Aptamer-Based Sensors for Detection of Proteins

April 1, 2007

Army Research Laboratory, Adelphi, Maryland

Molecular aptamer beacons (MABs) are being investigated for use as rapid-signaling probe compounds for detecting specific proteins of interest (target proteins). In the MAB approach, one exploits a combination of (1) the molecular- recognition capability and high affinity of aptamers (defined below) with respect to molecules of interest and (2) the fluorescent- signaling transduction method of molecular beacon probes (also defined below) to enable real-time monitoring of target proteins. MABs could help to satisfy the increasing need for rapid, sensitive biosensing in diverse endeavors that include medical diagnosis, discovery of drugs, and homeland security. For example, rapid biosensing could enable early diagnosis and treatment of disease or rapid response to a chemical or biological attack.

A biosensor fundamentally consists of the combination of (1) a bioreceptor C, a substance that recognizes and binds to a target, and (2) a transduction mechanism (which could include another substance) to convert the binding event to a measurable signal. Bioreceptors can function in a variety of ways, the most common being nucleic acid-based (hybridization) and antibody-based (immunological). In the case of nucleic acid hybridization, identification of a nucleic acid target is achieved through matching of complementary base pairs that often are genetic components of an organism. In the case of antibody-based sensing, identification of an organism is effected through binding of an antibody to a portion of an antigen molecule specific to, and located on the surface of, the organism. Transduction mechanisms also can function in a variety of ways; the transduction mechanisms of interest for the present purpose are based on observable changes in fluorescence.

The Recognition and Signaling Mechanisms of an aptamer and a molecular beacon probe are combined in a molecular aptamer beacon.

An aptamer is a special type of synthetic oligonucleotide [an oligonucleotide is a ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) molecule, typically of the order of tens to hundreds of bases long] that can bind to a target molecule such as a protein or a metabolite. Although not yet in common use, aptamers constitute the basis of an emerging chemical- and biologicalrecognition approach that offers advantages over approaches based on traditional bioreceptors. Aptamers can be formulated to be capable of binding to specific targets of a variety of types, including chemicals, viruses, toxins, cells, and spores. The binding occurs through a combination of shape complementarity and noncovalent chemical bonds, as depicted in the upper part of the figure. Although the mechanisms of specific binding are similar to those of antibody-antigen interactions, unlike antibodies, aptamers are amenable to mass production without use of animals. Other distinct advantages of aptamerover antibody-based sensing include greater stability for field use, and ease of chemical modification to enable use of different transduction mechanisms.

A molecular beacon (alternatively denoted a molecular beacon probe and depicted in the lower part of the figure) is a normally hairpin-shaped oligonucleotide, typically of the order of 25 bases long, to which is bound a fluorescent reporter molecular group on one end and a fluorescence-quenching molecular group at the other end. In the absence of a target molecule, the molecule retains its hairpin shape, causing the fluorescent and the fluorescence-quenching groups to be held in proximity and thereby causing quenching of the fluorescence. Upon binding to a target molecule, the molecular beacon becomes bent in such a way as to separate the fluorescent and the fluorescence- quenching groups by a distance sufficient to enable a significant increase in fluorescence. Hence, the increase in fluorescence can serve as a signal for detecting the target molecule.

For a demonstration of the MAB approach, an aptamer for recognizing thrombin was modified by attachment of a fluorophore at one end and a fluorescence quencher at the other end, and was further modified so that in the absence of thrombin, it would assume the stem-loop (hairpin) configuration and upon binding to thrombin, it would become bent in such a way as to separate the fluorophore from the quencher. In tests, introduction of an excess of thrombin into a solution containing the MAB molecules resulted in a maximum threefold increase in fluorescence intensity, 70 percent of the increase occurring during the first 15 minutes.

This work was done by Dimitra N. Stratis- Cullum of the Army Research Laboratory. For more information, download the Technical Support Package (free white paper) at www.defensetechbriefs.com/tsp under the Bio-Medical category. ARL-0002

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Aptamer-Based Sensors for Detection of Proteins

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Overview

The document titled "Feasibility of Aptamer-based Sensors for the Real-time Detection of Protein Targets," authored by Dimitra N. Stratis-Cullum and published in September 2006, investigates the potential of aptamer-based sensors for rapid and sensitive detection of protein targets. The research spans from January to October 2005 and addresses the growing need for efficient biosensing methodologies across various fields, including medical diagnostics, drug discovery, and homeland security.

The introduction emphasizes the importance of rapid biological detection, which can facilitate earlier diagnosis and treatment of diseases and enhance responses to biological threats. The document outlines the development of sensing methodologies that are not only quick but also capable of specific and sensitive biological identification, with the potential for multiplexed analysis on a single platform.

The methodology section details the materials and systems used in the experiments, including hybridization studies and thrombin studies. The results and discussion section presents findings on complement-induced and protein-induced probe signaling, highlighting the signal dependence on protein concentration and the rapid signaling capabilities of the aptamer-based sensors.

Key findings indicate that nucleic acid aptamers combined with a Förster Resonance Energy Transfer (FRET) signaling approach can enable rapid analysis of protein samples. The study observed a maximum three-fold change in fluorescence intensity upon the introduction of the thrombin target, compared to a twelve-fold change achieved with a complement control. This suggests that while the system shows promise, there is room for improvement in the signal-to-background ratio.

The conclusions drawn from the research support the effectiveness of aptamer-based sensors in real-time protein detection. Future work is suggested to focus on enhancing the signal quality, including exploring alternative quenching methods to improve the “off” state and advanced modeling studies to optimize the “on” state.

Overall, the document underscores the feasibility of using aptamer-based sensors for real-time detection of protein targets, paving the way for advancements in biosensing technologies that could significantly impact various applications in health and security.

Aptamer-Based Sensors for Detection of Proteins