DNA/RNA Sensors

Researchers: Ryan Hayman, Chak Chow, Maureen Regan
Past members: Linan Song, Kathryn Brogan, Soohyoun Ahn, Stacy Watkins

Background

Nucleic acid hybridization is a fundamental method of molecular biology and plays a central role in diagnostics. We have developed fiber-optic DNA microarrays using etched optical fiber bundles filled with oligonucleotide-functionalized microspheres. Employing fiber bundles comprised of thousands of individually addressable fibers enables massive parallel detection capabilities. This high-density array was fabricated to measure the hybridization of fluorescently labeled targets. Specific hybridization is detected by fluorescence only at the probe positions complementary to the targets.

Current research projects

• Harmful algae blooms
  
• Food / Water pathogens
  
• Saliva diagnostics
  
• Biowarfare agents
  
• Genotyping / Molecular Barcoding
  
• Molecular beacons

 

Oligonucleotide microarrays

A schematic of a fiber optic microsphere-based microarray is shown in Figure 1. Each circled number represents an oligonucleotide-functionalized microsphere, or bead, in an etched well. The fiber on the left has multiple replicates of different bead types (numbered 1-7). The fiber on the right represents an array after hybridization with a fluorescently-labeled oligonucleotide target solution. Only the probe sequences containing the perfect complement to that specific target (probes numbered 1) provides a positive signal response.

Figure 1 Adapted from Anal. Chem. ( 2000 ) 72, 5618-5624.

The individual detection elements can be regenerated with denaturants or high temperatures to release hybridized DNA targets from the array. This fiber optic microsphere-based array platform can be regenerated over 100 times with less than 8% standard deviation in signal intensity. Actual imaging fibers visualized with white light are shown below in Figure 2.

Figure 2 White light images of a 1 mm diameter imaging fiber bundle.

Figure 3 Bead-type registration (left) and pre- (center) and post- (right)
hybridization to the appropriate oligonucleotide target sequence.

Figure 3 shows three images from a fiber-optic DNA array. The image on the left is of encoded beads. This image enables the registration of each bead-type on the array. The middle image shows the background of a microsphere-based fiber array prior to hybridization. The image on the right is the same array hybridized to a target solution with complementary oligonucleotides labeled with a fluorescent dye. The images in Figure 3 were acquired with our custom-built imaging system and a charge-coupled device (CCD) camera.

Applications

Our DNA biosensor array can be used for gene expression analysis, single nucleotide polymorphism (SNP) discrimination, or detection of DNA linked to disease states. Samples containing specific targets have been positively detected in complex genomic mixtures. This fiber optic microarray platform has also been used to monitor DNA-protein interactions, for genomic bacterial discrimination, and to interrogate non-labeled targets with molecular beacons. Current applications studied in our group are as follows.

1. Sandwich assays for the detection of harmful algae blooms - Harmful algae blooms (HAB) are a serious threat to coastal resources, causing seafood contamination with potent toxins. HAB are often fatal to fish and marine animals. Pre-existing HAB monitoring systems have focused on plankton analysis, but the diversity of planktonic species poses a problem in discriminating toxic species from non-toxic with conventional detection methods.

In collaboration with the Woods Hole Oceanographic Institute (WHOI), we developed a sensitive sandwich hybridization assay that combines simple fiber optic microarrays with specific molecular probes to detect Alexandrium fundyense cells. Microarrays were prepared by loading probe-coupled microspheres onto the distal ends of chemically etched imaging fiber bundles. Hybridization of target rRNA from lysed A. fundyense to immobilized probes on the microspheres was visualized using Cy3-labeled secondary probes in a sandwich-type format (Figure 4).

Figure 4 Illustration of sandwich assay format used for the detection of harmful algal bloom.

We applied the sandwich hybridization for detection of A. fundyense cells in both cultured and field samples. In both cases, as few as 5 cells could be detected within 45 min without any separate amplification step. Our work with A. fundyense cells suggests that fiber-optic microarrays can provide rapid and sensitive HAB detection. In addition, the simplicity of the microarray format offers an opportunity to apply this technology in direct shipboard detection of HAB.  Ongoing efforts with WHOI are focused on expanding the number of species from the Gulf of Maine and around Cape Cod. 

 

2. Tufts Cummings School of Veterinary Medicine - We are developing an oligonucleotide microsphere (bead) array with sequences designed to detect a variety of viral and bacterial pathogens that are hamful to humans and livestock.

Organisms of interest include:

Campylobacter jejuni
Francisella tularensis
Vibrio cholerae
Escherichia coli
Shigella spp.
Salmonella spp.
Rotavirus
Adenovirus
Astrovirus
Norovirus

Multiple probes for each organism have been designed to detect specific genes responsible for pathogenesis such as adherence factors, virulence plasmids, and toxin production.  Using our bioinformatics software, the microarray probes are optimized to hybridize to fluorescently labeled PCR products.  In some cases, such as E. coli, strain determination has important ramifications on toxicity.  Here, the microarray has been designed to discrimate between Enteroaggreative, Enterotoxigenic, Enterohemorragic, Enteroinvasive, and Enteropathogenic strains (EAEC, ETEC, EHEC, EIEC, and EPEC, respectively).  Probes for virus detection were based on amplicons from RT-PCR primers designed by Dr. David Brown.  Adaptation to the Illumina platform will allow many samples to be processed in parallel.

Foodborne Pathogens - Escherichia coli O157:H7 and Salmonella spp. are important food pathogens in public health, causing severe illnesses associated with contamination of meat, poultry or dairy foods. Rapid and sensitive detection of these food pathogens is required for food safety surveillance.

We developed a multiplexed DNA microarray with oligonucleotide probes specific for virulence genes ( hly A and eae A for E. coli O157:H7, inv A and agf B for Salmonella spp.), which enables simultaneous detection of E. coli O157:H7 and Salmonella spp. Microarrays were prepared by randomly distributing DNA probe-functionalized microspheres (3.1 um diameter) into wells etched at the distal end of 500 micron diameter imaging fiber bundles. Target DNA was amplified from whole cells via single or multiplexed polymerase chain reaction (PCR), and PCR products were used for hybridization without further purification. Hybridization of PCR products to immobilized probes on the microspheres was visualized using Cy3-labeled reverse primers.

Our results show that this microarray format is more sensitive than gel electrophoresis, and that as few as ca. 1 CFU/mL is detected within 30 min for both E. coli O157:H7 and Salmonella spp., with 35 PCR cycles. The fiber optic DNA microarray provides high specificity and avoids false positive/negative signals that are common in other conventional detection methods. Since fiber optic microarrays can be prepared with different types of probe microspheres, this approach shows promise for the detection of multiple food pathogens.

 

3. Saliva diagnostics

 

4. Detection of biowarfare agents - Since September 11 and the subsequent Anthrax attacks in 2001, the threat posed by bioterrorism has become real and continues to grow. The need for accurate identification of threatening BWAs is now the focal point for countering bioterrorism. Timely and effective detection of released BWAs is crucial for an appropriate response to mitigate casualties caused by a bioterrorism attack.

We have developed a multiplexed, high-density DNA array capable of rapid, sensitive, and reliable identification of potential biological warfare agents (BWAs) including Bacillus anthracis, Yersinia pestis, Francisella tularensis, Brucella melitensis, Clostridium botulinum, Vaccinia virus, and Bacillus thuringiensis kurstaki (a BWA stimulant) in our laboratory. 50-mer single-stranded DNA sequences specific to the target BWAs were attached to 3.1-µm microspheres to make 18 different DNA probes. The microspheres were distributed into the microwells to form a randomized multiplexed high-density DNA array. Multiplex PCR was applied in conjunction with array hybridization, and the simultaneous detection of these BWAs in real environmental samples has been demonstrated with high sensitivity and specificity.

 

5. Pathogenic Organism Genotyping - Detection and discrimination of pathogenic microorganisms are important for effective treatment and prevention of widespread infection. Bacterial typing is particularly challenging because it is difficult to discriminate between normal and pathogenic serotypes of the same microorganisms.

Our lab has demonstrated the use of a fiber-optic array with cross reactive multilocus sequence typing response to characterize large numbers of closely related strains of E. coli. We are currently using a similar approach to develop an array that will enable the simultaneous serotyping of numerous pathogens based on the cross reactive array approach. To achieve greater numbers of organisms we are using an Illumina BeadStation, a commercial instrument based on the bead-in-fiber technology developed in our lab. This instrument will enable us to use up to 1500 different bead types and evaluate 96 samples simultaneously.

 

6. Molecular Beacons - The goal of this project is to design a reversible, fluorescent DNA probe that can be used to determine the dynamic concentration changes of single stranded DNA in solution. The probe consists of a single stranded oligonucleotide that adopts a stem-loop conformation in its non-hybridized state. The stem length and the length of the loop region that is complementary to the target were chosen to allow for reversible binding. A fluorescent indicator is attached to each end of the single strand probe to be used in a ratiometric measurement. The excitation and the emission wavelengths of the two labels, Cy3 and Cy5, allow for fluorescence resonance energy transfer (FRET) in the closed state. Upon hybridization to its complementary target, the stem-loop structure opens up, resulting in a fluorescence intensity increase of the Cy3. The ratio of the Cy5 to Cy3 fluorescence intensities, which is independent of the amount of probe, is a measure of the free target concentration.

 

 

“Fiber-Optic Microsphere-Based Arrays for Multiplexed Biological Warfare Agent Detection”, L. Song, S. Ahn, D.R. Walt. Analytical Chemistry , 2006 78(4),1023-1033

“Simultaneous Detection of Multiple Biological Warfare Agents in Environmental Samples Using Fiber-Optic Microsphere-Based Arrays”, L. Song, S. Ahn, D.R. Walt. Emerging Infectious Diseases , 2005 11, 1629-1632

“Development of a Microfluidic Platform with an Optical Imaging Microarray Capable of Attomolar Target DNA Detection”, M. Bowden, L. Song, D.R. Walt. Analytical Chemistry , 2005 77(17), 5583-5588

“Array-based Binary Analysis for Bacterial Typing,” J. E. Shepard, Y. Danin-Poleg, Y. Kashi, D.R. Walt, Analytical Chemistry , 2005 , 77 , 319-326.