Aptamer Arrays Research Articles

Programming DNA Aptamer Arrays of Prescribed Spatial Features with Enhanced Bioavailability and Cell Growth Modulation.

Epithelial cell adhesion molecules (EpCAMs) play pivotal roles in tumorigenesis in many cancer types, which is reported to reside within nano- to microscale membrane domains, forming small clusters. We propose that building multivalent ligands that spatially patch to EpCAM clusters may largely enhance their targeting capability. Herein, we assembled EpCAM aptamers into nanoscale arrays of prescribed valency and spatial arrangements by using a rectangular DNA pegboard. Our results revealed that EpCAM aptamer arrays exhibited significantly higher binding avidity to MCF-7 cells than free monovalent aptamers, which was affected by both valency and spatial arrangement of aptamers. Furthermore, EpCAM aptamer arrays showed improved tolerance against competing targets in an extracellular environment and potent bioavailability and targeting specificity in a xenograft tumor model in mice. This work may shed light on rationally designing multivalent ligand complexes of defined parameters with optimized binding avidity and targeting capability toward various applications in the biomedical fields.

Generation of small molecule-binding RNA arrays and their application to fluorogen-binding RNA aptamers.

The discovery and engineering of more and more functions of RNA has highlighted the utility of RNA-targeting small molecules. Recently, several fluorogen-binding RNA aptamers have been developed that have been applied to live cell imaging of RNA and metabolites as RNA tags or biosensors, respectively. Although the design and application of these fluorogen-binding RNA aptamer-based devices is straightforward in theory, in practice, careful optimisation is required. For this reason, high throughput in vitro screening techniques, capable of quantifying fluorogen-RNA aptamer interactions, would be beneficial. We recently developed a method for generating functional-RNA arrays and demonstrated that they could be used to detect fluorogen-RNA aptamer interactions. Specifically, we were able to visualise the interaction between malachite green and the malachite green-binding aptamer. Here we expand this study to demonstrate that functional-RNA arrays can be used to quantify fluorogen-aptamer interactions. As proof-of-concept, we provide detailed protocols for the production of malachite green-binding RNA aptamer and DFHBI-binding Spinach RNA aptamer arrays. Furthermore, we discuss the potential utility of the technology to fluorogen-binding RNA aptamers, including application as a molecular biosensor platform. We anticipate that functional-RNA array technology will be beneficial for a wide variety of biological disciplines.

Raman Spectroscopy and Aptamers for a Label-Free Approach: Diagnostic and Application Tools.

Raman spectroscopy is a powerful optical technique based on the inelastic scattering of incident light to assess the chemical composition of a sample, including biological ones. Medical diagnostic applications of Raman spectroscopy are constantly increasing to provide biochemical and structural information on several specimens, being not affected by water interference, and potentially avoiding the constraint of additional labelling procedures. New strategies have been recently developed to overcome some Raman limitations related, for instance, to the need to deal with an adequate quantity of the sample to perform a reliable analysis. In this regard, the use of metallic nanoparticles, the optimization of fiber optic probes, and other approaches can actually enhance the signal intensity compared to spontaneous Raman scattering. Moreover, to further increase the potential of this investigation technique, aptamers can be considered as a valuable means, being synthetic, short, single, or double-stranded oligonucleotides (RNAs or DNAs) that fold up into unique 3D structures to specifically bind to selected molecules, even at very low concentrations, and thus allowing an early diagnosis of a possible disease. Due to the paramount relevance of the topic, this review focuses on the main Raman spectroscopy techniques combined with aptamer arrays in the label-free mode, providing an overview on different applications to support healthcare management.

Sensitive colorimetric assay using insulin G-quadruplex aptamer arrays on DNA nanotubes coupled with magnetic nanoparticles.

Described here is a methodology for fabrication of a sensitive colorimetric nanoassay for measurement of insulin using G-quadruplex aptamer arrays on DNA nanotubes (DNTs) coupled with magnetic nanoparticles. The spectroscopic findings (e.g. visible spectra, velocity assay and limit of detection determination) indicated a highly sensitive performance of this new nanoassay in comparison to those results obtained from the insulin assay with non-arrayed aptamers. The clinical performance statistics (i.e. paired sample t-test, Bland–Altman plot and scatter diagram) from the newly developed assay and the enzyme-linked immunosorbent assay suggested its reliable precision and its acceptable repeatability for measurement of insulin in human sera. This is, to our knowledge, the first study for the application of magnetic nanoparticle-coupled DNTs for carrying G-quadruplex aptamers for detection of biomolecules (such as insulin) in human serum.

Advancements in Aptamer Discovery Technologies.

Affinity reagents that specifically bind to their target molecules are invaluable tools in nearly every field of modern biomedicine. Nucleic acid-based aptamers offer many advantages in this domain, because they are chemically synthesized, stable, and economical. Despite these compelling features, aptamers are currently not widely used in comparison to antibodies. This is primarily because conventional aptamer-discovery techniques such as SELEX are time-consuming and labor-intensive and often fail to produce aptamers with comparable binding performance to antibodies. This Account describes a body of work from our laboratory in developing advanced methods for consistently producing high-performance aptamers with higher efficiency, fewer resources, and, most importantly, a greater probability of success. We describe our efforts in systematically transforming each major step of the aptamer discovery process: selection, analysis, and characterization. To improve selection, we have developed microfluidic devices (M-SELEX) that enable discovery of high-affinity aptamers after a minimal number of selection rounds by precisely controlling the target concentration and washing stringency. In terms of improving aptamer pool analysis, our group was the first to use high-throughput sequencing (HTS) for the discovery of new aptamers. We showed that tracking the enrichment trajectory of individual aptamer sequences enables the identification of high-performing aptamers without requiring full convergence of the selected aptamer pool. HTS is now widely used for aptamer discovery, and open-source software has become available to facilitate analysis. To improve binding characterization, we used HTS data to design custom aptamer arrays to measure the affinity and specificity of up to ∼10(4) DNA aptamers in parallel as a means to rapidly discover high-quality aptamers. Most recently, our efforts have culminated in the invention of the "particle display" (PD) screening system, which transforms solution-phase aptamers into "aptamer particles" that can be individually screened at high-throughput via fluorescence-activated cell sorting. Using PD, we have shown the feasibility of rapidly generating aptamers with exceptional affinities, even for proteins that have previously proven intractable to aptamer discovery. We are confident that these advanced aptamer-discovery methods will accelerate the discovery of aptamer reagents with excellent affinities and specificities, perhaps even exceeding those of the best monoclonal antibodies. Since aptamers are reproducible, renewable, stable, and can be distributed as sequence information, we anticipate that these affinity reagents will become even more valuable tools for both research and clinical applications.

Tandem Spinach Array for mRNA Imaging in Living Bacterial Cells.

Live cell RNA imaging using genetically encoded fluorescent labels is an important tool for monitoring RNA activities. A recently reported RNA aptamer-fluorogen system, the Spinach, in which an RNA aptamer binds and induces the fluorescence of a GFP-like 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) ligand, can be readily tagged to the RNA of interest. Although the aptamer–fluorogen system is sufficient for imaging highly abundant non-coding RNAs (tRNAs, rRNAs, etc.), it performs poorly for mRNA imaging due to low brightness. In addition, whether the aptamer-fluorogen system may perturb the native RNA characteristics has not been systematically characterized at the levels of RNA transcription, translation and degradation. To increase the brightness of these aptamer-fluorogen systems, we constructed and tested tandem arrays containing multiple Spinach aptamers (8–64 aptamer repeats). Such arrays enhanced the brightness of the tagged mRNA molecules by up to ~17 fold in living cells. Strong laser excitation with pulsed illumination further increased the imaging sensitivity of Spinach array-tagged RNAs. Moreover, transcriptional fusion to the Spinach array did not affect mRNA transcription, translation or degradation, indicating that aptamer arrays might be a generalizable labeling method for high-performance and low-perturbation live cell RNA imaging.

Abstract 2227: Aptamer selection for cancer markers: High-throughput, quantitative selection and characterization of nucleic acid aptamers for human angiopoietin-2.

Abstract Abstract Nucleic acid-based aptamers represent a promising class of affinity reagents, as they can be chemically synthesized and modified, undergo reversible folding and exhibit excellent stability. Thus, the identification of aptamers that bind to specific cancer markers may serve as a powerful tool for early diagnosis and treatments of cancers. Unfortunately, conventional methods of aptamer generation remain inherently low-throughput in nature, and there is a pressing need for alternative technologies that can generate high quality aptamers in a high-throughput and economical manner. Towards the goal of accelerating aptamer discovery, we have developed the Quantitative Parallel Aptamer Selection System (QPASS) - an integrated platform that combines microfluidic selection, next-generation sequencing and in situ-synthesized aptamer arrays for parallel binding measurements. As a model, we used QPASS to select aptamers that specifically bind to human angiopoietin-2, an important mediator of angiogenesis and a biomarker of colon, prostate and breast cancers; after microfluidic selection and sequencing, we fabricated a custom aptamer array containing 120,000 sequences representing the most enriched aptamer candidates. Using this array, we simultaneously obtained equilibrium dissociation constants (Kd) and binding specificity data for these candidate aptamers in a massively parallel manner. Furthermore, we show that our array can operate directly in undiluted serum, enabling rapid identification of aptamers that perform optimally in complex mixtures. By exploiting the scalability and commercial availability of high-throughput sequencing and nucleic acid arrays, the QPASS platform offers the capability to simultaneously analyze and characterize unprecedented numbers of aptamer candidates—either against a common cancer marker, or for screening in parallel against multiple different biomarkers. Citation Format: Minseon Cho, Seung Soo Oh, Jeff Nie, Ron Stewart, Michael Eisenstein, James Chambers, Jamey D. Marth, James A. Thomson, Tom H. Soh. Aptamer selection for cancer markers: High-throughput, quantitative selection and characterization of nucleic acid aptamers for human angiopoietin-2. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2227. doi:10.1158/1538-7445.AM2013-2227

Identifying protein variants with cross-reactive aptamer arrays.

Much of the work directed at the creation of arrays for protein analysis relies on the use of highly selective molecules as receptors[1]. In these arrays, the receptors are immobilized and exposed to a mixture of target molecules[1d] or to heterogeneous biological samples such as serum from cancer patients[2]. However, such arrays generally only detect the molecules they were programmed to detect, and are ill-suited for the detection of protein variants, such as sequence changes, deletions, and insertions.

Nanoscale Patterning of Multicomponent Proteins by Bias‐Assisted Atomic Force Microscopy Nanolithography

Well-assisted nanopatterns: A new route to fabricate multicomponent protein nanopatterns by combining bias-assisted atomic force microscopy nanolithography and aptamer–protein recognition is presented. Two types of aptamer arrays for anchoring platelet-derived growth-factor-BB and thrombin (see picture) are introduced [Eg3-SH: 11-mercaptoundecyl-tri(ethylene glycol)].

Advances and perspectives in aptamer arrays

Aptamers are oligonucleotides (typically 10-60 bases in length) capable of binding target ligands with affinities similar to antibodies. The generation of high density multiplexed aptamer arrays for molecular diagnostics was first proposed nearly ten years ago for the quantification of the thousands of proteins within biological samples, including blood and urine. The tagless aptameric detection of small molecular compounds extends the application of such arrays to bioanalyses at the metabolite level. We present here a minireview on some existing technologies and highlight recent innovations that are being applied to this field, which may facilitate the vision of highly multi-parallelized arrays for the quantitative analysis of biological systems.

Biosensors

Biosensors

Optimization of aptamer microarray technology for multiple protein targets

Aptamer-based microarrays for the quantitation of multiple protein analytes have been developed. A multiplex aptamer microarray was generated by printing two RNA aptamers (anti-lysozyme and anti-ricin) and two DNA aptamers (anti-IgE and anti-thrombin) on to either streptavidin (SA) or neutravidin (NA)-coated glass slides. However, substantial optimization was required in order to ensure the simultaneous function of the aptamer:analyte pairs. The effects of protein labeling, assay buffer, surface coating, and immobilization chemistry and orientation were investigated. A single buffer (PBS buffer containing 5 mM MgCl 2 and 0.1% Tween 20) was found to work well with all the aptamers, even though this was not the buffer originally used in their selection, while neutravidin-coated slides yielded a lower detection limit, wider detection range, and more uniform background than streptavidin-coated slides. Incubation with Cy3-labeled proteins yielded sensitive, target-specific, and dose-dependent responses to each protein. Target protein concentrations as low as 72 pg/mL (5 pM, lysozyme), 15 ng/mL (0.5 nM, ricin), 1.9 ng/mL (0.01 nM, IgE), and 170 ng/mL (5 nM, thrombin) could be detected. These results show that aptamer arrays can potentially be used with numerous proteins in parallel, furthering the notion that aptamer arrays may be useful in proteomics.