Fundamental Understanding of DNAzymes, a New Class of Metalloenzymes and Their Applications as Sensing and Imaging Agents in Environmental Monitoring and Medical Diagnostics
For general reviews in this area, please see the following review articles (1) and an edited book (2).

One of the most important discoveries in the last decade is that DNA molecules are not only materials for genetic information storage, but also catalysts for a variety of biological reactions, and therefore DNA molecules with catalytic properties are called catalytic DNAs or DNAzymes. Since metal ions play essential roles in the structure and function of DNAzymes, their study has become a new frontier in bioinorganic chemistry (1a). Although many different classes of metal-specific proteins have been studied, similar information about metal-specific DNAzymes is not available.
Fundamental Understanding of Metallo-DNAzymes
General in vitro selection procedure (left), selected DNAzymes for metal binding (right)
The Lu group has made significant contributions to enriching our fundamental understandings of metallo-DNAzymes by carrying out in vitro selection to obtain new DNAzymes that are specific to a number of different metal ions (3) such as Zn2+ (4), Pb2+ (5), and UO22+ (6). By employing a negative selection strategy, we have shown that metal selectivity can be dramatically improved (6-7). In addition, we have carried out biochemical studies to define conserved sequences responsible for the metal ion selectivity (8), such as the presence of a GT wobble pair close to the metal-binding site (8a,b) and mass spectrometry to identify reaction intermediates and products (8a,b). Furthermore, spectroscopic studies of these DNAzymes (9) have elucidated metal-dependent folding and structures. For example, most previous studies indicate that a metal-dependent global folding step is required before DNA/RNAzyme reactions. In FRET studies of an 8-17 DNAzyme both in bulk solution and at the single molecule level, we discovered that, in contrast to observations from studies of ribozymes and even the same DNAzyme in the presence of less active Mg2+ and Zn2+, no folding step was observed in the presence of Pb2+, the most active metal ion (9c,d).
Lock and Key DNAzyme
Therefore, the DNAzyme may be prearranged to accept Pb2+ to catalyze the reaction (i.e., a lock and key type of mechanism), which may contribute to the remarkably fast Pb2+-dependent reaction (10). This observation has also been confirmed in another fast uranyl-specific DNAzyme (9g). The results strongly suggest that DNAzymes can use all modes of activation available to protein metalloenzymes.

Sensing and Imaging Applications
In addition to contributing fundamental understanding of the metallo-DNAzymes, we have also been at the forefront in their applications as sensing and imaging agents for environmental monitoring and medical diagnosis.(1c-f,11). Selective agents for metal ions and other targets such as toxins and cancer biomarkers are very useful in environmental monitoring, cellular imaging, and medical diagnostics. Despite much effort, few such agents are commercially available. For example, >60,000 papers have been published on metal ion sensors alone, and yet commercially available metal sensors are limited to only a few. We have identified challenges in both fundamental sciences and in technological developments and have made significant progresses in meeting these challenges.

In fundamental sciences, designing selective agents based on a single class of molecules for a broad range of targets with high sensitivity and selectivity remains a significant challenge. Most processes are on a trial and error basis where success in designing agents for one target (e.g., one metal ion) can be difficult to translate into success in designing agents for other targets (many other metal ions). To overcome these obstacles, we need to develop general strategies to:
Catalytic Beacon Sensors
  1. a) obtain molecules for any targets of interests;
  2. b) improve selectivity of the molecules;
  3. c) transform the binding into detectable signals, and
  4. d) tune dynamic range to match the concentration levels of the targets.
To meet these challenges, we have been able to
  1. a) use a combinatorial method called in vitro selection to obtain DNAzymes and aptamers, a new class of nucleic acids that can bind targets of choice strongly and specifically enough to rival antibodies (3-4,6);
  2. b) use negative selection strategy to improve the selectivity (6-7)
  3. c) develop general methodologies to transform these DNAzymes/aptamers into new classes of fluorescent sensors using a novel catalytic beacon approach (5,12). In addition, by coupling the DNAzymes/aptamers with gold nanoparticles (13), quantum dots (14), carbon nanotubes (15), supermagnetic iron oxide nanoparticles (16) and many other functional nanomaterials and devices (12b,17) we have developed new with colorimetric, fluorescent electrochemical, and MRI contrast agents for metal ions and a wide range of other targets with high sensitivity (down to 11 ppt, or 14 pM) and selectivity (> 1 million fold selectivity) (6).
  4. d) demonstrate a novel strategy to tune the dynamic range of detection to match those defined by US EPA and CDC (13a,18).
Creation of a User-Friendly Dipstick Sensor
We have recently further simplified DNAzymes/aptamers sensors by developing new label-free signal-reporting mechanisms (19) and developed new MRI contrast agents for non-invasive 3D imaging (16,20).
Furthermore, we have extended the methodology of target-recognition and stimuli-response to the imaging of metal ions and in living cells (21) as well as the selective delivery of imaging agents and anti-cancer drugs (such as cisplatin and doxorubicin) to cancerous cells in vitro and in vivo (22).

In technological development, there are still significant barriers from the public to adopt new devices or technologies developed in academic laboratories. We are exploring a way to overcome these barriers by
  1. a) developing dipstick tests,(18b,23) and by
  2. b) taking advantage of the wide availability and low cost of pocket-sized electrochemical devices such as glucose meters to detect many non-glucose targets ranging from toxic metal ions (e.g., lead and uranium) to recreational drugs (e.g., cocaine) and important biological cofactors (e.g., adenosine) and disease biomarkers (e.g., tuberculosis and prostate cancer) (24). Such methodology has been extended to detect viral DNA (25) and any targets that an antibody can recognize (26). This approach can be readily used by the general public to detect many other non-glucose targets at home and in the field. The Lu group’s patented technology has been licensed to two startup companies ( and who are working in the process of translating the research in the Lu group into products that many people in the world are able to use every day.