Molecular beacons: a real-time polymerase chain reaction assay for detecting Salmonella

Molecular beacons are oligonucleotide probes that become fluorescent upon hybridization. We developed a real-time PCR assay to detect the presence of Salmonella species using these fluorogenic reporter molecules. A 122-base-pair section of the himA was used as the amplification target. Molecular beacons were designed to recognize a 16-base-pair region on the amplicon. As few as 2 colony-forming unit (CFU) per PCR reaction could be detected. We also demonstrated the ability of the molecular beacons to discriminate between amplicons obtained from similar species such as Escherichia coli and Citrobacter freundii in real-time PCR assays. These assays could be carried out entirely in sealed PCR tubes, enabling fast and direct detection of Salmonella in a semiautomated format.     

Molecular recognition of cAMP by an RNA aptamer

Two classes of RNA aptamers that bind the second messenger adenosine 3',5'-cyclic monophosphate (cAMP; 1) were isolated from a random-sequence pool using in vitro selection. Class I and class II aptamers are formed by 33- and 31-nucleotide RNAs, respectively, and each is comprised of similar stem-loop and single-stranded structural elements. Class II aptamers, which dominate the final selected RNA population, require divalent cations for complex formation and display a dissociation constant (K(D)) for cAMP of approximately 10 microM. A representative class II aptamer exhibits substantial discrimination against 5'- and 3'-phosphorylated nucleosides such as ATP, 5'-AMP, and 3'-AMP. However, components of cAMP such as adenine and adenosine also are bound, indicating that the adenine moiety is the primary positive determinant of ligand binding. Specificity of cAMP binding appears to be established by hydrogen bonding interactions with the adenine base as well as by steric interactions with groups on the ribose moiety. In addition, the aptamer recognizes 8,5'-O-cycloadenosine (2) but not N(3), 5'-cycloadenosine (3), indicating that this RNA might selectively recognize the anti conformation of the N-glycosidic bond of cAMP.     

Molecular beacons

This opinion covers the field of molecular beacons (MBs), in which nucleic acids are molecularly engineered to have unique functions for the investigation of biomolecules. Molecular beacons have been used in a variety of formats, and this review discusses four: first, in vitro RNA and DNA monitoring; second, biosensors and biochips based on MBs; third, real-time monitoring of genes and gene expression in living systems; and finally, the next generation of molecular beacons that will be highly useful for studies with proteins, molecular beacon aptamers. These unique applications have shown that MBs holds great potential in genomics and proteomics where real-time molecular recognition with high sensitivity and excellent specificity is critical.     

Molecular beacons for detecting DNA binding proteins

We report here a simple, rapid, homogeneous fluorescence assay, the molecular beacon assay, for the detection and quantification of sequence-specific DNA-binding proteins. The central feature of the assay is the protein-dependent association of two DNA fragments each containing about half of a DNA sequence defining a protein-binding site. Protein-dependent association of DNA fragments can be detected by any proximity-based spectroscopic signal, such as fluorescence resonance energy transfer (FRET) between fluorochromes introduced into these DNA molecules. The assay is fully homogeneous and requires no manipulations aside from mixing of the sample and the test solution. It offers flexibility with respect to the mode of signal detection and the fluorescence probe, and is compatible with multicolor simultaneous detection of several proteins. The assay can be used in research and medical diagnosis and for high-throughput screening of drugs targeted to DNA-binding proteins.     

Molecular signaling aptamers for real-time fluorescence analysis of protein

Aptamers are a new class of nucleic acids that are selected in vitro for binding target molecules with high affinity and selectivity. They are promising protein-binding molecular probes that rival conventional antibodies for protein analysis. There have been recent advances in the development of molecular signaling aptamers that can transduce target protein binding to sensitive fluorescence signal changes. This facilitates the real time protein monitoring in homogenous solution as well as potentially in vivo. Different signaling strategies of using dual labeled aptamers based on fluorescence resonance energy transfer (FRET), one fluorophore labeled aptamers based on fluorescence anisotropy assay, or other label-free aptamers are reviewed.     

Molecular beacons with intrinsically fluorescent nucleotides

We report the design, synthesis and characterization of a novel molecular beacon (MB-FB) which uses the fluorescent bases (FB) 2-aminopurine (AP) and pyrrolo-dC (P-dC) as fluorophores. Because the quantum yield of these FB depend on hybridization with complementary target, the fluorescent properties of MB-FB were tuned by placing the FB site specifically within the MB such that hybridization with complementary sequence switches from single strand to double strand for AP and vice versa for P-dC. The MB-FB produces a ratiometric fluorescence increase (the fluorescence emission of P-dC over that of AP in the presence and absence of complementary sequence) of 8.5 when excited at 310 nm, the maximum absorption of AP. This ratiometric fluorescence is increased to 14 by further optimizing excitation (325 nm). The fluorescence lifetime is also affected by the addition of target, producing a change in the long-lived component from 6.5 to 8.7 ns (Exc. 310 nm, Em. 450 nm). Thermal denaturation profiles monitored at 450 nm (P-dC emission) show a cooperative denaturation of the MB-FB with a melting temperature of 53°C. The thermal denaturation profile of MB-FB hybridized with its target shows a marked fluorescence reduction at 53°C, consistent with a transition from double stranded helix to random coil DNA.     

Molecular beacons: fluorogenic probes for living cell study

Molecular beacons are a new class of fluorescent probes that can report the presence of specific nucleic acids with high sensitivity and excellent specificity. In addition to their current wide applications in monitoring the progress of polymerase chain reactions, their unique properties make them promising probes for the detection and visualization of target biomolecules in living cells. This article is focused on our recent research in exploring the potential of using molecular beacon for living-cell studies in three important areas: the monitoring of mRNA in living cells, the development of ultrasmall DNA/RNA biosensors, and the novel approach of combining molecular beacon's signal transduction mechanism with aptamer's specificity for real-time protein detection. These applications demonstrate molecular beacon's unique properties in bioanalysis and bioassay development.     

Semiautomated clone verification by real-time PCR using molecular beacons

Conventional, high-throughput PCR analysis of common elements utilizing numerous primer sets and template DNA requires multiple rounds of PCR to ensure optimal conditions. Laborious gel electrophoresis and staining is then necessary to visualize amplification products. We propose novel multicolor molecular beacons, to establish a high-throughput, PCR-based sequence tagged site (STS) detection system that swiftly and accurately confirms marker content in template containing common repeat elements. A simple, one-tube, real-time PCR assay system was developed to specifically detect regions containing CA and GATA repeats. Ninety-six samples can be confirmed for marker content in a closed-tube format in 3 h, eliminating product confirmation on agarose gels and avoiding crossover contamination. Multiple STSs can be detected simultaneously in the same reaction tube by utilizing molecular beacons labeled with multicolor fluorophores. Template DNA from 260 RPCI-11 bacterial artificial chromosome (BAC) clones was examined for the presence of CA and/or GATA repeats using molecular beacon PCR and compared with conventional PCR results of the same clones. Of the 205 clones containing CA and GATA repeats, we were able to identify 129 clones (CA, n = 99; GATA, n = 30) by using molecular beacons and only 121 clones (CA, n = 92; GATA, n = 29) by conventional PCR amplification. As anticipated, 55 clones that contained sequences other than CA or GATA failed molecular beacon detection. Molecular beacon PCR, employing beacons specific for tandem repeat elements, provides a fast, accurate, and sensitive multiplex detection assay that will expedite verification of marker content in a multitude of template containing these repeats.     

Molecular beacons for DNA biosensors with micrometer to submicrometer dimensions

Ultrasensitive molecular beacon (MB) DNA biosensors, with micrometer to submicrometer sizes, have been developed for DNA/RNA analysis. The fluorescence-based biosensors have been applied in DNA/ RNA detection without the need for a dye-labeled target molecule or an intercalation reagent in the testing solution. Molecular beacons are hairpin-shaped oligonucleotides that report the presence of specific nucleic acids. We have designed a surface-immobilizable biotinylated ssDNA molecular beacon for DNA hybridization at a liquid-solid interface. The MBs have been immobilized onto ultrasmall optical fiber probes through avidin-biotin binding. The MB DNA biosensor has been used directly to detect, in real time, its target DNA molecules without the need for a competitive assay. The biosensor is stable and reproducible. The MB DNA biosensor has selectivity with single base-pair mismatch identification capability. The concentration detection limits and mass detection limits are 0.3 nM and 15 amol for a 105-microm biosensor, and 10 nM and 0.27 amol for a submicrometer biosensor, respectively. We have also prepared molecular beacon DNA biosensor arrays for simultaneous analysis of multiple DNA sequences in the same solution. The newly developed DNA biosensors have been used for the precise quantification of a specific rat gamma-actin mRNA sequence amplified by the polymerase chain reaction.     

Molecular beacons for detecting DNA binding proteins: mechanism of action

New methodology for detecting sequence-specific DNA binding proteins has been recently developed (T. Heyduk, and E. Heyduk, Nat. Biotechnol. 20 (2002) 171). The central feature of this assay is protein-dependent association of two DNA fragments, each containing about half of a DNA sequence-defining the protein binding site. In this report we propose a physical model explaining the functioning of the assay. The model involves two linked equilibria: association between the two DNA fragments and binding of the protein exclusively to the complex between the two DNA fragments. Equilibrium and kinetic experiments provided evidence supporting the proposed model and showed that the model was sufficient to describe the behavior of the assay under a variety of conditions. Kinetic data identified the association between the two DNA half-sites as the rate-limiting step of the assay. Theoretical simulations based on the proposed model were used to investigate parameters important for the maximal sensitivity of the assay. Physical understanding of the assay will provide means for rational design of the assay for a variety of target proteins.     

Molecular beacons: an optimal multifunctional biological probe

Molecular beacon technology is set up based on fluorescence resonance energy transfer (FRET) and the complementary pairing principles. These fluorescent molecular probes, which are very highly specific and sensitive, have now become one important tool in medical and biological researches. This review introduces the molecular beacons structure, principle, the main impact factors, the labeling of the molecular beacons, and research progress on molecular beacons fluorescent-label in the polymerase chain reaction (PCR), DNA sequence analysis, gene dynamic detection in living cells, protein (enzyme)-nucleic acid interactions and applications in clinical medicine.     

Molecular recognition of fibroblast activation protein for diagnostic and therapeutic applications

Fibroblast activation protein (FAP) is a non-classical serine protease expressed predominantly in conditions accompanied by tissue remodeling, particularly cancer. Due to its plasma membrane localization, FAP represents a promising molecular target for tumor imaging and treatment. The unique enzymatic activity of FAP facilitates development of diagnostic and therapeutic tools based on molecular recognition of FAP by substrates and small-molecule inhibitors, in addition to conventional antibody-based strategies.In this review, we provide background on the pathophysiological role of FAP and discuss its potential for diagnostic and therapeutic applications. Furthermore, we present a detailed analysis of the structural patterns crucial for substrate and inhibitor recognition by the FAP active site and determinants of selectivity over the related proteases dipeptidyl peptidase IV and prolyl endopeptidase. We also review published data on targeting of the tumor microenvironment with FAP antibodies, FAP-targeted prodrugs, activity-based probes and small-molecule inhibitors. We describe use of a recently developed, selective FAP inhibitor with low-nanomolar potency in inhibitor-based targeting strategies including synthetic antibody mimetics based on hydrophilic polymers and inhibitor conjugates for PET imaging.In conclusion, recent advances in understanding of the molecular structure and function of FAP have significantly contributed to the development of several tools with potential for translation into clinical practice.     

Flavin recognition by an RNA aptamer targeted toward FAD

Flavin adenine dinucleotide (FAD) is one of the primary cofactors in biological redox reactions. Designing cofactor-dependent redox ribozymes could benefit from studies of new RNA-cofactor complexes, as would our understanding of ribozyme evolution during an RNA World. We have therefore used the SELEX method to identify RNA aptamers that recognize FAD. Functional analysis of mutant aptamers, S1 nuclease probing, and comparative sequence analysis identified a simple, 45 nt helical structure with several internal bulges as the core-binding element. These aptamers recognize with high specificity the isoalloxazine nucleus of FAD but do not distinguish FAD from FADH(2), nor are they removed from an FAD resin with UMP (which shares a pattern of hydrogen bond donors and acceptors along one face). Thus, these aptamers are structurally and functionally distinct from previously identified FMN and riboflavin aptamers. Circular dichroism data suggest a conformational change in the RNA upon FAD binding. These aptamers require magnesium and are active across a wide pH range (4.5-8.9). Since general acid-base catalysis plays a role in some flavin-dependent redox reaction mechanisms, these aptamers may be particularly well-suited to the design of new redox ribozymes.     

Energetic basis of molecular recognition in a DNA aptamer

The thermal stability and ligand binding properties of the L-argininamide-binding DNA aptamer (5'-GATCGAAACGTAGCGCCTTCGATC-3') were studied by spectroscopic and calorimetric methods. Differential calorimetric studies showed that the uncomplexed aptamer melted in a two-state reaction with a melting temperature T(m)=50.2+/-0.2 degrees C and a folding enthalpy DeltaH(0)(fold)=-49.0+/-2.1 kcal mol(-1). These values agree with values of T(m)=49.6 degrees C and DeltaH(0)(fold)=-51.2 kcal mol(-1) predicted for a simple hairpin structure. Melting of the uncomplexed aptamer was dependent upon salt concentration, but independent of strand concentration. The T(m) of aptamer melting was found to increase as L-argininamide concentrations increased. Analysis of circular dichroism titration data using a single-site binding model resulted in the determination of a binding free energy DeltaG(0)(bind)=-5.1 kcal mol(-1). Isothermal titration calorimetry studies revealed an exothermic binding reaction with DeltaH(0)(bind)=-8.7 kcal mol(-1). Combination of enthalpy and free energy produce an unfavorable entropy of -TDeltaS(0)=+3.6 kcal mol(-1). A molar heat capacity change of -116 cal mol(-1) K(-1) was determined from calorimetric measurements at four temperatures over the range of 15-40 degrees C. Molecular dynamics simulations were used to explore the structures of the unligated and ligated aptamer structures. From the calculated changes in solvent accessible surface areas of these structures a molar heat capacity change of -125 cal mol(-1) K(-1) was calculated, a value in excellent agreement with the experimental value. The thermodynamic signature, along with the coupled CD spectral changes, suggest that the binding of L-argininamide to its DNA aptamer is an induced-fit process in which the binding of the ligand is thermodynamically coupled to a conformational ordering of the nucleic acid.     

The intrinsic flexibility of the aptamer targeting the ribosomal protein S8 is a key factor for the molecular recognition

Background

Aptamers are RNA/DNA biomolecules representing an emerging class of protein interactors and regulators. Despite the growing interest in these molecules, current understanding of chemical-physical basis of their target recognition is limited. Recently, the characterization of the aptamer targeting the protein-S8 has suggested that flexibility plays important functional roles. We investigated the structural versatility of the S8-aptamer by molecular dynamics simulations.

Molecular beacons as probes of RNA unfolding under native conditions

Hybridization of fluorescent molecular beacons provides real-time detection of RNA secondary structure with high specificity. We used molecular beacons to measure folding and unfolding rates of the Tetrahymena group I ribozyme under native conditions. A molecular beacon targeted against 15 nt in the 5′ strand of the P3 helix specifically hybridized with misfolded forms of the ribozyme, without invading the native tertiary structure. The beacon associated with the misfolded ribozyme 300 times more slowly than with an unstructured oligonucleotide containing the same target sequence, suggesting that the misfolded ribozyme core remains structured in the absence of Mg²⁺. The rate of beacon hybridization under native conditions revealed a linear relationship between the free energy of unfolding and Mg²⁺ concentration. A small fraction of the RNA population unfolded very rapidly, suggesting parallel unfolding in one step or through misfolded intermediates.