Novel Probe for Quantitative Imaging of Gene Expression

Summary

Principal Investigator: Andrew Tsourkas
Abstract: We propose to develop a molecular imaging probe that will provide quantitative information on the[unreadable] expression level of mRNA with spatial and temporal resolution. Specifically, an oligonucleotide-based probe[unreadable] will be designed to form a stem-loop structure and will be labeled with a 'reporter' fluorophore at one end and[unreadable] a quencher at the other, analogous to a molecular beacon; however, the oligonucleotide will also be labeled[unreadable] with a second optically distinct 'reference' dye/nanoparticle, which will be selected such that it is unquenched[unreadable] regardless of the conformation of the probe. Fluorescently labeled neutravidin and quantum dots will be[unreadable] tested for their suitability in serving as the reference dye. We hypothesize that beneficial features of this[unreadable] novel probe compared with conventional molecular beacons will include (1) the ability to monitor transfection[unreadable] efficiency due to the presence of the unquenched reference dye. This will reduce false-negatives by[unreadable] allowing for the differentiation between untransfected cells and cells with low levels of gene expression. (2)[unreadable] The ability to remove via ratiometric imaging (i.e. reporter fluorscence/reference fluorescence) the impact of[unreadable] instrumental and experimental variability. (3) The ability to quantitatively compare variations in gene[unreadable] expression levels between samples, between cells within individual samples, and even between sub-cellular[unreadable] compartments by using the reference dye as a point of reference (4) The ability to quantify gene expression[unreadable] with spatial and temporal resolution since the covalent linkage between the reporter and reference dye[unreadable] ensures they exhibit an equivalent intracellular lifetime and co-localization pattern. (5) The ability to use the[unreadable] quantum dot/neutravidin as a platform to attach targeting agents, opening up the possibility for in vivo[unreadable] imaging. (6) The possibility of an improved signal-to-background due to quenching of the 'reporter' dye by[unreadable] both the quencher molecule and the 'reference' dye. To evaluate these features we will pursue two major[unreadable] aims during the proposed research: 1) We will design, synthesize and characterize the 'quantitative'[unreadable] molecular beacon (QMB) in terms of its signal-to-background and lower detection limit (in vitro and in vivo)[unreadable] and 2) we will evaluate the ability of the QMBs to quantify endogenous mRNA expression in breast cancer[unreadable] cells in real-time. It is envisioned that the approach proposed here will allow significant advancements in our[unreadable] understanding of human health and disease and could potentially prove to be a powerful diagnostic tool.
Funding Period: 2006-09-26 - 2009-08-31
more information: NIH RePORT

Top Publications

  1. pmc Imaging the directed transport of single engineered RNA transcripts in real-time using ratiometric bimolecular beacons
    Xuemei Zhang
    Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America
    PLoS ONE 9:e85813. 2014
  2. pmc Quantitative assessment of ratiometric bimolecular beacons as a tool for imaging single engineered RNA transcripts and measuring gene expression in living cells
    Xuemei Zhang
    Department of Bioengineering, University of Pennsylvania, 210 S 33rd Street, 240 Skirkanich Hall, Philadelphia, PA 19104, USA, Department of Biology, University of Pennsylvania, 433 S University Ave, 102 Leidy Laboratories, Philadelphia, PA 19104, USA and Integrated DNA Technologies, Inc, 1710 Commercial Park, Coralville, IA 52241, USA
    Nucleic Acids Res 41:e152. 2013
  3. ncbi Delivery of molecular beacons for live-cell imaging and analysis of RNA
    Antony K Chen
    Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA
    Methods Mol Biol 714:159-74. 2011
  4. ncbi Quantification of miRNA abundance in single cells using locked nucleic acid-FISH and enzyme-labeled fluorescence
    Jing Lu
    Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA
    Methods Mol Biol 680:77-88. 2011
  5. pmc Ratiometric bimolecular beacons for the sensitive detection of RNA in single living cells
    Antony K Chen
    Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA
    Nucleic Acids Res 38:e148. 2010
  6. pmc Sub-cellular trafficking and functionality of 2'-O-methyl and 2'-O-methyl-phosphorothioate molecular beacons
    Antony K Chen
    Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104 and Integrated DNA Technologies, Inc, Coralville, IA 52241, USA
    Nucleic Acids Res 37:e149. 2009
  7. pmc Imaging individual microRNAs in single mammalian cells in situ
    Jing Lu
    Department of Bioengineering, University of Pennsylvania School of Engineering and Applied Sciences, Philadelphia, PA 19104, USA
    Nucleic Acids Res 37:e100. 2009
  8. pmc Fluorescent probes for live-cell RNA detection
    Gang Bao
    Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia 30332, USA
    Annu Rev Biomed Eng 11:25-47. 2009
  9. pmc Assessing the sensitivity of commercially available fluorophores to the intracellular environment
    Antony K Chen
    Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA
    Anal Chem 80:7437-44. 2008
  10. pmc Avoiding false-positive signals with nuclease-vulnerable molecular beacons in single living cells
    Antony K Chen
    Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA
    Nucleic Acids Res 35:e105. 2007

Scientific Experts

  • Andrew Tsourkas
  • Gang Bao
  • Jing Lu
  • Antony K Chen
  • Mark A Behlke
  • Xuemei Zhang
  • Lingyan Huang
  • Allison L Zajac
  • Akash Y Shah
  • Virzhiniya Lekova
  • Ling Huang
  • Arjun Raj
  • Yang Song
  • Won Jong Rhee
  • Olga Davydenko
  • Zhiliang Cheng

Detail Information

Publications10

  1. pmc Imaging the directed transport of single engineered RNA transcripts in real-time using ratiometric bimolecular beacons
    Xuemei Zhang
    Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America
    PLoS ONE 9:e85813. 2014
    ..RNA motion was readily characterized by both mean squared displacement and moment scaling spectrum analyses. These analyses revealed clear examples of directed, Brownian, and subdiffusive movements. ..
  2. pmc Quantitative assessment of ratiometric bimolecular beacons as a tool for imaging single engineered RNA transcripts and measuring gene expression in living cells
    Xuemei Zhang
    Department of Bioengineering, University of Pennsylvania, 210 S 33rd Street, 240 Skirkanich Hall, Philadelphia, PA 19104, USA, Department of Biology, University of Pennsylvania, 433 S University Ave, 102 Leidy Laboratories, Philadelphia, PA 19104, USA and Integrated DNA Technologies, Inc, 1710 Commercial Park, Coralville, IA 52241, USA
    Nucleic Acids Res 41:e152. 2013
    ..Overall, these findings highlight the robustness and versatility of RBMBs as a tool for imaging RNA in live cells. We envision that the unique capabilities of RBMBs will open up new avenues for RNA research. ..
  3. ncbi Delivery of molecular beacons for live-cell imaging and analysis of RNA
    Antony K Chen
    Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA
    Methods Mol Biol 714:159-74. 2011
    ..Strategies for acquiring ratiometric measurements are also described...
  4. ncbi Quantification of miRNA abundance in single cells using locked nucleic acid-FISH and enzyme-labeled fluorescence
    Jing Lu
    Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA
    Methods Mol Biol 680:77-88. 2011
    ..The dynamic range was found to span over three orders of magnitude and the average miRNA copy number per cell was within 17.5% of measurements acquired by quantitative RT-PCR...
  5. pmc Ratiometric bimolecular beacons for the sensitive detection of RNA in single living cells
    Antony K Chen
    Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA
    Nucleic Acids Res 38:e148. 2010
    ..Combined, these attributes enabled RBMBs to exhibit an improved sensitivity for RNA detection in living cells...
  6. pmc Sub-cellular trafficking and functionality of 2'-O-methyl and 2'-O-methyl-phosphorothioate molecular beacons
    Antony K Chen
    Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104 and Integrated DNA Technologies, Inc, Coralville, IA 52241, USA
    Nucleic Acids Res 37:e149. 2009
    ..Overall, these results have significant implications for the design and applications of MBs for intracellular RNA measurement...
  7. pmc Imaging individual microRNAs in single mammalian cells in situ
    Jing Lu
    Department of Bioengineering, University of Pennsylvania School of Engineering and Applied Sciences, Philadelphia, PA 19104, USA
    Nucleic Acids Res 37:e100. 2009
    ..The dynamic range was found to span over three orders of magnitude and the average miRNA copy number per cell was within 17.5% of measurements acquired by quantitative RT-PCR...
  8. pmc Fluorescent probes for live-cell RNA detection
    Gang Bao
    Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia 30332, USA
    Annu Rev Biomed Eng 11:25-47. 2009
    ..It is expected that continued advancements in live cell imaging of RNA will open new and exciting opportunities in a wide range of biological and medical applications...
  9. pmc Assessing the sensitivity of commercially available fluorophores to the intracellular environment
    Antony K Chen
    Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA
    Anal Chem 80:7437-44. 2008
    ..Overall, the present study provides a means to select fluorophores for studies that require an absolute quantification of fluorescence in the intracellular environment...
  10. pmc Avoiding false-positive signals with nuclease-vulnerable molecular beacons in single living cells
    Antony K Chen
    Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA
    Nucleic Acids Res 35:e105. 2007
    ..Overall, these results provide evidence that accurate measurements of RNA levels can be made when MBs are retained in the cytoplasm...