Given the essential role of apurinic/apyrimidinic endonuclease (APE1) in gene repair and cancer progression, we report a novel approach for probing and regulating cellular APE1 activity by using DNA tetrahedrons.

The distinctive nuclease activity toward nucleic acid substrates enables various applications in analytical chemistry and dynamic DNA nanotechnology. λ Exonuclease is a widely used tool for the processing of PCR products, and DNA sequencing. This enzyme also shows promise for reducing the leakage (i.e., activation in absence of a correct input) in DNA-based analytical methods and nanotechnology due to its sensitivity to mismatches. However, the selectivity of λ exonuclease for single-mismatch in most applications is not high. Inspired by the increased specificity of dynamic probes in DNA nanotechnology, we enhanced the single-mismatch selectivity of λ exonuclease by using very short double-stranded DNA (dsDNA) as the substrate. From the bulk fluorescence measurements, short perfectly matched (PM) substrate which is as a correct input can be effectively digested, but the existence of single-mismatch drastically reduces the digestion rate. Real-time single-molecule kinetics analysis reveals that PM substrate can be selectively stabilized by the binding of λ exonuclease, which combines with the differential stability of transient hybridization of short substrates to yield high single-mismatch selectivity. An excellent selective assay for a single-nucleotide mutation in KRAS was demonstrated, which permits detecting this mutation from cell line at as low as 0.02%, holding potential for detecting rare mutations in circulating tumor DNA of early stage cancers.

Simultaneous analysis based on encoded fluorophores suffers from potential crosstalk between fluorophores and the limited number of colors that can be practically resolved. Inspired by nontrivial temporal patterns in living organisms, we developed a DNA-templated probe by utilizing DNA polymerase (DNAP) for multiplexed detection of nucleic acids. These probes use differential delay times of signaling by a DNAP-mediated extension to distinguish different targets, which serve as the primers. Taking advantage of the high processivity and the controllable kinetics of DNAP, we find that multiplexed detection can be achieved in homogeneous solution using a single fluorophore. As a proof of concept, we developed assays for genomic DNA from four different bacteria. In addition, we designed and implemented probes to undergo a single oscillation in signal as an alternative way for multiplexing. We anticipate this approach will find broad applications not only in sensing but also in synthetic DNA nanosystems.

We utilized site-specific and sequence-independent nucleases to engineer high-robustness DNA molecular circuits.

A DNA motor was operated in living cells by endogenous AP endonuclease.