Practical biophysics: Sensors for rapid detection of biological targets utilizing target-induced oligonucleotide annealing

Detection and quantitation of biomolecules is one of the most commonly performed measurements in biomedical research and clinical diagnostics. There is high demand for convenient, rapid and sensitive biomolecule detection methodologies. In this review we discuss a family of sensors that have been developed in our laboratory that share a common simple biophysical mechanism of action and that are capable of rapid detection of a diverse range of biological targets. The sensors generate fluorescence signal in the presence of the target molecule through target-induced association of short fluorochrome-labeled complementary oligonucleotides that are attached to target recognition elements of the sensors (antibodies, aptamers, etc.) via nanometer scale flexible linkers. This sensor design can be used for detecting proteins, antibodies, nucleic acids and whole cells. The assays using these sensors require only adding a sample to the sensor mix followed by simple fluorescence intensity readout. The simplicity, the speed of detection and the potential for miniaturization are the main assets of these sensors.     

Determination of interactions between structured nucleic acids by fluorescence resonance energy transfer (FRET): selection of target sites for functional nucleic acids.

We previously developed a method for monitoring the integrity of oligonucleotides in vitro and in vivo by quantitating fluorescence resonance energy transfer (FRET) between two different fluorochromes attached to a single oligonucleotide. As an extension of this analysis, we examined changes in the extent of FRET in the presence or absence of target nucleic acids with a specific sequence and a higher-ordered structure. In this system FRET was maximal when probes were free in solution and a decrease in FRET was evidence of successful hybridization. We used a single-stranded oligodeoxyribonucleotide labeled at its 5'-end and its 3'-end with 6-carboxyfluorescein and 6-carboxytetramethylrhodamine, respectively. Incubation of the probe with a single-stranded complementary oligonucleotide reduced the FRET. Moreover, a small change in FRET was also observed when the probe was incubated with an oligonucleotide in which the target site had been embedded in a stable hairpin structure. The decrease in the extent of FRET depended on the length of the stem region of the hairpin structure and also on the higher-ordered structure of the probe. These results indicate that this spectrofluorometric method and FRET probes can be used to estimate the efficacy of hybridization between a probe and its target site within highly ordered structures. This conclusion based on changes in FRET was confirmed by gel-shift assays.     

Synthesis and fluorescence studies of thiazole orange tethered onto oligonucleotide: development of a self-contained DNA biosensor on a fiber optic surface

Thiazole orange dyes were derivatized with ethylene glycol linkers of various lengths, and were covalently linked to the 5' end of the oligonucleotides after solid-phase synthesis. The labeled oligonucleotides exhibited enhanced fluorescence upon hybridization to complementary DNA sequences at the surfaces of optical fibers, providing for a self-contained labeling strategy. It was determined that the melt temperatures of DNA hybrids using one mixed polypyrimidine base oligonucleotide sequence were dependent on the length of the tethers, and that the melt temperature could be shifted by more than 10 degrees C when tethers were introduced.     

Excitonic heterodimer formation in an HIV-1 oligonucleotide labeled with a donor-acceptor pair used for fluorescence resonance energy transfer

In this study, we investigated the absorbance and fluorescence properties of cTAR, the complementary DNA sequence of the transactivation response element of the HIV-1 genome, doubly end-labeled by different dyes, 5(and 6)-carboxyfluorescein (Fl) and 5(and 6)-carboxytetramethylrhodamine (TMR), frequently used in fluorescence resonance energy transfer (FRET) studies. This oligonucleotide forms a stable stem-loop structure. The absorption spectrum of this species clearly differed from that of a doubly labeled cTAR derivative in which the terminal part of the stem is melted and from an equimolecular mixture of singly labeled species. Moreover, no significant TMR fluorescence change accompanies the dramatic Fl intensity increase when the doubly labeled native cTAR was melted by temperature or annealed with its complementary sequence. Both elements suggest the formation of an H-type ground-state heterodimer between Fl and TMR that may be described by the molecular exciton model. Moreover, time-resolved fluorescence further suggests that the nonfluorescent heterodimer is in equilibrium with a small population of partially melted species showing FRET. Based on the spectral shifts associated with heterodimer formation, an interchromophore distance of 7.7 A was calculated. Both the excitonic signal and the Fl fluorescence were used as sensitive tools to monitor the temperature-mediated and HIV nucleocapsid protein-mediated annealing of cTAR with its complementary sequence.     

Effect of salt and RNA structure on annealing and strand displacement by Hfq

The Sm-like protein Hfq promotes the association of small antisense RNAs (sRNAs) with their mRNA targets, but the mechanism of Hfq''s RNA chaperone activity is unknown. To investigate RNA annealing and strand displacement by Hfq, we used oligonucleotides that mimic functional sequences within DsrA sRNA and the complementary rpoS mRNA. Hfq accelerated at least 100-fold the annealing of a fluorescently labeled molecular beacon to a 16-nt RNA. The rate of strand exchange between the oligonucleotides increased 80-fold. Therefore, Hfq is very active in both helix formation and exchange. However, high concentrations of Hfq destabilize the duplex by preferentially binding the single-stranded RNA. RNA binding and annealing were completely inhibited by 0.5 M salt. The target site in DsrA sRNA was 1000-fold less accessible to the molecular beacon than an unstructured oligonucleotide, and Hfq accelerated annealing with DsrA only 2-fold. These and other results are consistent with recycling of Hfq during the annealing reaction, and suggest that the net reaction depends on the relative interaction of Hfq with the products and substrates.     

Highly sensitive detection of hybridization of oligonucleotides to specific sequences of nucleic acids by application of fluorescence resonance energy transfer

We show a new application of fluorescence resonance energy transfer (FRET) in two stages to detect specific sequences of nucleic acids. In the first stage, two fluorescently tagged oligonucleotides hybridize with a complementary target molecule to produce FRET. The sequences of the oligonucleotides and spectral properties of fluorophores are chosen to provide a basis for an efficient energy transfer. In the next step, the specificity of hybridization is tested by competition of labeled probes with an excess of unlabeled oligonucleotides of the same sequence. The resulting emission spectra, one obtained in the excess of unlabeled donor probe and the other produced in the excess of unlabeled acceptor probe, are compared with the spectrum from the first stage to look for differences in the emission pattern of the fluorescent labels. We show that it is possible to detect the existence of specific hybrids composed of the two probes and complementary target molecule even in very unfavorable conditions, such as the presence of unhybridized probes in the final reaction mixture, secondary nonacceptor quenching of donor probe fluorescence, and strong background emission of acceptor produced by its direct excitation with a donor excitation light.     

Slow non-specific accumulation of 2′-deoxy and 2′-O-methyl oligonucleotide probes at mitochondria in live cells

Molecular beacons (MBs) have the potential to provide a powerful tool for rapid RNA detection in living cells, as well as monitoring the dynamics of RNA expression in response to external stimuli. To exploit this potential, it is necessary to distinguish true signal from background signal due to non-specific interactions. Here, we show that, when cyanine-dye labeled 2′-deoxy and 2′-O-methyl oligonucleotide probes are inside living cells for >5 h, most of their signals co-localize with mitochondrial staining. These probes include random-sequence MB, dye-labeled single-strand linear oligonucleotide and dye-labeled double-stranded oligonucleotide. Using carbonyl cyanide m-chlorophenyl hydrazone treatment, we found that the non-specific accumulation of oligonucleotide probes at mitochondria was driven by mitochondrial membrane potential. We further demonstrated that the dye-labeled oligonucleotide probes were likely on/near the surface of mitochondria but not inside mitochondrial inner membrane. Interestingly, oligonucleotides probes labeled respectively with Alexa Fluor 488 and Alexa Fluor 546 did not accumulate at mitochondria, suggesting that the non-specific interaction between dye-labeled ODN probes and mitochondria is dye-specific. These results may help design and optimize fluorescence imaging probes for long-time RNA detection and monitoring in living cells.     

Fluorescent-labeled oligonucleotides that exhibit a measurable signal in the presence of complementary DNA.

Oligonucleotide derivatives with a fluorescent dye were designed for exhibiting a measurable signal only when they bind to complementary DNA in aqueous solution. The oligonucleotide with a dansyl group at the specific 2'-sugar residue was synthesized by using the protected 2'-dansylaminouridine phosphorobisamidite. The dansyl-oligonucleotide conjugate binds to its complementary DNA to form duplex with a normal stability and exhibits enhanced fluorescence together with a blue-shift in emission maxima after the hybridization. Another possible candidate involved the use of pyrene-excimer emission upon forming ternary complex between two pyrene-labeled oligonucleotide probes with target DNA. A new and general method for introduction of a pyrene fluorophore into the 3'- or 5'-terminal hydroxyl group of oligonucleotides via different linkers was developed.     

A new approach to SNP genotyping with fluorescently labeled mononucleotides

Fluorescence resonance energy transfer (FRET) is one of the most powerful and promising tools for single nucleotide polymorphism (SNP) genotyping. However, the present methods using FRET require expensive reagents such as fluorescently labeled oligonucleotides. Here, we describe a novel and cost-effective method for SNP genotyping using FRET. The technique is based on allele-specific primer extension using mononucleotides labeled with a green dye and a red dye. When the target DNA contains the sequence complementary to the primer, extension of the primer incorporates the green and red dye-labeled nucleotides into the strand, and red fluorescence is emitted by FRET. In contrast, when the 3′ end nucleotide of the primer is not complementary to the target DNA, there is no extension of the primer, or FRET signal. Therefore, discrimination among genotypes is achieved by measuring the intensity of red fluorescence after the extension reaction. We have validated this method with 11 SNPs, which were successfully determined by end-point measurements of fluorescence intensity. The new strategy is simple and cost-effective, because all steps of the preparation consist of simple additions of solutions and incubation, and the dye-labeled mononucleotides are applicable to all SNP analyses. This method will be suitable for large-scale genotyping.     

Fluorescence resonance energy transfer between donor-acceptor pair on two oligonucleotides hybridized adjacently to DNA template

We use fluorescein as the energy donor and rhodamine as the acceptor to measure the efficiency of fluorescence resonance energy transfer (FRET) in a set of hybridized DNA constructs. The two fluorophores are covalently attached via linkers to two separate oligonucleotides with fluorescein at the 3' end of one oligonucleotide and rhodamine at the 5' end or in the middle of another nucleotide. For the FRET analysis both fluorophore-labeled oligonucleotides are hybridized to adjacent sections of the same DNA template to form a three-component duplex with a one base gap between the two labeled oligonucleotides. A similar configuration is implemented for a quantitative real-time polymerase chain reaction (PCR) with LightCycler technology, where a 1-5 base separation between donor and acceptor is recommended to optimize energy transfer efficiencies. Our constructs cover donor-acceptor separations from 2 to 17 base pairs (approximately 10-70 A). The results show that, when the two fluorophores are located at close distances (less than 8 base separation), FRET efficiencies are above 80%, although there may be ground-state interactions between fluorophores when the separation is under about 6 bases. Modeling calculations are used to predict the structure of these three-component constructs. The duplex mostly retains a normal double helical structure, although slight bending may occur near the unpaired base in the DNA template. Stable and reproducible energy transfer is also observed over the distance range investigated here in real-time thermal cycling. The study identifies important parameters that determine FRET response in applications such as real-time PCR.     

Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations.

Fluorescent oligonucleotide hybridization probes were used to label bacterial cells for analysis by flow cytometry. The probes, complementary to short sequence elements within the 16S rRNA common to phylogenetically coherent assemblages of microorganisms, were labeled with tetramethylrhodamine and hybridized to suspensions of fixed cells. Flow cytometry was used to resolve individual target and nontarget bacteria (1 to 5 microns) via probe-conferred fluorescence. Target cells were quantified in an excess of nontarget cells. The intensity of fluorescence was increased additively by the combined use of two or three fluorescent probes complementary to different regions of the same 16S rRNA.     

Encoded metal nanoparticle-based molecular beacons for multiplexed detection of DNA

In this paper we describe a molecular beacon format assay in which encoded nanowire particles are used to achieve multiplexing. We demonstrate this principle with the detection of five viral pathogens; Hepatitis A virus, Hepatitis C virus, West Nile Virus, Human Immune Deficiency virus and Severe Acute Respiratory Syndrome virus. Oligonucleotides are designed complementary to a target sequence of interest containing a 3′ universal fluorescence dye. A 5′ thiol causes the oligonucleotides to self-assemble onto the metal nanowire. The single-stranded oligonucleotide contains a self-complementary hairpin stem sequence of 10 bases that forces the 3′ fluorophore to come into contact with the metallic nanowire surface, thereby quenching the fluorescence. Upon addition of target DNA, there is hybridization with the complementary oligonucleotides. The resulting DNA hybrid is rigid, unfolds the hairpin structure, and causes the fluorophore to be moved away from the surface such that it is no longer quenched. By using differently encoded nanowires, each conjugated with a different oligonucleotide sequence, multiplexed DNA assays are possible using a single fluorophore, from a multiplexed RT-PCR reaction.     

Bending and unwinding of nucleic acid by prion protein

Nucleic acid induces conformational changes in the prion protein (23-231 amino acids) to a structure resembling its pathological isoform. The prion protein, in turn, facilitates DNA strand transfer and acts as a DNA chaperone which is modulated by the N-terminal unstructured basic segment of the protein. Here we have studied the prion protein induced conformational changes in DNA using oligonucleotides covalently labeled with the energy donor fluorescein and the acceptor rhodamine moieties by fluorescence resonance energy transfer (FRET) and by thermal stability of the unlabeled oligonucleotides. The protein induces a strong FRET effect in the oligonucleotides evidenced from the simultaneous quenching of fluorescence intensity of the donor and increase in the fluorescence intensity of the acceptor, which indicate bending of the oligonucleotides by the prion protein. The energy transfer efficiency induced by the protein is greater for the larger oligonucleotide. The prion protein also induces significant structural destabilization of the oligonucleotides observed from the lowering of their melting temperatures in the presence of the protein. The truncated globular prion protein 121-231 fragment neither induces FRET effect on the oligonucleotides nor destabilizes their structures, indicating that the N-terminal segment of the prion protein is essential for the DNA bending process. Equilibrium binding and kinetics of FRET show that the protein binding to the oligonucleotides and their bending occur simultaneously. The DNA structural changes observed in the presence of the prion protein are similar to those caused by proteins involved in initiation and regulation for protein synthesis.     

Some Properties of Site-Specific Nickase BspD6I and the Possibility of Its Use in Hybridization Analysis of DNA

A new method for hybridization analysis of nucleic acids is proposed on the basis of the ability of site-specific nickases to cleave only one DNA strand. The method is based on the use of a labeled oligonucleotide with the recognition site of the nickase hybridized with the target (DNA or RNA) at an optimal temperature of the enzyme (55°C). The two shorter oligonucleotides formed after the cleavage with the nickase do not complex with the target. Thus, a multiple cleavage of the labeled oligonucleotide takes place on one target molecule. The cleavage of the nucleotide is recorded either by polyacrylamide gel electrophoresis (when a radioactive labeled oligonucleotide is used) or by fluorescence measurements (if the oligonucleotide has the structure of a molecular beacon). The new method was tested on nickase BspD6I and a radioactive oligonucleotide complementary to the polylinker region of the viral DNA strand in bacteriophage M13mp19. Unfortunately, nickase BspD6I does not cleave DNA in the RNA–DNA duplexes and therefore cannot be used for detection of RNA targets.     

Quantitative understanding of the energy transfer between fluorescent proteins connected via flexible peptide linkers

The fusion of different protein domains via peptide linkers is a powerful, modular approach to obtain proteins with new functions. A detailed understanding of the conformational behavior of peptide linkers is important for applications such as fluorescence resonance energy transfer (FRET)-based sensor proteins and multidomain proteins involved in multivalent interactions. To investigate the conformational behavior of flexible glycine- and serine-containing peptide linkers, we constructed a series of fusion proteins of enhanced cyan and yellow fluorescent proteins (ECFP-linker-EYFP) in which the linker length was systematically varied by incorporating between 1 and 9 GGSGGS repeats. As expected, both steady-state and time-resolved fluorescence measurements showed a decrease in energy transfer with increasing linker length. The amount of energy transfer observed in these fusion proteins can be quantitatively understood by simple models that describe the flexible linker as a worm-like chain with a persistence length of 4.5 A or a Gaussian chain with a characteristic ratio of 2.3. The implications of our results for understanding the properties of FRET-based sensors and other fusion proteins with Gly/Ser linkers are discussed.     

Oligonucleotide microchips as genosensors for determinative and environmental studies in microbiology.

The utility of parallel hybridization of environmental nucleic acids to many oligonucleotides immobilized in a matrix of polyacrylamide gel pads on a glass slide (oligonucleotide microchip) was evaluated. Oligonucleotides complementary to small-subunit rRNA sequences of selected microbial groups, encompassing key genera of nitrifying bacteria, were shown to selectively retain labeled target nucleic acid derived from either DNA or RNA forms of the target sequences. The utility of varying the probe concentration to normalize hybridization signals and the use of multicolor detection for simultaneous quantitation of multiple probe-target populations were demonstrated.