Book review of Aptamers in Bioanalysis

This book covers various applications of aptamers as biosensors. The chapters are organized in three major groups: (1) general aspects of aptamers and their selection, (2) aptamers as biosensors, and (3) applications of aptamer biosensors. It is clear even from the Table of Contents that aptamers have been utilized in many types of analytical techniques, including electrochemical, atomic force microscopy, capillary electrophoresis, spectrophotometric, and fluorometric. These applications are well described. However, the chapters tend to be repetitive, often reviewing the same applications and published works several times with little or no discussion of other approaches, such as the applications of aptamer biosensors in microcantilevers, photoinduced electron transfer, aptamer-coated SAW sensors, and evanescent-coupled field-guide wave sensors

Functional nucleic acid probes and uses thereof

The present invention provides functional nucleic acid probes, and methods of using functional nucleic acid probes, for binding a target to carry out a desired function. The probes have at least one functional nucleic acid, at least one regulating nucleic acid, and at least one attenuator. The functional nucleic acid is maintained in an inactive state by the attenuator and activated by the regulating nucleic acid only in the presence of a regulating nucleic acid target. In its activated state the functional nucleic acid can bind to its target to carry out a desired function, such as generating a signal, cleaving a nucleic acid, or catalyzing a reaction

Aptamer Functionalized Microcantilever Sensors for Cocaine Detection

A cocaine-specific aptamer was used as a receptor molecule in a microcantilever-based surface stress sensor for detection of cocaine molecules. An interferometric technique that relies on measuring differential displacement between two microcantilevers (a sensing/reference pair) was utilized to measure the cocaine/aptamer binding induced surface stress changes. Sensing experiments were performed for different concentrations of cocaine from 25 to 500 μM in order to determine the sensor response as a function of cocaine concentration. In the lower concentration range from 25 to 100 μM, surface stress values increased proportionally to coverage of aptamer/cocaine complexes from 11 to 26 mN/m. However, as the cocaine concentration was increased beyond 100 μM, the surface stress values demonstrated a weaker dependence on the affinity complex surface coverage. On the basis of a sensitivity of 3 mN/m for the surface stress measurement, the lowest detectable threshold for the cocaine concentration is estimated to be 5 μM. Sensing cantilevers could be regenerated and reused because of reversible thermal denaturation of aptamer

Computational and experimental analyses converge to reveal a coherent yet malleable aptamer structure that controls chemical reactivity

As short nucleic acids, aptamers in solution are believed to be structurally flexible. Consistent with this view, most aptamers examined for this property have been shown to bind their target molecules by mechanisms that can be described as induced fit . But, it is not known to what extent this structural flexibility affects the integrity of the target - aptamer interaction. Using the malachite green aptamer (MGA) as a model system, we show that the MGA can protect its bound target, malachite green (MG), from oxidation over several days. Protection is reversed by an oligonucleotide complementary to the MGA binding pocket. Computational cavity analysis of the MGA-MG structure predicted that MG oxidation is protected because a molecule as small as an OH- is sterically excluded from the C1 position of the bound MG. These results suggest that, while the MGA-MG interface is sufficiently coherent to prevent OH- penetration, the bases involved in the interaction are sufficiently mobile that they can exchange out of the MG binding interface to hybridize with a complementary oligonucleotide. The computational predictions were confirmed experimentally using variants of the MGA with single base changes in the binding pocket. This work demonstrates the successful application of molecular dynamics simulations and cavity analysis in determining the effects of sequence variations on the structure of a small single-stranded nucleic acid. It also shows that a nucleic acid aptamer can control access to specific chemical groups on its target, which suggests that aptamers might be applied for selectively protecting small molecules from modification

Detecting cells in time varying intensity images in confocal microscopy for gene expression studies in living cells

In this work we present a time-lapsed confocal microscopy image analysis technique for an automated gene expression study of multiple single living cells. Fluorescence Resonance Energy Transfer (FRET) is a technology by which molecule-to-molecule interactions are visualized. We analyzed a dynamic series of ~102 images obtained using confocal microscopy of fluorescence in yeast cells containing RNA reporters that give a FRET signal when the gene promoter is activated. For each time frame, separate images are available for three spectral channels and the integrated intensity snapshot of the system. A large number of time-lapsed frames must be analyzed to identify each cell individually across time and space, as it is moving in and out of the focal plane of the microscope. This makes it a difficult image processing problem. We have proposed an algorithm here, based on scale-space technique, which solves the problem satisfactorily. The algorithm has multiple directions for even further improvement. The ability to rapidly measure changes in gene expression simultaneously in many cells in a population will open the opportunity for real-time studies of the heterogeneity of genetic response in a living cell population and the interactions between cells that occur in a mixed population, such as the ones found in the organs and tissues of multicellular organisms

Parity is associated with an expanded macrophage population in the mammary gland

Pregnancy is a well established protective factor against breast cancer. One explanation for protection is the increased differentiation status of the parous epithelium. However, this does not explain the association of parity with increased aggressiveness of breast cancers, particularly cancers that occur soon after pregnancy. Because tumor aggressiveness can be influenced by the cell population that surrounds the mammary epithelium, we examined the potential role of the immune system in establishing a long-term difference between the mammary glands of primiparous and virgin animals. Specific mRNA levels, enzyme activities and antigen expressing cells were quantified in primiparous and virgin mammary glands from Sprague-Dawley rats in diestrous. Our results show that macrophages, but not neutrophils or B-cells, are specifically increased in fully involuted glands compared with age-matched virgin mammary glands. Macrophages play a dual role in tumor progression, both opposing and supporting the process. Our finding of an increased macrophage population in the primiparous mammary gland could explain the dichotomy of the reported association of parity with decreased breast cancer incidence and increased breast cancer aggressiveness

Aptamers for Diagnostics with Applications for Infectious Diseases

Aptamers are in vitro selected oligonucleotides (DNA, RNA, oligos with modified nucleotides) that can have high affinity and specificity for a broad range of potential targets with high affinity and specificity. Here we focus on their applications as biosensors in the diagnostic field, although they can also be used as therapeutic agents. A small number of peptide aptamers have also been identified. In analytical settings, aptamers have the potential to extend the limit of current techniques as they offer many advantages over antibodies and can be used for real-time biomarker detection, cancer clinical testing, and detection of infectious microorganisms and viruses. Once optimized and validated, aptasensor technologies are expected to be highly beneficial to clinicians by providing a larger range and more rapid output of diagnostic readings than current technologies and support personalized medicine and faster implementation of optimal treatments

Aptamers for Infectious Disease Diagnosis

Aptamers are in vitro-selected, nucleic acids with unique abilities to bind strongly and specifically to their selective targets (ligands) based on their three-dimensional structures. Target binding is generally associated with a change in aptamer structure, which provides a means of linking many output signals to the binding event. Being synthetic, aptamers are less expensive compared to antibodies. Aptamers are also more easily modified chemically or their sequence changed to optimize properties such target specificity, storability and stability. In this chapter we will discuss the potential benefits of applying aptamers to diagnostics with a focus on infectious disease and the unique challenges posed by aptamers for their successful incorporation into reliable aptasensors

An RNA Aptamer-Based Microcantilever Sensor To Detect the Inflammatory Marker, Mouse Lipocalin-2

Lipocalin-2 (Lcn2) is a biomarker for many inflammatory-based diseases, including acute kidney injury, cardiovascular stress, diabetes, and various cancers. Inflammatory transitions occur rapidly in kidney and cardiovascular disease, for which an in-line monitor could be beneficial. Microcantilever devices with aptamers as recognition elements can be effective and rapidly responsive sensors. Here, we have selected and characterized an RNA aptamer that specifically binds mouse Lcn2 (mLcn2) with a dissociation constant of 340 ± 70 nM in solution and 38 ± 22 nM when immobilized on a surface. The higher apparent affinity of the immobilized aptamer may result from its effective multivalency that decreases the off-rate. The aptamer competes with a catechol iron-siderophore, the natural ligand of mLcn2. This and the results of studies with mLcn2 mutants demonstrate that the aptamer binds to the siderophore binding pocket of the protein. A differential interferometer-based microcantilever sensor was developed with the aptamer as the recognition element in which the differential response between two adjacent cantilevers (a sensing/reference pair) is utilized to detect the binding between mLcn2 and the aptamer, ensuring that sensor response is independent of environmental influences, distance between sensing surface and detector and nonspecific binding. The system showed a detection limit of 4 nM. This novel microcantilever aptasensor has potential for development as an in-line monitoring system for mLcn2 in studies of animal models of acute diseases such as kidney and cardiac failure