High-throughput quantitative binding analysis of DNA aptamers using exonucleases

Aptamers are nucleic acid bioreceptors that have been used in various applications including medical diagnostics and as therapeutic agents. Identifying the most optimal aptamer for a particular application is very challenging. Here, we for the first time have developed a high-throughput method for accurately quantifying aptamer binding affinity, specificity, and cross-reactivity via the kinetics of aptamer digestion by exonucleases. We demonstrate the utility of this approach by isolating a set of new aptamers for fentanyl and its analogs, and then characterizing the binding properties of 655 aptamer–ligand pairs using our exonuclease digestion assay and validating the results with gold-standard methodologies. These data were used to select optimal aptamers for the development of new sensors that detect fentanyl and its analogs in different analytical contexts. Our approach dramatically accelerates the aptamer characterization process and streamlines sensor development, and if coupled with robotics, could enable high-throughput quantitative analysis of thousands of aptamer–ligand pairs.     

Selection and characterization of DNA aptamers with binding selectivity to Campylobacter jejuni using whole-cell SELEX

The need for pre-analytical sample processing prior to the application of rapid molecular-based detection of pathogens in food and environmental samples is well established. Although immunocapture has been applied in this regard, alternative ligands such as nucleic acid aptamers have advantages over antibodies such as low cost, ease of production and modification, and comparable stability. To identify DNA aptamers demonstrating binding specificity to Campylobacter jejuni cells, a whole-cell Systemic Evolution of Ligands by EXponential enrichment (SELEX) method was applied to a combinatorial library of FAM-labeled single-stranded DNA molecules. FAM-labeled aptamer sequences with high binding affinity to C. jejuni A9a as determined by flow cytometric analysis were identified. Aptamer ONS-23, which showed particularly high binding affinity in preliminary studies, was chosen for further characterization. This aptamer displayed a dissociation constant (K _d value) of 292.8 ± 53.1 nM with 47.27 ± 5.58% cells fluorescent (bound) in a 1.48-μM aptamer solution. Binding assays to assess the specificity of aptamer ONS-23 showed high binding affinity (25–36%) for all other C. jejuni strains screened (inclusivity) and low apparent binding affinity (1–5%) with non-C. jejuni strains (exclusivity). Whole-cell SELEX is a promising technique to design aptamer-based molecular probes for microbial pathogens without tedious isolation and purification of complex markers or targets.     

In vitro selection of DNA aptamers binding ethanolamine

We have identified aptamers (synthetic oligonucleotides) binding to the very small molecule ethanolamine with high affinity down to the low nanomolar range. These aptamers were selected for their ability to bind to ethanolamine immobilised on magnetic beads, from an 96mer library of initially about 1 x 10(16) randomised ssDNA molecules. The dissociation constants of these aptamers range between K(D)=6 and K(D)=19 nmol L(-1). The aim of the development of ethanolamine aptamers is their use for the detection of this substance in clinical and environmental analysis. Ethanolamine is associated with several diseases. Moreover, ethanolamine and its derivatives di- and tri-ethanolamine are used in chemical and cosmetic industries. The use of biosensors with ethanolamine aptamer as new molecular recognition element could be an innovative method for an easy and fast detection of ethanolamine.     

In vitro selection of hematoporphyrin binding DNA aptamers

DNA aptamers that bind to hematoporphyrin IX (HPIX) were isolated using an in vitro selection technique. Most aptamers obtained after the 7th and 10th rounds contained guanine-rich sequences. Binding assay using fluorescence polarization technique and structural analysis by CD spectra revealed that the parallel guanine-quartet structure of the aptamer participates in the recognition of HPIX.     

In vitro selection of DNA aptamers binding pesticide fluoroacetamide

Fluoroacetamide (Mw = 77.06) is a lethal rodenticide to humans and animals which is still frequently abused in food storage somewhere in China. The production of antibodies for fluoroacetamide is difficult due to its high toxicity to animals, which limits the application of immunoassay method in poison detection. In this work, aptamers targeting N-fluoroacetyl glycine as an analog of fluoroacetamide were selected by a specific systematic evolution of ligands by exponential enrichment (SELEX) strategy. The binding ability of the selected aptamers to fluoroacetamide was identified using surface plasmon resonance (SPR)-based assay. The estimated K_D values in the low micromolar range showed a good affinity of these aptamers to the target. Our work verified that the SELEX strategy has the potential for developing aptamers targeted to small molecular toxicants and aptamers can be employed as new recognition elements instead of antibodies for poison detection.     

Characterization of plant satellite DNA using restriction nucleases

The structural organization of satellite DNAs of mustard Brassica nigra and lemon Citrus limon has been studied by digestion with restriction nucleases. Analysis of DNA products produced by EcoRI and Bam I shows that two satellite DNAs contain long range periodicities belonging to several repeated sequences. The periodicities in two satellite DNAs differ characteristically, however, they have been found to contain common homologous sequences. Using the restriction nuclease Bsp I, a highly periodical fractions has been found in Citrus satellite DNA, composed of Bsp I fragments ranging from 80 to 1240 basepain. The major repeat units comprise five Bsp I fragments ranging from 80 to 200 bp. These fractions characterized by a high content of 5-methyl-cytosine.     

DNA affinity binding studies using a fluorescent dye displacement technique: the dichotomy of the binding site

We have observed a number of discrepancies and contradictions in the use of a fluorescent intercalator displacement assay in surveying the binding affinities of dinuclear polypyridyl ruthenium(II) complexes with DNA. By a modification of the assay using the fluorescent minor-groove binder 4′,6-diamidino-2-phenylindole, rather than intercalating dyes (ethidium bromide or thiazole orange), results were obtained for all complexes studied which were consistent with relative affinities and stereoselectivities observed with other techniques, including NMR, affinity chromatography and equilibrium dialysis. It is believed that the difference in binding mode between the minor groove-binding Ru(II) complexes and the intercalating fluorescent dyes they are displacing may contribute to these discrepancies. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Targeting and tracing antigens in live cells with fluorescent nanobodies

We fused the epitope-recognizing fragment of heavy-chain antibodies from Camelidae sp. with fluorescent proteins to generate fluorescent, antigen-binding nanobodies (chromobodies) that can be expressed in living cells. We demonstrate that chromobodies can recognize and trace antigens in different subcellular compartments throughout S phase and mitosis. Chromobodies should enable new functional studies, as potentially any antigenic structure can be targeted and traced in living cells in this fashion.     

Selection of DNA aptamers using atomic force microscopy

Atomic force microscopy (AFM) can detect the adhesion or affinity force between a sample surface and cantilever, dynamically. This feature is useful as a method for the selection of aptamers that bind to their targets with very high affinity. Therefore, we propose the Systematic Evolution of Ligands by an EXponential enrichment (SELEX) method using AFM to obtain aptamers that have a strong affinity for target molecules. In this study, thrombin was chosen as the target molecule, and an ‘AFM-SELEX’ cycle was performed. As a result, selected cycles were completed with only three rounds, and many of the obtained aptamers had a higher affinity to thrombin than the conventional thrombin aptamer. Moreover, one type of obtained aptamer had a high affinity to thrombin as well as the anti-thrombin antibody. AFM-SELEX is, therefore, considered to be an available method for the selection of DNA aptamers that have a high affinity for their target molecules.     

Deconvolution of a complex target using DNA aptamers

In vitro selection of single-stranded nucleic acid aptamers from large random sequence libraries is now a straightforward process particularly when screening with a single target molecule. These libraries contain considerable shape diversity as evident by the successful isolation of aptamers that bind with high affinity and specificity to chemically diverse targets. We propose that aptamer libraries contain sufficient shape diversity to allow deconvolution of a complex mixture of targets. Using unfractionated human plasma as our experimental model, we aim to develop methods to obtain aptamers against as many proteins as possible. To begin, it is critical that we understand how aptamer populations change with increasing rounds of in vitro selection when using complex mixtures. Our results show that sequence representation in the selected population changes dramatically with increasing rounds of selection. Certain aptamer families were apparent after only three selection rounds. Two additional cycles saw a decline in the relative abundance of these families and the emergence of yet another family that accounted for more than 60% of sequences in the pool. To overcome this population convergence, an aptamer-based target depletion method was developed, and the library screen was repeated. The previous dominant family effectively disappeared from the selected populations but was replaced by other aptamer families. Insights gained from these initial experiments are now being applied in the creation of second generation plasma protein screens and also to the analysis of other complex biological targets.     

Using azobenzene incorporated DNA aptamers to probe molecular binding interactions

The rational design of DNA/RNA aptamers for use as molecular probes depends on a clear understanding of their structural elements in relation to target-aptamer binding interactions. We present a simple method to create aptamer probes that can occupy two different structural states. Then, based on the difference in binding affinity between these states, target-aptamer binding interactions can be elucidated. The basis of our two-state system comes from the incorporation of azobenzene within the DNA strand. Azobenzene can be used to photoregulate the melting of DNA-duplex structures. When incorporated into aptamers, the light-regulated conformational change of azobenzene can be used to analyze how aptamer secondary structure is involved in target binding. Azobenzene-modified aptamers showed no change in target selectivity, but showed differences in binding affinity as a function of the number, position, and conformation of azobenzene modifications. Aptamer probes that can change binding affinity on demand may have future uses in targeted drug delivery and photodynamic therapy.     

Labelling of liposomes with intercalating perylene fluorescent dyes.

The high fluorescent potential and the exceptional photostability of lipophilic derivatives of perylene-3,4:9,10-bis(dicarboximides) are utilized for the fluorescence-labelling of liposomes. The preparation of the liposomes is effected by supersonic starting from a lipid mixture consisting of the matrix lipids soy lecithin, cholesterol, alpha-tocopherol and the perylene dyes. From a multitude of perylene derivatives investigated only those are optimally incorporated into the bilayer membrane of unilamellar liposomes which are substituted at both nitrogen atoms by one or two linear hydrocarbon groups. In order to attain an optimal fluorescent quantum yield, about 200 to 300 dye molecules can be incorporated per liposome. The liposomes thus obtained have a diameter of about 70 to 80 nm, are homogeneous and may be stored for more than seven months. Neither the fluorescent properties nor the stability of these liposomes are influenced by the additional incorporation of various ara C-derivatives and lipophilic anchor groups which subsequently enable the coupling of antibodies to the liposomes. As the water-insoluble perylene dyes are incorporated into the bilayer membrane, the aqueous inner volume of the liposomes remains available for a further utilization.     

Direct visualization of nucleolar G-quadruplexes in live cells by using a fluorescent light-up probe

Background

Direct detection of G-quadruplexes in human cells has become an important issue due to the vital role of G-quadruplex related to biological functions. Despite several probes have been developed for detection of the G-quadruplexes in cytoplasm or whole cells, the probe being used to monitor the nucleolar G-quadruplexes is still lacking.

Identification of fluorescent compounds with non-specific binding property via high throughput live cell microscopy

Introduction

Compounds exhibiting low non-specific intracellular binding or non-stickiness are concomitant with rapid clearing and in high demand for live-cell imaging assays because they allow for intracellular receptor localization with a high signal/noise ratio. The non-stickiness property is particularly important for imaging intracellular receptors due to the equilibria involved.

Method

Three mammalian cell lines with diverse genetic backgrounds were used to screen a combinatorial fluorescence library via high throughput live cell microscopy for potential ligands with high in- and out-flux properties. The binding properties of ligands identified from the first screen were subsequently validated on plant root hair. A correlative analysis was then performed between each ligand and its corresponding physiochemical and structural properties.

Results

The non-stickiness property of each ligand was quantified as a function of the temporal uptake and retention on a cell-by-cell basis. Our data shows that (i) mammalian systems can serve as a pre-screening tool for complex plant species that are not amenable to high-throughput imaging; (ii) retention and spatial localization of chemical compounds vary within and between each cell line; and (iii) the structural similarities of compounds can infer their non-specific binding properties.

Conclusion

We have validated a protocol for identifying chemical compounds with non-specific binding properties that is testable across diverse species. Further analysis reveals an overlap between the non-stickiness property and the structural similarity of compounds. The net result is a more robust screening assay for identifying desirable ligands that can be used to monitor intracellular localization. Several new applications of the screening protocol and results are also presented.     

Semi-automated selection of DNA aptamers using magnetic particle handling

We have developed a semi-automatic selection procedure for DNA aptamers. Employing a robotic workstation for magnetic particle handling, this method allows for a fast, reproducible, and parallelized selection of DNA aptamers. The selection protocol is designed to provide high flexibility and versatility in terms of choice of buffers and reagents, as well as stringency of selection. Using this procedure, we have successfully isolated ligand-specific, high-affinity DNA aptamers.     

Protection of HIV neutralizing aptamers against rectal and vaginal nucleases: implications for RNA-based therapeutics

RNA-based drugs are an emerging class of therapeutics. They have the potential to regulate proteins, chromatin, as well as bind to specific proteins of interest in the form of aptamers. These aptamers are protected from nuclease attack by chemical modifications that enhance their stability for in vivo usage. However, nucleases are ubiquitous, and as we have yet to characterize the entire human microbiome it is likely that many nucleases are yet to be identified. Any novel, unusual enzymes present in vivo might reduce the efficacy of RNA-based therapeutics, even when they are chemically modified. We have previously identified an RNA-based aptamer capable of neutralizing a broad spectrum of clinical HIV-1 isolates and are developing it as a vaginal and rectal microbicide candidate. As a first step we addressed aptamer stability in the milieu of proteins present in these environments. Here we uncover a number of different nucleases that are able to rapidly degrade 2'-F-modified RNA. We demonstrate that the aptamer can be protected from the nuclease(s) present in the vaginal setting, without affecting its antiviral activity, by replacement of key positions with 2'-O-Me-modified nucleotides. Finally, we show that the aptamer can be protected from all nucleases present in both vaginal and rectal compartments using Zn(2+) cations. In conclusion we have derived a stable, antiviral RNA-based aptamer that could form the basis of a pre-exposure microbicide or be a valuable addition to the current tenofovir-based microbicide candidate undergoing clinical trials.