Combinatorial fluorescence energy transfer molecular beacons for probing nucleic acid sequences.

We report the design, synthesis, and characterization of molecular beacons (MB) consisting of three distinct fluorophores, 6-carboxyfluorescein (Fam), N,N,N',N'-tetramethyl-6-carboxyrhodamine (Tam), and Cyanine-5 (Cy5). The primary light absorber/energy donor (Fam) is located on one terminus of the MB, whereas the primary energy acceptor/secondary donor (Tam) and secondary acceptor (Cy5) are located at the other terminus of the MB. In the absence of target DNA or RNA, the MB exists in the stem-closed form. Excitation of Fam initiates an energy transfer cascade from Fam to Tam and further to Cy5 generating unique fluorescence signatures defined as the ratio of the emission from each of the three fluorophores. This energy transfer cascade was investigated in detail by steady-state and time-resolved fluorescence spectroscopy, as well as fluorescence depolarization studies. In the presence of the complementary target DNA, the MB opened efficiently and hybridized with the target separating Fam and Tam by a large distance, so that energy transfer from Fam to Tam was blocked in the stem-open form. This opening of the MB generates a "bar code" fluorescence signature, which is different from the signature of the stem-closed MB. The fluorescence signature of this combinatorial fluorescence energy transfer MB can be tuned by variation of the spacer length between the individual fluorophores.     

Enzymatic amplification of DNA/RNA hybrid molecular beacon signaling in nucleic acid detection

A rapid assay operable under isothermal or nonisothermal conditions is described, where the sensitivity of a typical molecular beacon (MB) system is improved by using thermostable RNase H to enzymatically cleave an MB composed of a DNA stem and an RNA loop (R/D-MB). On hybridization of the R/D-MB to target DNA, there was a modest increase in fluorescence intensity (∼5.7× above background) due to an opening of the probe and a concomitant reduction in the Förster resonance energy transfer efficiency. The addition of thermostable RNase H resulted in the cleavage of the RNA loop, which eliminated energy transfer. The cleavage step also released bound target DNA, enabling it to bind to another R/D-MB probe and rendering the approach a cyclic amplification scheme. Full processing of R/D-MBs maximized the fluorescence signal to the fullest extent possible (12.9× above background), resulting in an approximately 2- to 2.8-fold increase in the signal-to-noise ratio observed isothermally at 50 °C following the addition of RNase H. The probe was also used to monitor real-time polymerase chain reactions by measuring enhancement of donor fluorescence on R/D-MB binding to amplified pUC19 template dilutions. Hence, the R/D-MB–RNase H scheme can be applied to a broad range of nucleic acid amplification methods.     

Feasibility of dual-contrast fluorescence imaging of pathological breast tissues

The combination of intravital dye, methylene blue (MB), with molecular cancer marker, pH low insertion peptide (pHLIP) conjugated with fluorescent Alexa532 (Alexa532-pHLIP), was evaluated for enhancing contrast of pathological breast tissue ex vivo. Fresh, thick breast specimens were stained sequentially with Alexa532-pHLIP and aqueous MB and imaged using dual-channel fluorescence microscopy. MB and Alexa532-pHLIP accumulated in the nuclei and cytoplasm of cancer cells, respectively. MB also stained nuclei of normal cells. Some Alexa532-pHLIP fluorescence emission was detected from connective tissue and benign cell membranes. Overall, Alexa532-pHLIP showed high affinity to cancer, while MB highlighted tissue morphology. The results indicate that MB and Alexa532-pHLIP provide complementary information and show promise for the detection of breast cancer.     

Development of a novel FRET immunosensor technique

We report on a novel technique to develop an optical immunosensor based on fluorescence resonance energy transfer (FRET). IgG antibodies were labeled with acceptor fluorophores while one of three carrier molecules (protein A, protein G, or F(ab')2 fragment) was labeled with donor fluorophores. The carrier molecule was incubated with the antibody to allow specific binding to the Fc portion. The labeled antibody-protein complex was then exposed to specific and nonspecific antigens, and experiments were designed to determine the 'in solution' response. The paper reports the results of three different donor-acceptor FRET pairs, fluorescein isothiocyanate/tetramethylrhodamine isothiocyanate, Texas Red/Cy5, and Alexa Fluor 546/Alexa Fluor 594. The effects of the fluorophore to protein conjugation ratio (F/P ratio) and acceptor to donor fluorophore ratios between the antibody and protein (A/D ratio) were examined. In the presence of specific antigens, the antibodies underwent a conformational change, resulting in an energy transfer from the donor to the acceptor fluorophore as measured by a change in fluorescence. The non-specific antigens elicited little or no changes. The Alexa Fluor FRET pair demonstrated the largest change in fluorescence, resulting in a 35% change. The F/P and A/D ratio will affect the efficiency of energy transfer, but there exists a suitable range of A/D and F/P ratios for the FRET pairs. The feasibility of the FRET immunosensor technique was established; however, it will be necessary to immobilize the complexes onto optical substrates so that consistent trends can be obtained that would allow calibration plots.     

Denaturation and condensation of intracellular nucleic acids monitored by fluorescence depolarization of intercalating dyes in individual cells

The intercalating binding of planar aromatic dye molecules to nucleic acids can be analyzed using fluorescence depolarization measurements of the dye molecules excited by linearly polarized light. In this study, we investigated the conformational changes of the intracellular DNA-dye complex in single cells. Flow cytometry, combined with a newly developed double-beam autocompensation technique, permitted rapid high-precision fluorescence depolarization measurements on a large number of individual cells. The dyes ethidium bromide (EB), propidium iodide (PI), and acridine orange (AO) were used in this study. Depending on the dye-to-phosphate ratio of the nuclear acid-dye complex, as well as on the spatial dye structure itself, internal and external binding sites can be monitored by fluorescence depolarization analysis. Both energy transfer and rotation and vibration of the dye molecules cause depolarization of the fluorescence emission. Differences in the concentration-dependent dye fluorescence depolarization values between PI and EB on one side and AO on the other side can be interpreted as a denaturation and condensation of double-stranded DNA regions by AO. We further show that the fluorescence polarization measurement technique can be used in an alternative way to monitor thermal denaturation of cellular DNA.     

Nonenzymatic autoligation in direct three-color detection of RNA and DNA point mutations

Enzymatic ligation methods are useful in diagnostic detection of DNA sequences. Here we describe the investigation of nonenzymatic phosphorothioate-iodide DNA autoligation chemistry as a method for detection and identification of both RNA and DNA sequences. Combining ligation specificity with the hybridization specificity of the ligated product is shown to yield discrimination of a point mutation as high as >10(4)-fold. Unlike enzymatic ligations, this reaction is found to be equally efficient on RNA or DNA templates. The reaction is also shown to exhibit a significant level of self-amplification, with the template acting in catalytic fashion to ligate multiple pairs of probes. A strategy for fluorescence labeling of three autoligating energy transfer (ALET) probes and directly competing them for autoligation on a target sequence is described. The method is tested in several formats, including solution phase, gel, and blot assays. The ALET probe design offers direct RNA detection, combining high sequence specificity with an easily detectable color change by fluorescence resonance energy transfer (FRET).     

Proteolytic stability of beta-peptide bonds probed using quenched fluorescent substrates incorporating a hemoglobin cleavage site

Anthrax lethal toxin is a binary bacterial toxin consisting of two proteins, protective antigen (PA) and lethal factor (LF), that self-assemble on receptor-bearing eukaryotic cells to form toxic, non-covalent complexes. PA₆₃, a proteolytically activated form of PA, spontaneously oligomerizes to form ring-shaped heptamers that bind LF and translocate it into the cell. Site-directed mutagenesis was used to substitute cysteine for each of three residues (N209, E614 and E733) at various levels on the lateral face of the PA₆₃ heptamer and for one residue (E126) on LF_N, the 30 kDa N-terminal PA binding domain of LF. Cysteine residues in PA were labeled with IAEDANS and that in LF_N was labeled with Alexa 488 maleimide. The mutagenesis and labeling did not significantly affect function. Time-resolved fluorescence methods were used to study fluorescence resonance energy transfer (FRET) between the AEDANS and Alexa 488 probes after the complex assembled in solution. The results clearly indicate energy transfer between AEDANS labeled at residue N209C on PA and the Alexa 488-labeled LF_N, whereas transfer from residue E614C on PA was slight, and none was observed from residue E733C. These results support a model in which LF_N binds near the top of the ring-shaped (PA₆₃)₇ heptamer.     

Efficient detection of secondary structure folded nucleic acids related to Alzheimer's disease based on junction probes

Single stranded DNA often forms stable secondary structures under physiological conditions. These DNA secondary structures play important physiological roles. However, the analysis of such secondary structure folded DNA is often complicated because of its high thermodynamic stability and slow hybridization kinetics. In this article, we demonstrate that Y-shaped junction probes could be used for rapid and highly efficient detection of secondary structure folded DNA. Our approach contained a molecular beacon (MB) probe and an assistant probe. In the absence of target, the MB probe failed to hybridize with the assistant probe. Whereas, the MB probe and the assistant probe could cooperatively unwind the secondary structure folded DNA target to form a ternary Y-shaped junction structure. In this condition, the MB probe was also opened, resulting in separating the fluorophores from the quenching moiety and emitting the fluorescence signal. This approach allowed for the highly sensitive detection of secondary structure folded DNA target, such as a tau specific DNA fragment related to Alzheimer's disease in this case. Additionally, this approach showed strong SNPs identifying capability. Furthermore, it was noteworthy that this newly proposed approach was capable of detecting secondary structure folded DNA target in cell lysate samples.     

Operating Cooperatively (OC) Sensor for Highly Specific Recognition of Nucleic Acids

Molecular Beacon (MB) probes have been extensively used for nucleic acid analysis because of their ability to produce fluorescent signal in solution instantly after hybridization. The indirect binding of MB probe to a target analyte offers several advantages, including: improved genotyping accuracy and the possibility to analyse folded nucleic acids. Here we report on a new design for MB-based sensor, called ‘Operating Cooperatively’ (OC), which takes advantage of indirect binding of MB probe to a target analyte. The sensor consists of two unmodified DNA strands, which hybridize to a universal MB probe and a nucleic acid analyte to form a fluorescent complex. OC sensors were designed to analyze two human SNPs and E.coli 16S rRNA. High specificity of the approach was demonstrated by the detection of true analyte in over 100 times excess amount of single base substituted analytes. Taking into account the flexibility in the design and the simplicity in optimization, we conclude that OC sensors may become versatile and efficient tools for instant DNA and RNA analysis in homogeneous solution.     

Real-time monitoring of RNA helicase activity using fluorescence resonance energy transfer in vitro

We have developed a continuous fluorescence assay based on fluorescence resonance energy transfer (FRET) for the monitoring of RNA helicase activity in vitro. The assay is tested using the hepatitis C virus (HCV) NS3 helicase as a model. We prepared a double-stranded RNA (dsRNA) substrate with a 5′ fluorophore-labeled strand hybridized to a 3′ quencher-labeled strand. When the dsRNA is unwound by helicase, the fluorescence of the fluorophore is emitted following the separation of the strands. Unlike in conventional gel-based assays, this new assay eliminates the complex and time-consuming steps, and can be used to simply measure the real-time kinetics in a single helicase reaction. Our results demonstrate that Alexa Fluor 488 and BHQ1 are an effective fluorophore-quencher pair, and this assay is suitable for the quantitative measurement of the RNA helicase activity of HCV NS3. Moreover, we found that several extracts of marine organisms exhibited different inhibitory effects on the RNA and DNA helicase activities of HCV NS3. We propose that this assay will be useful for monitoring the detailed kinetics of RNA unwinding mechanisms and screening RNA helicase inhibitors at high throughput.     

Target accessibility and signal specificity in live-cell detection of BMP-4 mRNA using molecular beacons

The ability to visualize mRNA in single living cells and monitor in real-time the changes of mRNA level and localization can provide unprecedented opportunities for biological and disease studies. However, the mRNA detection specificity and sensitivity are critically dependent on the selection of target sequences and their accessibility. We carried out an extensive study of the target accessibility of BMP-4 mRNA using 10 different designs of molecular beacons (MBs), and identified the optimal beacon design. Specifically, for MB design 1 and 8 (MB1 and MB8), the fluorescent intensities from BMP-4 mRNA correlated well with the GFP signal after upregulating BMP-4 and co-expressing GFP using adenovirus, and the knockdown of BMP-4 mRNA using siRNA significantly reduced the beacon signals, demonstrating detection specificity. The beacon specificity was further confirmed using blocking RNA and in situ hybridization. We found that fluorescence signal from MBs depends critically on target sequences; the target sequences corresponding to siRNA sites may not be good sites for beacon-based mRNA detection, and vice versa. Possible beacon design rules are identified and approaches for enhancing target accessibility are discussed. This has significant implications to MB design for live cell mRNA detection.     

Trajectory of nucleosomal linker DNA studied by fluorescence resonance energy transfer

While the structure of the nucleosome core is known in atomic detail, the precise geometry of the DNA beyond the core particle is still unknown. We have used fluorescence resonance energy transfer (FRET) for determining the end-to-end distance of DNA fragments assembled with histones into nucleosomes. The DNA of a length of 150-220 bp was labeled with rhodamine-X on one end and fluorescein or Alexa 488 on the other. Assembling nucleosomes on these DNA fragments leads to a measurable energy transfer. The end-to-end distance computed from the FRET increases from 60 +/- 5 A at 150 bp to 75 +/- 5 A at 170 bp without measurable change above it. These distances are compatible with different geometries of the linker DNA, all having in common that no crossing can be observed up to 220 bp. Addition of H1 histone leads to an increase in energy transfer, indicating a compaction of the linker DNA toward the nucleosome.     

Possible sources of dye-related signal correlation bias in two-color DNA microarray assays

DNA microarray analyses commonly use two spectrally distinct fluorescent labels to simultaneously compare different mRNA pools. Signal correlation bias currently limits accepted resolution to twofold changes in gene expression. This bias was investigated by (i) examining fluorescence and absorption spectra and changes in relative fluorescence of DNAs labeled with the Cy3, Cy5, Alexa Fluor 555, and Alexa Fluor 647 dyes and by (ii) using homotypic hybridization assays to compare the Cy dye pair with the Alexa Fluor dye pair. Cy3 or Cy5 dye-labeled DNA exhibited reduced fluorescence and absorption anomalies that were eliminated by nuclease treatment, consistent with fluorescence quenching that arises from dye-dye or dye-DNA-dye interactions. Alexa Fluor 555 and Alexa Fluor 647 dye-labeled DNA exhibited little or no such anomalies. In microarray hybridization, the Alexa Fluor dye pair provided higher signal correlation coefficients (R2) than did the Cy dye pair; at the 95% prediction level, a 1.3-fold change in gene expression was significant using the Alexa Fluor dye pair. Lowered signal correlation of the Cy dye pair was associated with high variance in Cy5 dye signals. These results indicate that fluorescence quenching may be a source of signal bias associated with the Cy dye pair.     

Characterization and applications of CataCleave probe in real-time detection assays

Cycling probe technology (CPT), which utilizes a chimeric DNA-RNA-DNA probe and RNase H, is a rapid, isothermal probe amplification system for the detection of target DNA. Upon hybridization of the probe to its target DNA, RNase H cleaves the RNA portion of the DNA/RNA hybrid. Utilizing CPT, we designed a catalytically cleavable fluorescence probe (CataCleave probe) containing two internal fluorophores. Fluorescence intensity of the probe itself was weak due to F?rster resonance energy transfer. Cleavage of the probe by RNase H in the presence of its target DNA caused enhancement of donor fluorescence, but this was not observed with nonspecific target DNA. Further, RNase H reactions with CataCleave probe exhibit a catalytic dose-dependent response to target DNA. This confirms the capability for the direct detection of specific target DNA through a signal amplification process. Moreover, CataCleave probe is also ideal for detecting DNA amplification processes, such as polymerase chain reaction (PCR) and isothermal rolling circle amplification (RCA). In fact, we observed signal enhancement proportional to the amount of RCA product formed. We were also able to monitor real-time PCR by measuring enhancement of donor fluorescence. Hence, CataCleave probe is useful for real-time monitoring of both isothermal and temperature-cycling nucleic acid amplification methods.     

Multiplex real-time single nucleotide polymorphism detection and quantification by quencher extension

Multiplex quencher extension (multiplex-QEXT) is a novel closed tube single-step method for detection and quantification of several single nucleotide polymorphisms (SNPs) simultaneously. The principle of multiplex-QEXT is that 5' reporter-labeled probes are 3' single-base-extended with TAMRA dideoxy nucleotides if the respective SNP alleles are present. TAMRA can serve as either an energy acceptor (quencher-based detection) or donor [fluorescence resonance energy transfer (FRET)-based detection] for a wide range of different reporter fluorochromes. The extension can therefore be recorded by the respective reporter fluorescence change. We evaluated multiplex-QEXT, analyzing four different SNP loci in the Listeria monocytogenes inlA gene. Probes labeled with the reporters 6-FAM, TET, VIC, and Alexa Fluor 594 were used. Responses for the fluorochromes 6-FAM, TET, and VIC were detected by quenching (decreased fluorescence), while the response for Alexa Fluor 594 was detected by FRET (increased fluorescence). We evaluated the SNP-allele pattern in 252 different L. monocytogenes strains. Multiplex-QEXT gave a good resolution, detecting seven major and five minor groups of L. monocytogenes. Comparison with serotyping showed that multiplex-QEXT gave better resolution. We also evaluated the quantitative aspects of multiplex-QEXT. Quantitative information was obtained for all the fluorochrome/probe combinations in the sample pools. The detection limits for 6-FAM, TET and Alexa Fluor 594 were the presence of the 10% target SNP alleles (P < 0.05), while the detection limit for VIC was the presence of the 5% target SNP alleles (P < 0.05). Currently, overlap in the fluorescence emission spectra is the limiting factor for the multiplexing potential of QEXT. With the emergence of new fluorochromes with narrow emission spectra, we foresee great potential for increasing the multiplex level in the future.     

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.     

An OR logic gate based on two molecular beacons

Design of elementary molecular logic gates is the key and the fundamental of performing complicated Boolean calculations. Herein, we report a strategy for constructing a DNA-based OR gate by using the mechanism of sequence recognition and the principle of fluorescence resonance energy transfer (FRET). In this system, the gate is entirely composed of a single strand of DNA (A, B and C) and the inputs are the molecular beacon probes (MB1 and MB2). Changes in fluorescence intensity confirm the realization of the OR logic operation and electrophoresis experiments verify these results. Our successful application of DNA to perform the binary operation represents that DNA can serve as an efficient biomaterial for designing molecular logic gates and devices.     

Fluorescence anisotropy of DNA/DAPI complex: torsional dynamics and geometry of the complex.

Fluorescence depolarization of synthetic polydeoxynucleotide/4'-6-diamidino-2-phenylindole dihydrochloride complexes has been investigated as a function of dye/polymer coverage. At low coverage, fluorescence depolarization is due to local torsional motions of the DNA segment where the dye resides. At relatively high coverage, fluorescence depolarization is dominated by energy transfer to other dye molecules along the DNA. The extent of the observed depolarization due to torsional motion depends on the angle the dye molecule forms with the DNA helical axis. A large torsional motion and a small angle produce the same depolarization as a small torsional motion and a large projection angle. Furthermore, the extent of transfer critically depends on the relative orientation of dye molecules along the DNA. The effect of multiple transfer is examined using a Monte Carlo approach. The measurement of depolarization with transfer, at high coverage, allows determination of the dye orientation about the DNA helical axis. The value of the torsional spring constant is then determined, at very low coverage, for few selected polydeoxynucleotides.     

A molecular beacon DNA microarray system for rapid detection of E. coli O157:H7 that eliminates the risk of a false negative signal

A DNA hybridization based optical detection platform for the detection of foodborne pathogens has been developed with virtually zero probability of the false negative signal. This portable, low-cost and real-time assaying detection platform utilizes the color changing molecular beacon as a probe for the optical detection of the target sequence. The computer-controlled detection platform exploits the target hybridization induced change of fluorescence color due to the F?rster (fluorescence) resonance energy transfer (FRET) between a pair of spectrally shifted fluorophores conjugated to the opposite ends of a beacon (oligonucleotide probe). Unlike the traditional fluorophore-quencher beacon design, the presence of two fluorescence molecules allows to actively visualize both hybridized and unhybridized states of the beacon. This eliminates false negative signal detection characteristic for the fluorophore-quencher beacon where bleaching of the fluorophore or washout of a beacon is indistinguishable from the absence of the target DNA sequence. In perspective, the two-color design allows also to quantify the concentration of the target DNA in a sample down to < =1 ng/microl. The new design is suitable for simultaneous reliable detection of hundreds of DNA target sequences in one test run using a series of beacons immobilized on a single substrate in a spatial format.