An official website of the United States government
Abstract Aptamers are short oligonucleotides isolated in vitro from randomized libraries that can bind to specific molecules with high affinity, and offer a number of advantages relative to antibodies as biorecognition elements in biosensors. However, it remains difficult and labor‐intensive to develop aptamer‐based sensors for small‐molecule detection. Here, we review the challenges and advances in the isolation and characterization of small‐molecule‐binding DNA aptamers and their use in sensors. First, we discuss in vitro methodologies for the isolation of aptamers, and provide guidance on selecting the appropriate strategy for generating aptamers with optimal binding properties for a given application. We next examine techniques for characterizing aptamer–target binding and structure. Afterwards, we discuss various small‐molecule sensing platforms based on original or engineered aptamers, and their detection applications. Finally, we conclude with a general workflow to develop aptamer‐based small‐molecule sensors for real‐world applications.
more »
« less
RE-SELEX: restriction enzyme-based evolution of structure-switching aptamer biosensors
Aptamers are widely employed as recognition elements in small molecule biosensors due to their ability to recognize small molecule targets with high affinity and selectivity. Structure-switching aptamers are particularly promising for biosensing applications because target-induced conformational change can be directly linked to a functional output. However, traditional evolution methods do not select for the significant conformational change needed to create structure-switching biosensors. Modified selection methods have been described to select for structure-switching architectures, but these remain limited by the need for immobilization. Herein we describe the first homogenous, structure-switching aptamer selection that directly reports on biosensor capacity for the target. We exploit the activity of restriction enzymes to isolate aptamer candidates that undergo target-induced displacement of a short complementary strand. As an initial demonstration of the utility of this approach, we performed selection against kanamycin A. Four enriched candidate sequences were successfully characterized as structure-switching biosensors for detection of kanamycin A. Optimization of biosensor conditions afforded facile detection of kanamycin A (90 μM to 10 mM) with high selectivity over three other aminoglycosides. This research demonstrates a general method to directly select for structure-switching biosensors and can be applied to a broad range of small-molecule targets.
more »
« less
- Award ID(s):
- 1904885
- PAR ID:
- 10355111
- Date Published:
- Journal Name:
- Chemical Science
- Volume:
- 12
- Issue:
- 35
- ISSN:
- 2041-6520
- Page Range / eLocation ID:
- 11692 to 11702
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Abstract Aptamers are promising biorecognition elements for sensors. However, aptamer‐based assays often lack the requisite levels of sensitivity and/or selectivity because they typically employ structure‐switching aptamers with attenuated affinity and/or utilize reporters that require aptamer labeling or which are susceptible to false positives. Dye‐displacement assays offer a label‐free, sensitive means for overcoming these issues, wherein target binding liberates a dye that is complexed with the aptamer, producing an optical readout. However, broad utilization of these assays has been limited. Here, we demonstrate a rational approach to develop colorimetric cyanine dye‐displacement assays that can be broadly applied to DNA aptamers regardless of their structure, sequence, affinity, or the physicochemical properties of their targets. Our approach should accelerate the development of mix‐and‐measure assays that could be applied for diverse analytical applications.more » « less
-
The fast, accurate detection of biomolecules, ranging from nucleic acids and small molecules to proteins and cellular secretions, plays an essential role in various biomedical applications. These include disease diagnostics and prognostics, environmental monitoring, public health, and food safety. Aptamer recognition (DNA or RNA) has gained extensive attention for biomolecular detection due to its high selectivity, affinity, reproducibility, and robustness. Concurrently, biosensing with nanoparticles has been widely used for its high carrier capacity, stability and feasibility of incorporating optical and catalytic activity, and enhanced diffusivity. Biosensors based on aptamers and nanoparticles utilize the combination of their advantages and have become a promising technology for detecting of a wide variety of biomolecules with high sensitivity, reliability, specificity, and detection speed. Via various sensing mechanisms, target biomolecules have been quantified in terms of optical (e.g., colorimetric and fluorometric), magnetic, and electrical signals. In this review, we summarize the recent advances in and compare different aptamer–nanoparticle-based biosensors by nanoparticle types and detection mechanisms. We also share our views on the highlights and challenges of the different nanoparticle-aptamer-based biosensors.more » « less
-
Detection of analytes by means of field-effect transistors bearing ligand-specific receptors is fundamentally limited by the shielding created by the electrical double layer (the “Debye length” limitation). We detected small molecules under physiological high–ionic strength conditions by modifying printed ultrathin metal-oxide field-effect transistor arrays with deoxyribonucleotide aptamers selected to bind their targets adaptively. Target-induced conformational changes of negatively charged aptamer phosphodiester backbones in close proximity to semiconductor channels gated conductance in physiological buffers, resulting in highly sensitive detection. Sensing of charged and electroneutral targets (serotonin, dopamine, glucose, and sphingosine-1-phosphate) was enabled by specifically isolated aptameric stem-loop receptors.more » « less
-
null (Ed.)Rapid and accurate diagnosis of various biomarkers associated with medical conditions including early detection of viruses and bacteria with highly sensitive biosensors is currently a research priority. Aptamer is a chemically derived recognition molecule capable of detecting and binding small molecules with high specificity and its fast preparation time, cost effectiveness, ease of modification, stability at high temperature and pH are some of the advantages it has over traditional detection methods such as High Performance Liquid Chromatography (HPLC), Enzyme-linked Immunosorbent Assay (ELISA), Polymerase Chain Reaction (PCR). Higher sensitivity and selectivity can further be achieved via coupling of aptamers with nanomaterials and these conjugates called “aptasensors” are receiving greater attention in early diagnosis and therapy. This review will highlight the selection protocol of aptamers based on Traditional Systematic Evolution of Ligands by EXponential enrichment (SELEX) and the various types of modified SELEX. We further identify both the advantages and drawbacks associated with the modified version of SELEX. Furthermore, we describe the current advances in aptasensor development and the quality of signal types, which are dependent on surface area and other specific properties of the selected nanomaterials, are also reviewed.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/D1SC02715H
