding-induced DNA assembly, or BINDA (Anal. Chem. 2012, DOI: 10.1021/ac203207g). The researchers start with two different molecules, such as antibodies, that bind the protein of interest. They then attach these molecules to two separate strands of DNA with stretches of a complementary sequence. When the molecules bind their target protein, the two DNA strands come into close contact, allowing the complementary sequences to hybridize.

Through this hybridization, the strands assemble to reveal a DNA sequence complementary to that of a third DNA strand. This third piece of DNA, which contains a fluorophore, then binds to the complex and generates a fluorescent signal. Over time, this signal gets amplified because a DNA-snipping enzyme cuts the third strand and releases it from the DNA complex, allowing other third strands to move in.

Although the method seems complicated, it’s really quite simple to operate, Zhang says. Researchers mix all reagents in a single tube at 37 °C, wait two hours, and then read the signal on a fluorescence plate reader. Zhang and his colleagues used the method to detect picomolar concentrations of two proteins: streptavidin and prostate-specific antigen in diluted human serum.

“The sensitivity of the method is comparable to ELISA, but the operation is much easier,” Zhang says.



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