Enzymatic signal amplification of molecular beacons for sensitive DNA detection

Molecular beacons represent a new family of fluorescent probes for nucleic acids, and have found broad applications in recent years due to their unique advantages over traditional probes. Detection of nucleic acids using molecular beacons has been based on hybridization between target molecules and molecular beacons in a 1:1 stoichiometric ratio. The stoichiometric hybridization, however, puts an intrinsic limitation on detection sensitivity, because one target molecule converts only one beacon molecule to its fluorescent form. To increase the detection sensitivity, a conventional strategy has been target amplification through polymerase chain reaction. Instead of target amplification, here we introduce a scheme of signal amplification, nicking enzyme signal amplification, to increase the detection sensitivity of molecular beacons. The mechanism of the signal amplification lies in target-dependent cleavage of molecular beacons by a DNA nicking enzyme, through which one target DNA can open many beacon molecules, giving rise to amplification of fluorescent signal. Our results indicate that one target DNA leads to cleavage of hundreds of beacon molecules, increasing detection sensitivity by nearly three orders of magnitude. We designed two versions of signal amplification. The basic version, though simple, requires that nicking enzyme recognition sequence be present in the target DNA. The extended version allows detection of target of any sequence by incorporating rolling circle amplification. Moreover, the extended version provides one additional level of signal amplification, bringing the detection limit down to tens of femtomolar, nearly five orders of magnitude lower than that of conventional hybridization assay.     

P-loop catalytically assisting the enzymatic cleavage of single-stranded DNA

We demonstrated that a P-loop, a looped complex formed inside duplex DNA by adding peptide nucleic acids (PNA), acts catalytically as a template for enzymatic cleavage of single-stranded probe oligodeoxynucleotides (ODN). A PD-loop complex formed from P-loop and probe ODN was digested efficiently by a restriction enzyme, and the truncated probe ODN was released. The P-loop nicked by the enzyme can form PD-loop again with another probe ODN, and then assisted the enzymatic cleavage of an excess of probe ODN. In addition, by using dumbbell-formed ODN as a probe ODN, the efficiency of the P-loop-assisted ODN cleavage was enhanced considerably as compared with that of linear ODN. Thus, the method utilizing P-loop will make it possible to amplify the sequence information of duplex DNA via a catalytic cleavage of probe ODNs.     

Enzymatic production of single-stranded DNA as a target for fluorescence in situ hybridization

This study demonstrates that Exonuclease III (Exo III) can be used to produce sufficient single-stranded (ss)DNA in chromosomes and cells to allow in situ hybridization. In this study, all of the probes were modified with biotin and the probe binding was visualized with fluorescein-labeled avidin. Exo III digestion starting at naturally occurring breaks in methanol-acetic acid preparations produced enough ssDNA for strong hybridization when human genomic DNA was used to probe human chromosomes. Pretreatment with the endonucleases EcoRI, Hind III and BamHI was used to produce more sites for initiation of Exo III digestion when using a chromosome-specific repetitive probe specific to a small chromosomal subregion near the telomere of human chromosome 1(1p36). The fluorescence intensity following hybridization to Exo Ill-treated targets was roughly equal to that following hybridization to thermally denatured targets, but background fluorescence was lower.     

The use of DNA molecular beacons as nanoscale temperature probes for microchip-based biosensors

Today's biosensors and drug delivery devices are increasingly incorporating lithographically patterned circuitry that is placed within microns of the biological molecules to be detected or released. Elevated temperatures due to Joule heating from the underlying circuitry cannot only reduce device performance, but also alter the biological activity of such molecules (i.e. binding, enzymatic, folding). As a consequence, biochip design and characterization will increasingly require local measurements of the temperature and temperature gradients on the biofunctionalized surface. We have developed a technique to address this challenge based on the use of DNA molecular beacons as a nanoscale temperature probe. The surface of fused-silica chips with lithographically patterned, current-carrying gold rings have been functionalized with a layer of molecular beacons. We utilize the temperature dependence of the molecular beacons to calibrate the temperature at the center of the rings as a function of applied current from 25 to 50 degrees C. The fluorescent images of the rings reveal the extent of heating to the surrounding chip due to the applied current while resolving temperature gradients over length scales of less than 500nm. Finite element analysis and analytic calculations of the distribution of heat in the vicinity of the current-carrying rings agree well with the experimental results. Thus, molecular beacons are shown to be a viable tool for temperature calibration of micron-sized circuitry and the visualization of submicron temperature gradients.     

Pyrene-labeled deoxyguanosine as a fluorescence sensor to discriminate single and double stranded DNA structures: Design of ends free molecular beacons

A novel fluorescent DNA probe containing pyrene-labeled C8 alkylamino-substituted 2′-deoxyguanosine was designed in order to discriminate single stranded and double stranded regions in DNA. This fluorescent sensor was used for the design of practically useful 3′- and 5′-ends free self-quenched molecular beacon (MB). Unique MB detectable by pyrene excimer fluorescence was also demonstrated.     

Fluorescent single-stranded DNA binding protein as a probe for sensitive, real-time assays of helicase activity

The formation and maintenance of single-stranded DNA (ssDNA) are essential parts of many processes involving DNA. For example, strand separation of double-stranded DNA (dsDNA) is catalyzed by helicases, and this exposure of the bases on the DNA allows further processing, such as replication, recombination, or repair. Assays of helicase activity and probes for their mechanism are essential for understanding related biological processes. Here we describe the development and use of a fluorescent probe to measure ssDNA formation specifically and in real time, with high sensitivity and time resolution. The reagentless biosensor is based on the ssDNA binding protein (SSB) from Escherichia coli, labeled at a specific site with a coumarin fluorophore. Its use in the study of DNA manipulations involving ssDNA intermediates is demonstrated in assays for DNA unwinding, catalyzed by DNA helicases.     

Surface-attached molecular beacons light the way for DNA sequencing

Rapid testing of DNA and RNA nucleotide sequences is required for various research protocols including wide-scale genetic testing, diagnostics, fast detection of biological warfare agents, environmental testing and forensic medicine. At present many laboratories are interested in research and development of an inexpensive, easy-to-use, fast-response device for this purpose. Various methods based on acoustic, electronic and optical detection of the DNA hybridization event have been reported.     

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.     

Molecular beacons for isothermal fluorescence enhancement by the cleavage of RNase HII from Chlamydia pneumoniae

This article describes a new assay for isothermal enhancement of fluorescence intensity. The assay is based on the cleavage of duplexes formed by the chimeric DNA-rN(1)-DNA molecular beacon (cMB) and target DNA with Chlamydia pneumoniae RNase HII (CpRNase HII). The loop sequence of the cMB, which was designed according to the target sequence, contains a single ribonucleotide. The combination of CpRNase HII cleavage and cMB (RHMB) permitted a 90-fold increase in fluorescence intensity change compared with the hybridization reaction in the presence of the same amount of target DNA. These results indicate that the RHMB assay can enhance the fluorescence signal in real-time monitoring of the target DNA.     

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.     

Removal of pyrimidine dimers from Saccharomyces cerevisiae nuclear DNA under nongrowth conditions as detected by a sensitive, enzymatic assay.

A sensitive and quantitative procedure for the detection of pyrimidine dimers in yesast nuclear DNA is described. The assay employs dimer-specific, endonuclease activities from Micrococcus luteus together with DNA sedimentation through calibrated, alkaline sucrose gradients to detect endonuclease-induced, single-strand breaks. Breaks were induced in a dose-dependent manner from 0 to 80 J m-2 at 254 nm and in numbers equivalent to the numbers of dimers induced by similar doses (Unrau et al., Biochim. Biophys. Acta, 312 (1973) 626--632). This procedure also allows the use of [6-3H] uridine to label cellular nucleic acids, but dose not require extensive DNA purification to eliminate concomitantly labeled RNA. Endonuclease-sensitive sites in the wild-type, haploid strain S288C, after irradiation with 5 J m-2 (254 nm), were removed in less than 5 min when cells were incubated in buffer (pH 7.0) at 28 degrees C. After irradiation with doses from 30 to 100 Jm-2 site removal in S288C required longer postirradiation incubations and was about 90% complete. In a radiation-sensitive strain carrying the mutant allele rad4-3 the number of endonuclease-sensitive sites remained constant for 6 h after irradiation with 5 Jm-2. The retention of sites in this strain indicates that it is defective in the excision of pyrimidine dimers.     

Two-wavelength fluorescence assay for DNA repair

A simple and reliable quantitative assay for measuring cellular DNA repair capacity has been developed. It is based on the host cell reactivation of the UV-irradiated plasmid pEGFP carrying the marker gene for the enhanced green fluorescent protein (EGFP). As a reference we used the plasmid pEYFP carrying the gene for a red-shifted fluorescent protein (EYFP). Both proteins can be excited by visible light with a maximum at 488 nm, but EGFP emits with a maximum at 509 nm, while EYFP emits with a maximum at 527 nm. This makes it possible to monitor the expression of the two genes simultaneously by measuring the fluorescence at two wavelengths. HEK293 cells were cotransfected with a mixture of UV-irradiated pEGFP and undamaged pEYFP. At different time intervals after transfection the fluorescence of EGFP was determined relative to the fluorescence of EYFP to compensate for any differences in the transfection efficiency or other experimental variables. It was used to calculate the number of UV lesions in DNA and hence the repair capacity of the host cells. It was found that HEK293 cells were able to repair approximately 1.4 UV lesions per 1000 nucleotides DNA for 12 h on the average.     

Adaptation of an enzymatic fluorescence assay for L-glutamic acid decarboxylase

The activity of L-glutamic acid decarboxylase (GAD) is commonly estimated by several radiometric methods, whereas a fluorimetric assay based on an enzymatic formation of NADPH as described by Y. Okada and C. Shimada [(1975) Brain Res. 98, 202-206] has been given little attention in biochemical and pharmacological investigations. A simple modification of this assay is presented to permit rapid and sensitive GAD measurements in unpurified tissue homogenates. This method, employing a linear NADPH standard curve, is demonstrated to be a valid assay system for a pharmacological approach using 3-mercaptopropionic acid.     

A sensitive enzymatic assay for determination of cholesterol in lipid extracts

A procedure for the determination of free and total cholesterol in lipid extracts is described. The method for free cholesterol employs cholesterol oxidase to generate H2O2 and peroxidase to catalyze the reaction of H2O2 with o-dianisidine to yield a colored product. For the determination of total cholesterol, cholesterol ester hydrolase is included.     

Exciton interaction in molecular beacons: a sensitive sensor for short range modifications of the nucleic acid structure

Molecular beacons are hairpin-shaped, single-stranded oligonucleotides constituting sensitive fluorescent DNA probes widely used to report the presence of specific nucleic acids. In its closed form the stem of the hairpin holds the fluorophore covalently attached to one end, close to the quencher, which is covalently attached to the other end. Here we report that in the closed form the fluorophore and the quencher form a ground state intramolecular heterodimer whose spectral properties can be described by exciton theory. Formation of the heterodimers was found to be poorly sensitive to the stem sequence, the respective positions of the dyes and the nature of the nucleic acid (DNA or RNA). The heterodimer allows strong coupling between the transition dipoles of the two chromophores, leading to dramatic changes in the absorption spectrum that are not compatible with a Förster-type fluorescence resonance energy transfer (FRET) mechanism. The excitonic heterodimer and its associated absorption spectrum are extremely sensitive to the orientation of and distance between the dyes. Accordingly, the application of molecular beacons can be extended to monitoring short range modifications of the stem structure. Moreover, the excitonic interaction was also found to operate for doubly end-labeled duplexes.