NBTC: bda22

Project Title
A Novel Aptamer-Based Approach for Label-Free Protein-Sensing Microarrays

Research Program
Biomolecular Devices and Analysis

Project #
BDA24

Participating Faculty:	S. Jaffrey and S. Blanchard
NBTC Students/Postdocs:	Jeremy Paige, Richa Dave
Other Students/Postdocs:

Objectives
The ability to measure proteins in biological samples is fundamental to all biomedical research yet is impractical or not feasible for many types of studies. This proposal describes the development of a generalizable approach to generate simple, sensitive, and specific label-free sensors of proteins. These sensors would be useful in any setting where protein detection is important, such as essentially all biomedical research laboratories, clinical testing laboratories, and industrial screening facilities. Furthermore, we describe experiments that will lead to simple protein sensing microarrays, a long-standing goal in biomedical research. Together, the experiments in this proposal will result in a novel and powerful new method for detecting proteins in a microarray format.

Methods
This project uses novel in vitro selection techniques to identify novel RNA sequences that bind and fluoresce upon binding specific proteins in cells or biological samples. A multiplexed detection strategy using total internal reflection fluorescence microscopy will be used to develop novel protein-sensing microarrays.

Summary

Detecting proteins is one of the most fundamental requirements in biomedical research. However, endogenous proteins cannot be detected in living cells in real time. Furthermore, simple, reagentless techniques for protein quantification in biological samples are not available. We have developed novel oligonucleotide-based sensors that become fluorescent upon exposure to specific analytes. This application describes a simple, sensitive, and specific method for using these sensors for detecting and quantifying proteins in a miniaturized microarray format. These experiments will result in a powerful new nanotechnology based protein detection device that will markedly enhance and expand biomedical research.

Goals & Accomplishments

To develop a method for the generation of fluorescent sensors to detect proteins
To develop protein-sensing microarrays

Fig 1. Switch on fluorescent dyes that resemble the fluorophore in GFP. (A) HBI, the GFP fluorophore, is nonfluorescent in solution due to light-dependent conformational changes dissipating the energy of the excited state. (B) HBI is part of the backbone of GFP, as indicated. The protein (in pink), imposes steric constraints that force the excited state energy to be dissipated in a radiative, i.e., fluorescence, mechanism. (C) DMHBI, 3,5-difluoro-HBI, and 2-HBI are shown and the color of the RNA-fluorophore complex they generate is indicated.
Fig 2. RNA aptamers switch on the fluorescence of DMHBI. (A) An RNA aptamer robustly switches on the fluorescence of DMHBI. Each tube contained the indicated solution and was irradiated at 365 nm. The tube containing the RNA and fluorophore is intensely fluorescent, demonstrating that an RNA can adopt a structure that is capable of rigidifying DMHBI and switching on its fluorescence. (B) The excitation-emission spectra of the 13-2-DMHBI complex shows an emission peak at 535 nm, which is in the green/yellow range.
Fig 3. Design of a fluorescent ATP sensor. (A) Aptamer 13-2 was modified by fusing it to a previously-described ATP-aptamer (right). (B) An ATP-dependent fluorescent sensor. In the absence of ATP, there is minimal fluorescence, even in the presence of DMHBI (red). However, after addition of 1 mM ATP, the fluorescence increases nearly 30-fold (green). The molar fluorescence intensity of the ATP-bound ATP aptamer was ~20% of the 13-2 parent aptamer, demonstrating that ATP binding was able to significantly restore the DMHBI-binding property of the 13-2 portion of the sensor. These data indicate that 13-2 can be used for the design of analyte-dependent sensors.

This material is based upon work supported in part by the STC Program of the National Science Foundation under Agreement No. ECS-9876771. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.

Home

Facilities