Fundamentals of DNAzymes and Sensor Development for environmental monitoring and medical diagnostics
One of the most important discoveries in the last decade is that DNA and RNA are not only materials for genetic information storage and transfer, but also catalysts for a variety of biological reactions, and thus called catalytic DNA or DNAzymes. Metal ions play essential roles in the structure and function of DNAzymes, thus their study has become a new frontier in bioinorganic chemistry. Although many different classes of metal-specific proteins have been studied, similar information about metal-specific DNA/RNA interactions is not available. Using in-vitro selection methods, we have been able to obtain new DNAzymes that can carry out catalysis in the presence of a specific transition metal ion of choice with high selectivity (1-3). These DNAzymes are highly active and versatile (4, 5). Furthermore, they are amenable to spectroscopic studies, allowing in-depth study as in metalloenzymes (6, 7).
By using an in vitro selection method, we have demonstrated that we can obtain DNAzymes specific for any metal ion of interest, such as Pb2+ (4, 8), UO22+ (3) and Hg2+ (9). By employing a negative selection strategy, we have shown that metal selectivity can be dramatically improved (10).
General in vitro selection procedure (left), selected DNAzymes for metal binding (right)
Fundamental studies to further understanding of the selected DNAzymes is of great interest. The majority of previous studies indicate that a metal-dependent global folding step (induced fit) is required before enzymatic reactions. In FRET studies of a 8-17 DNAzyme both in bulk solution and at the single molecule level, we discovered that, in contrast to those observed in 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 (11). 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. The results strongly suggest that DNAzymes can use all modes of activation as protein metalloenzymes use. Other studies using biochemical and biophysical characterization to enhance understanding of various DNAzymes using techniques such as CD and FRET are ongoing (12-15).
Lock and Key DNAzyme
In addition to fundamental studies of this new class of metalloenzymes, we have also been at the forefront of practical applications of the DNAzymes that contribute to technological developments in other fields, such as sensors and materials chemistry. For example, one of the biggest challenges in the field of metal sensors is that the method for developing a sensor for one metal ion (e.g., Ca2+) cannot be generally applicable to designing sensors for other metal ions. Therefore, much trial-and-error is required, making it difficult to design sensors for any metal ion of choice. The issues include a lack of general strategies to:
- a) obtain the sensing molecules;
- b) improve selectivity of the sensing molecules;
- c) convert the molecular recognition events into detectable signals;
- d) fine-tune the dynamic range.
Catalytic Beacon Sensor
By inventing a patented catalytic beacon method of attaching fluorophore/quencher pairs to DNAzymes, we have demonstrated highly sensitive and selective sensors for Pb2+ (8, 10, 16), UO22+ (3), and Hg2+ (17, 18) that are among the best in the field. In some cases, such as in UO22+, the sensitivity (45 pM or 11 ppt) and selectivity (over million fold) rivals that of expensive analytical instruments (e.g., ICP-MS has a detection limit of 420 pM for UO22+). We have extended the methodology to sensing paramagnetic metal ions such as Cu2+ (19), meeting a significant challenge in the metal sensor field, since paramagnetic metal ions often quench fluorescence signals from fluorophores. Recently, a new approach designated Catalytic And Molecular Beacon (CAMB) has been developed, combining efficient quenching with catalytic capability (20). In addition, a classic lead DNAzyme has been reimagined as a highly selective Pb2+ sensor (21)
Colorimetric, MRI-based, and Enantioselective Sensing
By attaching gold nanoparticles or iron oxide nanoparticles to DNAzymes, we have also transformed the DNAzymes and DNA aptamers into colorimetric sensors (22-28) or MRI contrast agents (25, 29), respectively. We have extended the methodology to sensing non-metal ions, such as organic molecules, proteins and viruses (30-33). Aptamer-based sensors capable of chiral discrimination were developed for adenosine and arginine allowing rapid, and separation free, detection (34).
Creation of a User-Friendly Dipstick Sensor
Enhanced Sensitivity and Temperature Tolerance
Recently we have expanded our sensor development to "label-free" methodology, allowing lower cost detection and improved detection limits.(35) By using a variant of DNAzyme, we have demonstrated fine-tuning the dynamic range of the sensors. This unique feature has allowed us to fine-tune the sharpest color change of Pb2+ sensor right at the federal toxic threshold level for lead in paint (10).
We have made the sensors even more user-friendly by converting the colorimetric sensors into a dipstick test (33). The sensor work has already resulted in two issued patents, more than 10 pending patents, and a startup company.
Though excellent systems, sensor designs incorporating fluorophore-labeled DNA can be expensive. As such, we have worked towards label-free sensing using a vacant site approach as well as using the malachite green aptamer. (36, 37, 38)
For a more in-depth overview please see the reviews and book chapters have been authored by the group on many of the topics described above and provide general background on these fields of study (39-43).
- (1) Bruesehoff, P., J.; Li, J.; Augustine, A. J.; Lu, Y. Comb. Chem. High Throughput Screen. 2002, 5, 327-335.
- (2) Nelson, K. E.; Bruesehoff, P. J.; Lu, Y. J. Mol. Evol. 2005, 61, 216-225.
- (3) Liu, J.; Brown, A. K.; Meng, X.; Cropek, D. M.; Istok, J. D.; Watson, D. B.; Lu, Y. Proc. Nat. Acad. Sci. U. S. A. 2007, 104, 2056-2061.
- (4) Li, J.; Zheng, W.; Kwon, A. H.; Lu, Y. Nucleic Acids Res. 2000, 28, 481-488.
- (5) Brown, A. K.; Li, J.; Pavot, C. M. -.; Lu, Y. Biochemistry 2003, 42, 7152-7161.
- (6) Liu, J.; Lu, Y. J. Am. Chem. Soc. 2002, 124, 15208-15216.
- (7) Kim, H.; Liu, J.; Li, J.; Nagraj, N.; Li, M.; Pavot, C. M. -.; Lu, Y. J. Am. Chem. Soc. 2007, 129, 6896-6902.
- (8) Liu, J.; Lu, Y. Angew. Chem. Int. Ed. 2007, 46, 7587-7590.
- (9) Liu, J. W.; Lu, Y. Anal. Chem. 2003, 75, 6666-6672.
- (10) Liu, J.; Lu, Y. J. Am. Chem. Soc. 2007, 129, 9838-9839.
- (11) Li, J.; Lu, Y. J. Am. Chem. Soc. 2000, 122, 10466-10467.
- (12) Kim, H.; Rasnik, I.; Liu, J.; Ha, T.; Lu, Y. Nat. Chem. Biol. 2007, 3, 763-768.
- (13) Kim, H. K.; Li, J.; Nagraj, N.; Lu, Y. Chemistry 2008.
- (14) Brown, A. K.; Liu, J.; He, Y.; Lu, Y. ChemBioChem 2009, 10, 486-492.
- (15) Mazumdar, D.; Nagraj, N.; Kim, H.; Meng, X.; Brown, A. K.; Sun, Q.; Li, W.; Lu, Y. J. Am. Chem. Soc. 2009, 131, 5506-5515.
- (16) Nagraj, N.; Liu, J.; Sterling, S.; Wu, J.; Lu, Y. Chem. Commun. 2009, 4103-4105.
- (17) Liu, J.; Lu, Y. Anal. Chem. 2003, 75, 6666-6672.
- (18) Wang, Z.; Heon Lee, J.; Lu, Y. Chem. Commun. 2008, 6005-6007.
- (19) Liu, J.; Lu, Y. J. Am. Chem. Soc. 2003, 125, 6642-6643.
- (20) Zhang, X.-B.; Wang, Z.; Xing, H.; Xiang, Y.; Lu, Y. Anal. Chem. 2010 82, 5005-5011
- (21) Lan, T.; Furuya, K.; Lu, Y. Chem. Commun. 2010 46, 3896-3898
- (22) Liu, J.; Lu, Y. Chem. Mater. 2004, 16, 3231-3238.
- (23) Liu, J.; Lu, Y. J. Am. Chem. Soc. 2004, 126, 12298-12305.
- (24) Liu, J.; Lu, Y. J. Am. Chem. Soc. 2005, 127, 12677-12683.
- (25) Liu, J.; Lu, Y. Anal. Chem. 2004, 76, 1627-1632.
- (26) Yigit, M. V.; Mazumdar, D.; Kim, H. K.; Lee, J. H.; Odintsov, B.; Lu, Y. Chembiochem 2007, 8, 1675-1678.
- (27) Lee, J. H.; Wang, Z.; Liu, J.; Lu, Y. J. Am. Chem. Soc. 2008 130, 14217-14226
- (28) Liu, J.; Lu, Y. Chem. Commun. 2007, 4872-4874.
- (29) Yigit, M. V.; Mazumdar, D.; Lu, Y. Bioconjug. Chem. 2008, 19, 412-417.
- (30) Liu, J. W.; Lu, Y. J. Fluoresc. 2004, 14, 343-354.
- (31) Liu, J.; Lu, Y. Angew. Chem. Int. Ed. 2006, 45, 90-94.
- (32) Liu, J.; Lu, Y. Nat. Protoc. 2006, 1, 246-252.
- (33) Liu, J.; Mazumdar, D.; Lu, Y. Angew. Chem. Int. Ed. 2006, 45, 7955-7959.
- (34) Null, E. L.; Lu, Y. Analyst 2010 135, 419-422
- (35) Wang, Z.; Lee, J. H.; Lu, Y. Adv. Mater. 2008, 20, 3263-3267.
- (36) Xiang, Y.; Tong, A.; Lu, Y. J. Am. Chem. Soc. 2009 131, 15352-15357
- (37) Xu, W.; Lu, Y. Anal. Chem. 2010 82, 574-578
- (38) Xiang, Y.; Wang, Z.; Xing, H.; Wong, N. Y.; Lu, Y. Anal. Chem. 2010 82, 4122-4129
- (39) Wernette, D. P.; Liu, J.; Bohn, P. W.; Lu, Y. MRS Bull 2008, 33, 34-41.
- (40) Mazumdar, D.; Liu, J.; Lu, Y. Nanotechnol. Appl. Clean Water 2009, 427-446.
- (41) Wang, Z.; Lu, Y. J. Mater. Chem. 2009, 19, 1788-1798.
- (42) Lu, Y.; Liu, J.; Mazumdar, D. Methods Mol. Biol. 2009, 535, 223-239.
- (43) Liu, J.; Cao, Z.; Lu, Y. Chem. Rev. 2009, 109, 1948-1998.