We propose to develop advanced fluorogenic probes that use the principle of RNA- templated chemistry to report on the presence of specific disease-relevant RNAs in solution and in cells. The analysis of cellular RNAs is increasingly important to diagnosis, treatment and prognosis of disease;however, current clinical methods for detection and identification of native RNAs are laborious and expensive, and cannot be applied in intact cells or tissues. Our recent work has developed designs for fluorogenic probes that offer the highest sequence specificity and lowest background of any probes yet reported, and thus offers much promise for solving these problems. However, further development is required in lowering background signal for the rarest targets, and for addressing folded target structure. Moreover, as with most DNA probes, the issue of probe delivery needs to be addressed (for whole-cell applications) by taking advantage of recently developed next-generation delivery agents. Our proposed work will address these issues by (a) developing novel chemistries and strategies that focus and intensify signal and lower background markedly;(b) exploring new molecular transporters, based on oligoguanidine-lipid conjugates, for enhancing cellular uptake, and by (c) collaboratively studying the detection and identification of type A influenza RNAs in vitro and directly in cells. In the near term covered by this proposal, this work is significant for its development of new chemistries that could lower background and enhance signal for RNA detection markedly. Moreover, it will address some of the most important and widely recognized problems that are faced in the nucleic acids field, including the problems of target accessibility and cell permeability. The work is novel as well: we will develop previously unreported strategies for signaling including template-triggered fluorophore release and capture, and templated chemiluminescence, as well as previously untested molecular strategies for cell uptake. In the longer term, the development of low-background fluorogenic templated probes could allow for unprecedented and widespread applications in medicine.

Public Health Relevance

Rapid, simple and inexpensive methods are needed for identifying cellular RNAs, many of which are directly connected to human disease. We propose to develop templated chemistry, in which two modified DNA probes bind side-by-side on the RNA target, triggering a chemical reaction to detect and identify these RNAs by a fluorescence signal. The planned work will provide useful new tools for biomedical research and for diagnosis of disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM068122-10
Application #
8687666
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Preusch, Peter
Project Start
2003-06-01
Project End
2017-04-30
Budget Start
2014-05-01
Budget End
2015-04-30
Support Year
10
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Stanford University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
City
Stanford
State
CA
Country
United States
Zip Code
94304
Oertell, Keriann; Harcourt, Emily M; Mohsen, Michael G et al. (2016) Kinetic selection vs. free energy of DNA base pairing in control of polymerase fidelity. Proc Natl Acad Sci U S A 113:E2277-85
Flynn, Ryan A; Do, Brian T; Rubin, Adam J et al. (2016) 7SK-BAF axis controls pervasive transcription at enhancers. Nat Struct Mol Biol 23:231-8
Ji, Debin; Mohsen, Michael G; Harcourt, Emily M et al. (2016) ATP-Releasing Nucleotides: Linking DNA Synthesis to Luciferase Signaling. Angew Chem Int Ed Engl 55:2087-91
Roost, Caroline; Lynch, Stephen R; Batista, Pedro J et al. (2015) Structure and thermodynamics of N6-methyladenosine in RNA: a spring-loaded base modification. J Am Chem Soc 137:2107-15
Larsen, Dennis; Pittelkow, Michael; Karmakar, Saswata et al. (2015) New organocatalyst scaffolds with high activity in promoting hydrazone and oxime formation at neutral pH. Org Lett 17:274-7
Karmakar, Saswata; Harcourt, Emily M; Hewings, David S et al. (2015) Organocatalytic removal of formaldehyde adducts from RNA and DNA bases. Nat Chem 7:752-8
Xu, Liang; Da, Linati; Plouffe, Steven W et al. (2014) Molecular basis of transcriptional fidelity and DNA lesion-induced transcriptional mutagenesis. DNA Repair (Amst) 19:71-83
Kool, Eric T; Crisalli, Pete; Chan, Ke Min (2014) Fast alpha nucleophiles: structures that undergo rapid hydrazone/oxime formation at neutral pH. Org Lett 16:1454-7
Spitale, Robert C; Flynn, Ryan A; Torre, Eduardo A et al. (2014) RNA structural analysis by evolving SHAPE chemistry. Wiley Interdiscip Rev RNA 5:867-81
Xu, Liang; Butler, Kyle Vincent; Chong, Jenny et al. (2014) Dissecting the chemical interactions and substrate structural signatures governing RNA polymerase II trigger loop closure by synthetic nucleic acid analogues. Nucleic Acids Res 42:5863-70

Showing the most recent 10 out of 36 publications