1Department of Biochemistry and Biomedical Sciences, McMaster University, 2Department of Chemistry and Chemical Biology, McMaster University
Aguirre, S. D., Ali, M. M., Kanda, P., Li, Y. Detection of Bacteria Using Fluorogenic DNAzymes. J. Vis. Exp. (63), e3961, doi:10.3791/3961 (2012).
Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses each and every year. Prevention of such outbreaks and minimization of the impact of an ongoing epidemic place an ever-increasing demand for analytical methods that can accurately identify culprit pathogens at the earliest stage. Although there is a large array of effective methods for pathogen detection, none of them can satisfy all the following five premier requirements embodied for an ideal detection method: high specificity (detecting only the bacterium of interest), high sensitivity (capable of detecting as low as a single live bacterial cell), short time-to-results (minutes to hours), great operational simplicity (no need for lengthy sampling procedures and the use of specialized equipment), and cost effectiveness. For example, classical microbiological methods are highly specific but require a long time (days to weeks) to acquire a definitive result.1 PCR- and antibody-based techniques offer shorter waiting times (hours to days), but they require the use of expensive reagents and/or sophisticated equipment.2-4 Consequently, there is still a great demand for scientific research towards developing innovative bacterial detection methods that offer improved characteristics in one or more of the aforementioned requirements. Our laboratory is interested in examining the potential of DNAzymes as a novel class of molecular probes for biosensing applications including bacterial detection.5
DNAzymes (also known as deoxyribozymes or DNA enzymes) are man-made single-stranded DNA molecules with the capability of catalyzing chemical reactions.6-8 These molecules can be isolated from a vast random-sequence DNA pool (which contains as many as 1016 individual sequences) by a process known as "in vitro selection" or "SELEX" (systematic evolution of ligands by exponential enrichment).9-16 These special DNA molecules have been widely examined in recent years as molecular tools for biosensing applications.6-8
Our laboratory has established in vitro selection procedures for isolating RNA-cleaving fluorescent DNAzymes (RFDs; Fig. 1) and investigated the use of RFDs as analytical tools.17-29 RFDs catalyze the cleavage of a DNA-RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by a fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the uncleaved substrate minimal fluorescence. However, the cleavage event leads to the separation of F and Q, which is accompanied by significant increase of fluorescence intensity.
More recently, we developed a method of isolating RFDs for bacterial detection.5 These special RFDs were isolated to "light up" in the presence of the crude extracellular mixture (CEM) left behind by a specific type of bacteria in their environment or in the media they are cultured (Fig. 1). The use of crude mixture circumvents the tedious process of purifying and identifying a suitable target from the microbe of interest for biosensor development (which could take months or years to complete). The use of extracellular targets means the assaying procedure is simple because there is no need for steps to obtain intracellular targets.
Using the above approach, we derived an RFD that cleaves its substrate (FS1; Fig. 2A) only in the presence of the CEM produced by E. coli (CEM-EC).5 This E. coli-sensing RFD, named RFD-EC1 (Fig. 2A), was found to be strictly responsive to CEM-EC but nonresponsive to CEMs from a host of other bacteria (Fig. 3).
Here we present the key experimental procedures for setting up E. coli detection assays using RFD-EC1 and representative results.
1. Preparation of Chemical Solutions
2. Construction of RFD-EC1 and RFSS1 by Template Mediated Enzymatic Ligation
RFD-EC1 (Fig. 2A) is the featured DNAzyme. It consists of the catalytic sequence EC1 and the substrate sequence FS1 (indicated by black and green lines in Fig. 2A). RFSS1 (Fig. 2A) is a scrambled version of RFD-EC1 where the catalytic sequence EC1 is partially shuffled into SS1 but the FS1 portion remains unchanged. RFD-EC1 and RFSS1 were made by template mediated enzymatic ligation of the oligonucleotide FS1 with oligonucleotide EC1 or SS1 in the presence of LT1 as the ligation template (see the inserted box in Fig. 2A). The procedure for conducting the ligation reaction is provided below. FS1 was obtained from Keck Oligo Synthesis Facilities at Yale University, deprotected and purified by gel electrophoresis following a previously established protocol.17-24 EC1, SS1 and LT1 were purchased from Integrated DNA Technologies and purified by gel electrophoresis.
3. Preparation of 10% dPAGE Gel
The following steps briefly describe the apparatus of gel electrophoresis and its set-up. For greater details about the apparatus, settings and handling, please refer to our previously published protocols.30,31
4. Purification of Ligated RFD-EC1 and RFSS1 by 10% dPAGE Gel
5. Preparation of Bacteria
6. Preparation of Crude Extracellular Mixtures (CEMs)
7. Detection using Fluorescence Spectrophotometer
8. Detection by Gel Electrophoresis
The same reaction mixtures prepared in step 7.4 can be used for analysis by gel electrophoresis; alternatively new reactions can be prepared similarly and incubated in 1.5 mL microcentrifuge tubes. In either case:
9. Detection Specificity
10. Single Cell Detection
Prepare a 1 mL E. coli glycerol stock of 2 CFU/mL (CFU: colony-forming unit) by serial dilution and confirm CFU concentration by plating.5 This stock should contain 0.2 CFU/100 μL. Store at -80 °C until use.
11. Concept and Representative Results
The concept of exploiting an RNA-cleaving fluorescent DNAzyme (RFD) for bacterial detection is illustrated in Fig. 1. The RFD cleaves a chimeric DNA/RNA substrate at a lone RNA linkage (blue R) flanked by two nucleotides labeled with a fluorophore (F) and a quencher (Q), respectively. As a bacterium of interest (such as E. coli) grows in media, it will leave behind a crude extracellular mixture (CEM). This CEM as a whole is then used in a in vitro selection experiment to obtain an RFD that is responsive specifically to the CEM; presumably the RFD interacts with a specific molecule (purple star) in the CEM that is a signature molecule of the bacterium. When the CEM is added to the reaction solution containing the RFD, it triggers the RNA-cleaving activity of the RFD. The cleavage event separates F from Q, resulting in a fluorescent signal that can be detected either using a fluorimeter or by gel electrophoresis.
The experimental validation of the above concept was done with the CEM from E. coli (CEM-EC). We obtained 3 RFD molecules via in vitro selection, and the most efficient one was designated as RFD-EC1 (Fig. 2A).5 We tested the cleavage activity of RFD-EC1 (along with a mutant sequence named RFSS1) in response to CEM-EC. Both RFD-EC1 and RFSS1 were prepared by enzymatic ligation of the DNAzyme portions to the substrate FS1 (all sequences are shown in Fig. 2A). In the fluorescence measurement experiment (Fig. 2B), CEM-EC was incubated alone for 5 min, followed by the addition of RFD-EC1 or RFSS1, and by further incubation for 55 more min. The fluorescence intensity of the solution was continuously read every minute and the data was used to calculate relative fluorescence (RF; calculated as the ratio of the fluorescence intensity at time t vs. the fluorescence intensity at time 0). The RF values vs. the time of incubation are plotted as Fig. 2B. It was found that RFD-EC1 produced a high level of fluorescence signal upon the addition of CEM-EC; in stark contrast, RFSS1 did not produce a strong fluorescence signal. Thus, the fluorescence-producing function of RFD-EC1 upon contacting CEM-EC is sequence-specific.
In order to verify that observed fluorescence increases are due to the cleavage of the RNA linkage, we analyzed reaction mixtures by dPAGE. Cleavage of RFD-EC1 is expected to generate two DNA fragments, a 5' fragment retaining the fluorophore and a 3' fragment retaining the quencher. Only uncleaved RFD-EC1 (unclv) and the 5′ fragment (clv) could be detected by fluorescence imaging. The dPAGE result shown in Fig. 2C reveals that the reaction mixture of RFD-EC1 and CEM-EC indeed produced the expected cleavage product, while the RFSS1/CEM-EC mixture did not.
The specificity of RFD-EC1 was examined using CEMs collected from several other gram negative and gram positive bacteria and the data is shown in Fig. 3A. Only the sample containing CEM-EC (blue curve) produced an increase in fluorescence. The lack of cross-reactivity with CEMs from the other bacteria indicates that RFD-EC1 is highly selective for E. coli.
We also examined the time needed for culturing a single E. coli cell in order to produce sufficient CEM that can induce the cleavage of RFD-EC1. For this experiment, a E. coli sample containing defined CFU (colony-forming units) was adequately diluted to achieve the concentration of 1 CFU/mL. This was followed by mixing 100 μL of the diluted bacterial sample with growth media and culturing it for 4, 8, 12, 16 and 24 h. CEMs were then collected for each timepoint and tested for inducing the cleavage activity of RFD-EC1. The dPAGE result shown in Fig. 3B indicates that a culturing time of 12 h is needed.
It is important to note that the initial small signal increase observed in fluorescence measurements after the addition of RFSS1 sequence (as a negative control) to CEM-EC (Fig. 2B; red curve) or RFD-EC1 to other bacterial CEMs (Fig. 3B; all curves except blue) is attributed to the intrinsic fluorescence of the FRQ module (due to incomplete quenching of F by Q). Thus, it is expected that the addition of F- and Q-labeled sequences would produce an initial fluorescence increase. However, only RFD-EC1/CEM-EC mixtures are capable of producing a high level of fluorescence over time.
Figure 1. Schematic illustration of the RNA-cleaving fluorescent DNAzyme (RFD) probe that fluoresces upon contact with the crude extracellular mixture (CEM) produced by specific bacterial cells of interest. The RFD cleaves a chimeric DNA/RNA substrate at a lone RNA linkage (blue R) flanked by two nucleotides labeled with a fluorophore (F) and a quencher (Q), respectively. Before the cleavage reaction, the fluorescence level of the RFD is minimal due to the close proximity of F and Q. Upon cleavage, Q departs from F; as a result, a strong fluorescence signal is produced.
Figure 2. The E. coli-sensing RFD. (A) RFD-EC1 is the DNAzyme probe that can be activated by CEM-EC. RFSS1 is a scrambled sequence of RFD-EC1 used as a control. RFD-EC1 and RFSS1 were produced by ligating FS1 with EC1 and SS1, respectively, in the presence of LT1 as the template. F: fluorescein-modified deoxythymidine. Q: Dabcyl-modified deoxythymidine. R: adenine ribonucleotide. (B) Fluorescence signaling profiles of RFD-EC1 and RFSS1 in the presence of CEM-EC. (C) dPAGE analysis of the cleavage reaction mixtures in B (reaction time: 60 min). Pictured is a fluorescence image of the dPAGE gel obtained with by Typhoon scanner. Lane NC: RFD-EC1 or RFSS1 in the reaction buffer alone; Lane CEM-EC: RFD-EC1 or RFSS1 in the reaction buffer containing CEM-EC. Marker: RFD-CE1 treated with 0.25 N NaOH, a procedure known to cause full cleavage of RNA. unclv: uncleaved RFD-EC1. clv: the cleavage fragment containing the fluorophore.
Figure 3. (A) Fluorescence signaling profile of RFD-EC1 in CEMs prepared from various bacterial cells. EC: Escherichia coli-K12; PP: Pseudomonas peli; BD: Brevundimonas diminuta; HA: Hafnia alvei; YR: Yersinia ruckeri; OG: Ochrobactrum grignonese; AX: Achromobacter xylosoxidans; MO: Moraxella osloensis; AI: Acinetobacter lwoffi; SF: Serratia fonticola; BS: Bacillus subtilis; LM: Leuconostoc mesenteroides; LP: Lactobacillus planturum; PA: Pediococcus acidilactici; AO: Actinomyces orientalis. Each CEM sample was incubated for 5 min followed by the addition of RFD-EC1. (B) dPAGE analysis of RFD-EC1/CEM-EC mixtures after a 60-min reaction. Lane NC1: RFD-EC1 in the reaction buffer alone. Lane NC2: RFD-EC1 in the reaction buffer containing CEM-BS (the CEM prepared from Bacillus subtilis). The lanes labeled with 4, 8, 12, 16 and 24: RFD-EC1 in the reaction buffer containing CEM-EC taken from the bacterial culture containing a single E. coli cell following a growing period of 4, 8, 12, 16 and 24 h, respectively.
Most of the common bacterial detection methods today are either slow (classic microbial) or technically demanding (antibody, PCR). Thus, we believe that the next generation of detection tools should cater toward speed and simplicity. To this end, we have created an RNA-cleaving and fluorescence-signaling DNAzyme that can be used to develop simple assays to report the presence of bacteria through the generation of a fluorescence signal. The featured DNAzyme probe, RFD-EC1, is activated by the CEM produced during the growth of E. coli in culture media. Since our method uses crude extracellular mixtures of a bacterium as the target of detection and bypasses the laborious target extraction and amplification steps, it can be used to set up very simple, "mix-and-read" type of assays for bacterial detection. The use of our DNAzyme is not restricted to fluorescence based detection method. For example, colorimetric detection using the same DNAzyme system assay can be designed using a previously reported method that exploits rolling circle amplification in conjunction with an organic dye.32 We foresee the use of DNAzymes for bacterial detection as an attractive avenue to generate new bacterial biosensors with greater operational simplicity.
No conflicts of interest declared.
Funding for this work was provided by the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Sentinel Bioactive Paper Network.
|Ammonium persulfate (APS)||BioShop Canada||AMP001|
|Acrylamide/Bis-acrylamide (40%, 29:1)||BioShop Canada||ACR004|
|Boric acid||BioShop Canada||BOR001|
|EDTA||Electron Microscopy Sciences||EXO539-1|
|DNA concentrator||Thermo Fisher Scientific, Inc.||Savant DNA SpeedVac 120|
|Millex filter unit||EMD Millipore||SLGP033RS|
|Gel loading tips||Diamed||TEC200EX-K|
|ImageQuant software||Molecular Dynamics||Version 5.0|
|Mini Vortexer||VWR international||58816-121|
|Parafilm||Pechiney Plastic Packaging||PM996|
|Petri dishes||Fisher Scientific||Fisherbrand 08-757-12|
|Stripettor Plus (Pipette gun)||Corning||07764714|
|Quartz cuvettes||Varian Inc., Agilent||66-100216-00|
|Shaker/Incubator||New Brunswick Scientific||Classic Series C24|
|Typhoon Scanner||GE Healthcare||9200 Variable mode|
|Centrifuge||Beckman Coulter Inc.||Allegra X22-R|
|UV Spectrophotometer||Thermo Fisher Scientific, Inc.||GenesysUV 10|
|Fluorescence Spectrophotometer||Varian Inc., Agilent||Cary Eclipse|