Welcome to the McNamara Lab!
Our research group is focused on developing rapid diagnostic assays that detect disease-associated nuclease activities. The molecular foundation of our approach is quenched fluorescent oligonucleotide substrates that are engineered (via sequence and nucleotide modifications) to be selectively activatable by disease-specific target nucleases. This format entails labeling of the engineered oligonucleotide with a fluorophore on one end and a fluorescence quencher on the other end (see Figure).
The fluorophore is not fluorescent in the intact oligonucleotide due to its close proximity to the quencher. Upon digestion of the oligonucleotide, the quencher diffuses from the fluorophore which then exhibits fluorescence. The source of the target nuclease depends on the nature of the disease. For instance, in the case of infectious diseases, we are targeting nucleases produced by bacterial or viral pathogens; for cancer, we are targeting cancer-specific endogenous nucleases. Current projects include
development of a non-invasive imaging approach for focal Staphylococcus aureus (S. aureus) infections (see Hernandez et al., Nature Medicine, 2014);
creation of a rapid diagnostic assay for S. aureus bacteremia (see Burghardt et al., PLoS One, 2016);
generation of a rapid diagnostic assay for E. coli urinary tract infections (initial manuscript in preparation);
development of a rapid diagnostic assay for breast cancer via detection of circulating tumor cell- associated nucleases (initial manuscript in preparation).
Among the more mature projects is an oligonucleotide-based approach for non-invasive imaging of bacterial infections. This approach exploits the ability of bacterial nucleases to degrade synthetic oligonucleotides that, due to particular chemical modifications, are resistant to degradation by mammalian serum nucleases (see Hernandez et al., Nature Medicine, 2014). Our recent results demonstrate that we can detect and localize focal S. aureus infections in mice in less than 30 minutes with this approach. These results compare favorably with the current methods (biopsy and culture) which take at least a day to detect S. aureus in such infections. We have also developed methods for detecting bacteria in vitro in various clinical samples, such as blood and urine. This includes development of rapid assays for S. aureus bacteremia and E. coli urinary tract infections. S. aureus bacteremia is a substantial clinical problem with a high mortality rate. Current diagnostic methods require blood culturing methods that take more than a day to identify the causative pathogen. Our latest assay can rapidly identify S. aureus in blood without the need for culture (see Burghardt, et al., PLoS One, 2016). Similarly, current methods for reliably diagnosing urinary tract infections (the most common infection) require urine culturing methods that take more than a day. Our current assay for E. coli (the most common uropathogen) can detect this species in patient urine in under 3 hours. We anticipate that these approaches can address important unmet needs for infectious disease diagnostics. In the context of these ongoing projects, we are developing the basis for producing more advanced probes and more complex assays that we expect will greatly facilitate our ability to detect and identify target nucleases. For instance, we have identified the target nuclease of E. coli. In collaboration with Dr. Catherine Musselman, a structural biologist here, we have also generated diffactable crystals of a recombinant form of the protein and have developed a preliminary structural model. We anticipate that this structure may facilitate development of more specific assays for the nuclease. Finally, we have plans to develop multiplexed assays in which we use multiple probes with different-colored fluorophores to simultaneously detect multiple, distinct nucleases. Altogether, we expect these approaches will greatly enhance our ability to rapidly detect and identify a variety of disease-specific target nucleases, and to thereby provide the means of addressing a variety of unmet clinical diagnostic needs.
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Our group currently includes a graduate student from the Cellular and Molecular Biology Program and two research assistants. We share a common interest in developing molecular tools that will expedite the initiation of effective therapies for patients with bacterial infections.
- Rosman CWK, Romero Pastrana F, Buist G, Heuker M, van Oosten M, McNamara JO, van Dam GM, van Dijl JM. Ex Vivo Tracer Efficacy in Optical Imaging of Staphylococcus Aureus Nuclease Activity. Sci Rep. 2018 Jan 22;8(1):1305. doi: 10.1038/s41598-018-19289-y. PMID: 29358617
Kruspe S, Dickey DD, Urak KT, Blanco GN, Miller MJ, Clark KC, Burghardt E, Gutierrez WR, Phadke SD, Kamboj S, Ginader T, Smith BJ, Grimm SK, Schappet J, Ozer H, Thomas A, McNamara JOII, Chan CH, Giangrande PH. Rapid and Sensitive Detection of Breast Cancer Cells in Patient Blood with Nuclease-Activated Probe Technology. Molecular Therapy Nucleic Acids. August 28, 2017. DOI: http://dx.doi.org/10.1016/j.omtn.2017.08.004.
Flenker KS, Burghardt EL, Dutta N, Burns WJ, Grover JM, Kenkel EJ, Weaver TM, Mills J, Kim H, Huang L, Owczarzy R, Musselman CA, Behlke MA, Ford B, McNamara JO 2nd. Rapid Detection of Urinary Tract Infections via Bacterial Nuclease Activity. Mol Ther. 2017 Apr 5. pii: S1525-0016(17)30113-2. doi: 10.1016/j.ymthe.2017.03.015. [Epub ahead of print]
Tiet P, Clark KC, McNamara JO, Berlin JM. Colorimetric Detection of Staphylococcus aureus Contaminated Solutions without Purification. Bioconjug Chem. 2017 Jan 18;28(1):183-193. doi: 10.1021/acs.bioconjchem.6b00571.
Burghardt EL, Flenker KS, Clark KC, Miguel J, Ince D, Winokur P, Ford B, McNamara JO.Rapid, Culture-free Detection of Staphylococcus aureus Bacteremia. PLoS One. 11(6):e015723.
Hernandez FJ, Huang L, Olson ME, Powers KM, Hernandez LI, Meyerholz DK, Thedens DR, Behlke MA, Horswill AR, McNamara JO. Noninvasive Imaging of Staphylococcus aureus Infections with a Nuclease-Activated Probe. Nature Medicine. 20(3):301-306.
Huang YZ, Hernandez FJ, Gu B, Stockdale KR, Nanapaneni K, Scheetz TE, Behlke MA, Peek AS, Bair T, Giangrande PH, McNamara JO. RNA Aptamer-based Functional Ligands of the Neurotrophin Receptor, TrkB. Molecular Pharmacology. 82(4):623-35.
Hernandez FJ, Stockdale KR, Huang L, Horswill AR, Behlke MA, McNamara JO. Degradation of Nuclease-Stabilized RNA Oligonucleotides in Mycoplasma-Contaminated Cell Culture Media. Nucleic Acid Therapeutics. 22:58-68.
McNamara JO, Kolonias D, Pastor F, Mittler RS, Chen L, Giangrande PH, Sullenger B, Gilboa E. Multivalent 4-1BB binding aptamers costimulate CD8+ T cells and inhibit tumor growth in mice. Journal of Clinical Investigation. 118(1):376-86.
McNamara JO, Andrechek E, Wang Y, Viles KD, Rempel RE, Gilboa E, Sullenger BA, Giangrande PH. Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras.Nature Biotechnology. 24(8):1005-1015.