Turning a kinase deoxyribozyme into a sensor.

TitleTurning a kinase deoxyribozyme into a sensor.
Publication TypeJournal Article
Year of Publication2013
AuthorsMcManus SA, Li Y
JournalJournal of the American Chemical Society
Volume135
Issue19
Pagination7181-6
Date Published2013 May 15
Abstract

The vast majority of deoxyribozyme-based sensors are designed using modified RNA-cleaving deoxyribozymes and detect analytes that act as allosteric regulators of their catalytic activity. These sensors are susceptible to background signals due to catalytic activity in the absence of target or contaminant molecules that cleave the RNA substrate, mimicking the deoxyribozyme reaction. In this manuscript, we introduce a novel system that avoids these problems by using the analyte as the substrate for a deoxyribozyme catalyzed self-phosphorylation reaction. This reaction creates a modified deoxyribozyme product that can be circularized and subjected to massive signal amplification by rolling circle amplification, leading to a sensor system with high sensitivity and low background, which can be coupled to numerous reporter systems. As an example of the potential of this system, we used the self-phosphorylating deoxyribozyme Dk2 to detect as little as 25 nM GTP even in the presence of 1 mM ATP, a potential contaminant. To demonstrate the adaptive properties of this system, we appended another DNA sequence to Dk2, which, once amplified by RCA, codes for a fluorescence generating deoxyribozyme. This two-deoxyribozyme system was able to report the presence of GTP from 4 μM to 1 mM, with specificity over other NTP molecules. Using this model system, we were able to show that small molecule modifying deoxyribozymes can be converted to analyte sensors by coupling their catalytic activity to signal amplification and reporting.

DOI10.1074/jbc.M113.510966
Alternate JournalJ. Am. Chem. Soc.