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
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).
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:
- a) obtain molecules for any targets of interests;
- b) improve selectivity of the molecules;
- c) transform the binding into detectable signals, and
- d) tune dynamic range to match the concentration levels of the targets.
- a) use a combinatorial method called
in vitroselection 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);
- b) use negative selection strategy to improve the selectivity (6-7)
- 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).
- d) demonstrate a novel strategy to tune the dynamic range of detection to match those defined by US EPA and CDC (13a,18).
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
- a) developing dipstick tests,(18b,23) and by
- 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 (www.ANDalyze.com and www.GlucoSentient.com) 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.
*Click to Expand Table*
- 1. (a) Y. Lu Chemistry 2002, 8, 4588-4596 (b) Zehui Cao; Yi Lu In Metal Complex–DNA Interactions; John Wiley & Sons, Ltd, 2009 (c) Tian Lan; Yi Lu In Interplay between Metal Ions and Nucleic Acids; Astrid Sigel, Helmut Sigel, Roland K. O. Sigel, Eds.; Springer Netherlands, 2012; Vol. 10 (d) J. W. Liu; Z. H. Cao; Y. Lu Chemical Reviews 2009, 109, 1948-1998 (e) H. Xing; N. Y. Wong; Y. Xiang; Y. Lu Current Opinion in Chemical Biology 2012, 16, 429-435 (f) Lele Li; Yi Lu In DNA Nanotechnology; Chunhai Fan, Ed.; Springer Berlin Heidelberg, 2013.
- 2.Functional Nucleic Acids for Sensing and Other Analytical Applications; Yingfu Li; Yi Lu, Eds.; Springer: New York, NY, 2009.
- 3. H. E. Ihms; Y. Lu Methods Mol Biol 2012, 848, 297-316.
- 4. (a) J. Li; W. C. Zheng; A. H. Kwon; Y. Lu Nucleic Acids Research 2000, 28, 481-488 (b) K. E. Nelson; P. J. Bruesehoff; Y. Lu Journal of Molecular Evolution 2005, 61, 216-225.
- 5. J. Li; Y. Lu Journal of the American Chemical Society 2000, 122, 10466-10467.
- 6. J. W. Liu; A. K. Brown; X. L. Meng; D. M. Cropek; J. D. Istok; D. B. Watson; Y. Lu Proceedings of the National Academy of Sciences of the United States of America 2007, 104, 2056-2061.
- 7. P. J. Bruesehoff; J. Li; A. J. Angustine; Y. Lu Combinatorial Chemistry & High Throughput Screening 2002, 5, 327-335.
- 8. (a) A. K. Brown; J. Li; C. M. B. Pavot; Y. Lu Biochemistry 2003, 42, 7152-7161 (b) A. K. Brown; J. W. Liu; Y. He; Y. Lu Chembiochem 2009, 10, 486-492 (c) K. E. Nelson; H. E. Ihms; D. Mazumdar; P. J. Bruesehoff; Y. Lu Chembiochem 2012, 13, 381-391 (d) M. Cepeda-Plaza; E. L. Null; Y. Lu Nucleic acids research 2013.
- 9. (a) J. Liu; Y. Lu Journal of the American Chemical Society 2002, 124, 15208-15216 (b) J. Liu; Y. Lu Methods in molecular biology 2006, 335, 257-271 (c) H. K. Kim; J. W. Liu; J. Li; N. Nagraj; M. X. Li; C. M. B. Pavot; Y. Lu Journal of the American Chemical Society 2007, 129, 6896-6902 (d) H. K. Kim; I. Rasnik; J. W. Liu; T. J. Ha; Y. Lu Nature Chemical Biology 2007, 3, 763-768 (e) B. Ravel; S. C. Slimmer; X. Meng; G. C. L. Wong; Y. Lu Radiation Physics and Chemistry 2009, 78, S75-S79 (f) D. Mazumdar; N. Nagraj; H. K. Kim; X. L. Meng; A. K. Brown; Q. Sun; W. Li; Y. Lu Journal of the American Chemical Society 2009, 131, 5506-5515 (g) Y. He; Y. Lu Chemistry-a European Journal 2011, 17, 13732-13742.
- 10. H. K. Kim; J. Li; N. Nagraj; Y. Lu Chemistry-a European Journal 2008, 14, 8696-8703.
- 11. (a) Y. Lu; J. W. Liu; J. Li; P. J. Bruesehoff; C. M. B. Pavot; A. K. Brown Biosensors & Bioelectronics 2003, 18, 529-540 (b) H. Liu; Y. Xiang; Y. Lu; R. M. Crooks Angewandte Chemie-International Edition 2012, 51, 6925-6928 (c) D. P. Wernette; J. W. Liu; P. W. Bohn; Y. Lu Mrs Bulletin 2008, 33, 34-41 (d) Y. Lu; J. W. Liu Wiley Interdisciplinary Reviews-Nanomedicine and Nanobiotechnology 2009, 1, 35-46 (e) Z. D. Wang; Y. Lu Journal of Materials Chemistry 2009, 19, 1788-1798 (f) Juewen Liu; Yi Lu In Functional Nucleic Acids for Analytical Applications; Li Yingfu, Lu Yi, Eds.; Springer New York, 2009 (g) D. Mazumdar; J. W. Liu; Y. Lu Nanotechnology Applications for Clean Water 2009, 427-446 (h) X. B. Zhang; R. M. Kong; Y. Lu Annual Review of Analytical Chemistry, Vol 4 2011, 4, 105-128 (i) Nandini Nagraj; Yi Lu In Nucleic Acid Biosensors for Environmental Pollution Monitoring; The Royal Society of Chemistry, 2011 (j) J. H. Lee; M. V. Yigit; D. Mazumdar; Y. Lu Advanced Drug Delivery Reviews 2010, 62, 592-605 (k) JungHeon Lee; Zidong Wang; Yi Lu In Molecular Biological Technologies for Ocean Sensing; Sonia M. Tiquia-Arashiro, Ed.; Humana Press, 2012 (l) Yu Xiang; Peiwen Wu; LiHuey Tan; Yi Lu; Springer Berlin Heidelberg, 2013.
- 12. (a) T. Lan; K. Furuya; Y. Lu Chemical Communications 2010, 46, 3896-3898 (b) C. B. Swearingen; D. P. Wernette; D. M. Cropek; Y. Lu; J. V. Sweedler; P. W. Bohn Analytical Chemistry 2005, 77, 442-448 (c) J. W. Liu; Y. Lu Analytical Chemistry 2003, 75, 6666-6672 (d) J. Liu; Y. Lu Methods in molecular biology 2006, 335, 275-288 (e) J. Liu; Y. Lu Journal of the American Chemical Society 2007, 129, 9838-9839 (f) J. Liu; Y. Lu Angewandte Chemie-International Edition 2007, 46, 7587-7590 (g) Z. D. Wang; J. H. Lee; Y. Lu Chemical Communications 2008, 6005-6007 (h) N. Nagraj; J. W. Liu; S. Sterling; J. Wu; Y. Lu Chemical Communications 2009, 4103-4105 (i) X. B. Zhang; Z. D. Wang; H. Xing; Y. Xiang; Y. Lu Analytical Chemistry 2010, 82, 5005-5011 (j) E. L. Null; Y. Lu Analyst 2010, 135, 419-422 (k) Y. Xiang; Y. Lu Analytical Chemistry 2012, 84, 9981-9987.
- 13. (a) J. W. Liu; Y. Lu Journal of the American Chemical Society 2003, 125, 6642-6643 (b) J. W. Liu; Y. Lu Analytical Chemistry 2004, 76, 1627-1632 (c) J. W. Liu; Y. Lu Journal of Fluorescence 2004, 14, 343-354 (d) J. W. Liu; Y. Lu Chemistry of Materials 2004, 16, 3231-3238 (e) J. W. Liu; Y. Lu Journal of the American Chemical Society 2004, 126, 12298-12305 (f) J. Liu; Y. Lu Journal of the American Chemical Society 2005, 127, 12677-12683 (g) J. Liu; Y. Lu Nature Protocols 2006, 1, 246-252 (h) J. W. Liu; Y. Lu Angewandte Chemie-International Edition 2006, 45, 90-94(i) J. W. Liu; Y. Lu Advanced Materials 2006, 18, 1667-1671 (j) J. Liu; Y. Lu Chemical communications 2007, 4872-4874 (k) J. W. Liu; Y. Lu Journal of the American Chemical Society 2007, 129, 8634-8643 (l) J. H. Lee; Z. D. Wang; J. W. Liu; Y. Lu Journal of the American Chemical Society 2008, 130, 14217-14226.
- 14. J. W. Liu; J. H. Lee; Y. Lu Analytical Chemistry 2007, 79, 4120-4125.
- 15. T. J. Yim; J. W. Liu; Y. Lu; R. S. Kane; J. S. Dordick Journal of the American Chemical Society 2005, 127, 12200-12201.
- 16. (a) M. V. Yigit; D. Mazumdar; H. K. Kim; J. H. Lee; B. Dintsov; Y. Lu Chembiochem 2007, 8, 1675-1678 (b) M. V. Yigit; D. Mazumdar; Y. Lu Bioconjugate Chemistry 2008, 19, 412-417.
- 17. (a) I. H. Chang; J. J. Tulock; J. Liu; W. S. Kim; D. M. Cannon, Jr.; Y. Lu; P. W. Bohn; J. V. Sweedler; D. M. Cropek Environmental science & technology 2005, 39, 3756-3761 (b) D. P. Wernette; C. B. Swearingen; D. M. Cropek; Y. Lu; J. V. Sweedler; P. W. Bohn Analyst 2006, 131, 41-47(c) D. P. Wernette; C. Mead; P. W. Bohn; Y. Lu Langmuir 2007, 23, 9513-9521 (d) T. S. Dalavoy; D. P. Wernette; M. J. Gong; J. V. Sweedler; Y. Lu; B. R. Flachsbart; M. A. Shannon; P. W. Bohn; D. M. Cropek Lab on a Chip 2008, 8, 786-793 (e) Y. Lu; J. W. Liu Current Opinion in Biotechnology 2006, 17, 580-588 (f) L. L. Li; P. H. Ge; P. R. Selvin; Y. Lu Analytical Chemistry 2012, 84, 7852-7856 (g) Longhua Tang; Ik Su Chun; Zidong Wang; Jinghong Li; Xiuling Li; Yi Lu Analytical Chemistry 2013, 85, 9522-9527.
- 18. J. W. Liu; Y. Lu Organic & Biomolecular Chemistry 2006, 4, 3435-3441 (b) D. Mazumdar; J. W. Liu; G. Lu; J. Z. Zhou; Y. Lu Chemical Communications 2010, 46, 1416-1418.
- 19. (a) Z. D. Wang; J. H. Lee; Y. Lu Advanced Materials 2008, 20, 3263-3267 (b) Y. Xiang; A. J. Tong; Y. Lu Journal of the American Chemical Society 2009, 131, 15352-15357 (c) W. C. Xu; Y. Lu Analytical Chemistry 2010, 82, 574-578 (d) P. S. Song; Y. Xiang; H. Xing; Z. J. Zhou; A. J. Tong; Y. Lu Analytical Chemistry 2012, 84, 2916-2922.
- 20. (a) (a) W. C. Xu; Y. Lu Chemical Communications 2011, 47, 4998-5000 (b) Weichen Xu; Hang Xing; Yi Lu Analyst 2013, 138, 6266-6269.
- 21. (a) M. V. Yigit; A. Mishra; R. Tong; J. J. Cheng; G. C. L. Wong; Y. Lu Chemistry & Biology 2009, 16, 937-942 (b) P. W. Wu; K. V. Hwang; T. Lan; Y. Lu Journal of the American Chemical Society 2013, 135, 5254-5257.
- 22. (a) Z. H. Cao; R. Tong; A. Mishra; W. C. Xu; G. C. L. Wong; J. J. Cheng; Y. Lu Angewandte Chemie-International Edition 2009, 48, 6494-6498 (b) Hang Xing; Li Tang; Xujuan Yang; Kevin Hwang; Wendan Wang; Qian Yin; Ngo Yin Wong; Lawrence W. Dobrucki; Norio Yasui; John A. Katzenellenbogen; William G. Helferich; Jianjun Cheng; Yi Lu Journal of Materials Chemistry B 2013, 1, 5288-5297 (c) L. L. Li; Q. Yin; J. J. Cheng; Y. Lu Advanced Healthcare Materials 2012, 1, 567-572 (d) L. L. Li; M. Y. Xie; J. Wang; X. Y. Li; C. Wang; Q. Yuan; D. W. Pang; Y. Lu; W. H. Tan Chemical Communications 2013, 49, 5823-5825 (e) L. L. Li; P. W. Wu; K. Hwang; Y. Lu Journal of the American Chemical Society 2013, 135, 2411-2414.
- 23. (a) J. W. Liu; D. Mazumdar; Y. Lu Angewandte Chemie-International Edition 2006, 45, 7955-7959 (b) Yi Lu; Juewen Liu; Debapriya Mazumdar In Nucleic Acid and Peptide Aptamers; Günter Mayer, Ed.; Humana Press, 2009; Vol. 535 (c) S. F. Torabi; Y. Lu Faraday Discussions 2011, 149, 125-135.
- 24. (a) Y. Xiang; Y. Lu Nature Chemistry 2011, 3, 697-703 (b) Y. Xiang; Y. Lu Chemical Communications 2013, 49, 585-587.
- 25. Y. Xiang; Y. Lu Analytical Chemistry 2012, 84, 1975-1980.
- 26. Y. Xiang; Y. Lu Analytical Chemistry 2012, 84, 4174-4178.