Abstract
This thesis describes the development and use of molecular beacons as sensitive and specific nucleic acid hybridization probes. It also introduces novel variants of these probes, called wavelength-shifting molecular beacons, that increase the number of different targets that can be detected simultaneously.
A number of formats for nucleic acid hybridization have been developed to identify DNA and RNA sequences that are involved in cellular processes and that aid in the diagnosis of genetic and infectious diseases. The introduction of hybridization probes with interactive fluorophore pairs has enabled the development of homogeneous hybridization assays for the direct identification of nucleic acids. A change in the fluorescence of these probes indicates the presence of a target nucleic acid, and there is no need to separate unbound probes from hybridized probes. The advantages of homogeneous hybridization assays are their speed and simplicity. In addition, homogeneous assays can be combined with nucleic acid amplification, enabling the detection of rare target nucleic acids. These assays can be followed in real time, providing quantitative determination of target nucleic acids over a broad range of concentrations. The General Introduction provides a brief history of the development of nucleic acid hybridization, a description of fluorescence energy transfer, and a discussion of the properties and uses of different types of fluorescent hybridization probes.
An important factor in the design of fluorescent hybridization probes that determines their sensitivity is the efficiency of energy transfer between the fluorophore and the quencher. Chapter 2 describes a study to determine the efficiency of energy transfer for both resonance energy transfer and the static mode of fluorescence quenching. A simple method was developed that enables the determination of quenching efficiency for a large number of combinations of commonly used fluorophores and quenchers. The results confirm that an overlap between the emission spectrum of the fluorophore and the absorption spectrum of the quencher is required for efficient quenching by fluorescence energy transfer. However, when the fluorophore and the quencher come within contact distance of each other, spectral overlap is not a significant determinant of quenching efficiency and all fluorophores are equally well quenched. In addition, it was found that some fluorophore-quencher pairs form stable complexes that result in highly quenched probes.
This thesis explores the properties of molecular beacons that affect nucleic acid detection. Molecular beacons are single-stranded oligonucleotide probes that possess a stem-and-loop (hairpin) structure. The loop contains the probe sequence, which is embedded between two complementary arm sequences that can form a hairpin stem. A fluorophore is covalently linked to the end of one arm, and a quencher is covalently linked to the end of the other arm. When the probe sequence is not hybridized to its nucleic acid target sequence, the arm sequences bind to each other, forming a hairpin stem, which brings the fluorophore and the quencher in contact with each other. In this hairpin structure, the fluorescence of the fluorophore is quenched by the static mode of quenching. However, when the probe sequence anneals to its target sequence, the rigidity of the hybrid that is formed forces the hairpin stem to unwind, separating the fluorophore from the quencher, and restoring fluorescence. Molecular beacons can possess a wide variety of differently colored fluorophores, all quenched by the same non-fluorescent quencher. This property allows multiple targets to be distinguished in the same solution, using several different molecular beacons, each designed to detect a different target, and each labeled with a differently colored fluorophore. Furthermore, the hairpin structure enables the detection of single nucleotide polymorphisms with higher specificity than corresponding linear hybridization probes. Chapter 3 describes in detail how to design, synthesize, characterize, and use molecular beacons for identifying nucleic acids, with an emphasis on genotyping single nucleotide polymorphisms.
Chapter 4 describes a study that determines how specific molecular beacons can be used to distinguish single nucleotide polymorphisms. To demonstrate the specificity of molecular beacons, four DNA templates that differed from one another by a single nucleotide variation at one position were constructed. In addition, four differently colored molecular beacons were designed, each specific for one of the four nucleotide variations. Four polymerase chain reactions were carried out, each containing all four molecular beacons, but each initiated with only one of the four DNA templates. The results demonstrated that in each reaction, the only fluorescent signal that developed was from the molecular beacon that was perfectly complementary to the amplified DNA, and did not arise from the three molecular beacons whose probe sequence mismatched the target sequence.
Many instruments utilize a monochromatic light source for the excitation of fluorophores in fluorescent hybridization probes. Monochromatic light sources excite some fluorophores optimally, but excite other fluorophores less well, or not at all. To overcome this limitation, wavelength-shifting molecular beacons were developed in which a “harvester” fluorophore strongly absorbs energy in the wavelength range of the monochromatic light source and then transfers its energy to a second “emitter” fluorophore that then fluoresces strongly in its own characteristic emission color. As in conventional molecular beacons, when the probe is not hybridized to its target, a non-fluorescent quencher inhibits fluorescence from the harvester fluorophore. Chapter 5 describes the concept of wavelength-shifting molecular, and describes experiments that demonstrate that wavelength-shifting molecular beacons enhance the multiplexing capacity of nucleic acid detection assays and allows those assays to be performed on simpler devices.
Chapter 6 demonstrates the simultaneous use of both conventional molecular beacons and wavelength-shifting molecular beacons in an extremely sensitive, high-throughput, single-tube polymerase chain reaction assay for the identification of rifampin-resistant Mycobacterium tuberculosis. The presence of any mutation in the core region of the rpoB gene of M. tuberculosis results in resistance to rifampin. For this assay, five different probes were designed that together bind to the entire core region of the rpoB gene. Each probe was perfectly complementary to the rifampin-susceptible rpoB sequence, and each probe was labeled with a differently colored fluorophore. When a mutation is present anywhere in the core region of the rpoB gene of the bacilli, it prevents the hybridization of one of the probes, resulting in the absence of one of the five fluorescent colors. The results that are obtained from this assay indicate whether a patient is infected with M. tuberculosis, what concentration of bacilli is present in the clinical sample, and whether the bacilli are rifampin resistant.
In order to develop new drugs that inhibit particular RNA polymerases involved in disease processes, it would be helpful to have an assay that monitors the rate of RNA synthesis under different conditions. Chapter 7 describes the development of a simple method to measure RNA synthesis in real time. It was observed that DNA-directed RNA polymerases of both bacteriophage and bacterial origin have the ability to transcribe molecular beacons possessing a deoxyribonucleotide backbone in a promoter-independent manner, resulting in the synthesis of transcripts that are complementary to the molecular beacons. These transcripts then hybridize to the molecular beacons, generating a nonspecific increase in fluorescence intensity. To overcome this problem, molecular beacons that possess a 2’-O-methylribonucleotide backbone, which does not serve as a template for DNA-directed RNA polymerases, were utilized to monitor transcription reactions. The results show that quantitative comparisons of polymerase activity can be made among a variety of RNA polymerases, and the effects of different inhibitors on these activities can be determined.
Although the development of novel hybridization probes has resulted in assays that possess enhanced sensitivity and specificity, new strategies should enable even higher degrees of multiplexing, and alternative assay designs should increase assay sensitivity. In addition, the use of modified oligonucleotides, such as the 2’-O-methylribonucleotides and peptide nucleic acids, should enhance the affinity and specificity of probes for their targets.
In conclusion, this thesis discusses <span style="color: black;">the development and utilization of molecular beacons, and describes experiments that demonstrate the usefulness of these probes in sensitive, accurate, robust, high-throughput nucleic acid assays for research and clinical applications. Molecular beacons are ideal for in vitro, in vivo, and in situ nucleic acid hybridization assays, in which it is not desired, and frequently impossible, to separate unbound probes from probe-target hybrids.