DNA Detection and Signal Amplification via an Engineered Allosteric Enzyme

Abstract

Rapid, sensitive, and sequence-specific DNA detection can be achieved in one step using an engineered intrasterically regulated enzyme. The semi-synthetic inhibitor-DNA-enzyme (IDE) construct (left) rests in the inactive state, but upon exposure to a complementary DNA sequence undergoes a DNA hybridization-triggered allosteric enzyme activation (right). The ensuing rapid substrate turnover provides the built-in signal amplification mechanism for detecting approximately 10 femtomoles of DNA in less than three minutes under physiological conditions. We describe the design, synthesis, and functional characterization of an intrasterically regulated semi-synthetic enzyme and its application in sequence specific DNA detection. 1-2 The system is composed of covalently associated inhibitor-DNA-enzyme (IDE) modules and functions via DNA hybridization-triggered allosteric enzyme activation and signal amplification through substrate turnover. 3 Its functional capacity is highlighted by the sequence specific detection of approximately 10 femtomoles of DNA in less than three minutes under physiological conditions. These studies suggest that rationally designed intrasterically regulated enzymes may constitute a promising new class of reagents for highly sensitive, rapid, and PCR-independent one-step detection of label-free DNA sequences.The principles that govern intrasteric regulation in natural enzymes 4 are firmly rooted in the physicochemical advantages of intramolecular binding. These enzymes often use an appending N-or C-terminal polypeptide pseudosubstrate to block access to their active sites. Enzyme regulation (activation) occurs at an allosteric site, typically the site between the enzyme and pseudosubstrate, where a conformational change or cleavage event removes the pseudosubstrate from the active site. We sought to mimic the intrasteric regulatory features of natural enzymes by designing novel semi-synthetic constructs that can be activated in the presence of complementary DNA sequences. The IDE complex described employs a single stranded DNA probe to covalently tether Cereus neutral protease 5 (CNP) to its small molecule NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript phosphoramidite inhibitor. 6 We anticipated that the conformational flexibility of the ss-DNA tether in an appropriately functionalized system would allow facile intramolecular binding of the inhibitor to the enzyme active site to furnish the inactive state of the IDE (Figure 1). Likewise, we postulated that IDE should rapidly convert to an active state in the presence of its complementary DNA sequence because formation of a high-affinity DNA duplex structure was expected to drastically alter the conformation of the tether and favor the liberation of the inhibitor from the enzyme active site. Once activated, the enzyme would act upon its fluorogenic substrate, present in situ, producing an optical output signal. Moreover, because the enzyme turns over many copies of the substrate, in principle each probe hybridiza...

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