Turning a Kinase Deoxyribozyme into a Sensor

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.

Noncanonical Self-Assembly of Multifunctional DNA Nanoflowers for Biomedical Applications

“…1–5 The sequence programmability, automated controllable synthesis, high stability and intrinsic functionalities make DNA nanostructures advantageous over other counterparts in many biomedical applications. Conventional approaches to DNA nanostructure construction typically rely on Watson-Crick base-pairing between short DNA building blocks.…”

DNA Catalysts with Tyrosine Kinase Activity

“…Deoxyribozymes are specific DNA sequences with catalytic activity that have been used to catalyze a variety of reactions, 7 including self-5′-phosphorylation. 8 We have initiated a research program to use deoxyribozymes to perform covalent modification of amino acid side chains in peptide and protein substrates. 9 In the present study, we report that deoxyribozymes can be identified for phosphorylation of tyrosine residues in peptide substrates, using as the phosphoryl donor either a 5′-triphosphorylated RNA oligonucleotide (pppRNA) or guanosine 5′-triphosphate (GTP).…”

DNAzymes: Selected for Applications

“…The first example, reported by Andrew Ellington's group in 2005, used a two‐piece DNA assembly with an ATP‐dependent DNA‐ligating DNAzyme and its substrate arranged in a special way such that binding of ATP to the aptamer domain activates the ligase DNAzyme, which creates the needed circular DNA template for subsequent RCA reaction . The second example of making the requisite circular template for RCA was reported by our group, with a goal of detecting guanosine‐5′‐triphosphates (GTP) . In this case, a self‐phosphorylating DNAzyme first transfers a γ‐phosphate from GTP to the 5′‐end of the DNAzyme.…”