The recent explosion of genomic data has offered an unprecedented opportunity to study the molecular basis for developmental and disease processes. Although microarray techniques for profiling the genes expressed in a given tissue have become commonplace, methods for determining the exact spatial and temporal extent of gene expression are still in need of improvement. In situ hybridization techniques for gene expression studies often have limited sensitivity and significant background signal even in locations where no target genes are present. Furthermore, existing technologies do not fully exploit the potential for imaging many genes simultaneously. The goal of the proposed research is to adapt a newly developed nanosensor technology for multiplexed in situ amplification of gene expression. This amplification tool, termed hybridization chain reaction (HCR), reduces background by activating only when a probe molecule binds specifically to its target. This event triggers the self-assembly of a tethered 'polymer' from fluorescently-labeled DMA hairpins. Parallel multiplexing can be achieved simply by using independent HCR amplifiers for each unique target species. The research plan involves the design, validation and application of in situ HCR amplifiers.
Specific aims are: 1: Design triggered, multiplexed, nonlinear HCR amplifiers, and refine the computational tools for the design of HCR amplifiers. 2: Validate the spatial localization, sensitivity, specificity and multiplexing of the amplifiers in situ using nanolithography techniques to precisely pattern target molecules on surfaces. 3: Apply HCR to in situ hybridization of patterning genes in avian embryos, testing for the co- expression of genetic markers predicted by previous studies. The objective is to develop in situ HCR amplifiers that will enable the sensitive and simultaneous detection of numerous targets in biological specimens. If successful, this nanotechnology will serve as an important adjunct to modern genomic and proteomic tools in settings ranging from biological experiments to tissue biopsies.

Agency
National Institute of Health (NIH)
Institute
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Type
Research Project (R01)
Project #
5R01EB006192-04
Application #
7448652
Study Section
Special Emphasis Panel (ZRG1-BCMB-A (50))
Program Officer
Korte, Brenda
Project Start
2005-09-22
Project End
2010-07-31
Budget Start
2008-08-01
Budget End
2010-07-31
Support Year
4
Fiscal Year
2008
Total Cost
$233,139
Indirect Cost
Name
California Institute of Technology
Department
Engineering (All Types)
Type
Schools of Engineering
DUNS #
009584210
City
Pasadena
State
CA
Country
United States
Zip Code
91125
Choi, Harry M T; Schwarzkopf, Maayan; Fornace, Mark E et al. (2018) Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust. Development 145:
Trivedi, Vikas; Choi, Harry M T; Fraser, Scott E et al. (2018) Multidimensional quantitative analysis of mRNA expression within intact vertebrate embryos. Development 145:
Choi, Harry M T; Calvert, Colby R; Husain, Naeem et al. (2016) Mapping a multiplexed zoo of mRNA expression. Development 143:3632-3637
Schwarzkopf, Maayan; Pierce, Niles A (2016) Multiplexed miRNA northern blots via hybridization chain reaction. Nucleic Acids Res 44:e129
Shah, Sheel; Lubeck, Eric; Schwarzkopf, Maayan et al. (2016) Single-molecule RNA detection at depth by hybridization chain reaction and tissue hydrogel embedding and clearing. Development 143:2862-7
Huss, David; Choi, Harry M T; Readhead, Carol et al. (2015) Combinatorial analysis of mRNA expression patterns in mouse embryos using hybridization chain reaction. Cold Spring Harb Protoc 2015:259-68
Choi, Harry M T; Beck, Victor A; Pierce, Niles A (2014) Next-generation in situ hybridization chain reaction: higher gain, lower cost, greater durability. ACS Nano 8:4284-94
Rosenthal, Adam Z; Zhang, Xinning; Lucey, Kaitlyn S et al. (2013) Localizing transcripts to single cells suggests an important role of uncultured deltaproteobacteria in the termite gut hydrogen economy. Proc Natl Acad Sci U S A 110:16163-8
Tu, Chuqiao; Osborne, Elizabeth A; Louie, Angelique Y (2011) Activatable T? and T? magnetic resonance imaging contrast agents. Ann Biomed Eng 39:1335-48
Tu, Chuqiao; Ma, Xuchu; Pantazis, Periklis et al. (2010) Paramagnetic, silicon quantum dots for magnetic resonance and two-photon imaging of macrophages. J Am Chem Soc 132:2016-23

Showing the most recent 10 out of 11 publications