Aptamer Binding Studies Using MicroScale Thermophoresis

Breitsprecher, Dennis; Schlinck, Nina; Witte, David P.; Duhr, Stefan; Philipp, B.; Schubert, Thomas

doi:10.1007/978-1-4939-3197-2_8

Cited by 29 publications

(25 citation statements)

References 10 publications

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“…The Soret coefficient ST: chot/ccold = exp(-ST ΔT) provides a quantitative measure for this effect and is depending on the size, charge and hydration shell of the molecules. Upon molecular interaction, at least one of these parameters is changed, resulting in distinct thermophoretic movements of the unbound and bound states [31]. …”

Section: Resultsmentioning

confidence: 99%

The monoclonal S9.6 antibody exhibits highly variable binding affinities towards different R-loop sequences

König¹,

Schubert²,

Längst³

2017

PLoS ONE

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The monoclonal antibody S9.6 is a widely-used tool to purify, analyse and quantify R-loop structures in cells. A previous study using the surface plasmon resonance technology and a single-chain variable fragment (scFv) of S9.6 showed high affinity (0.6 nM) for DNA—RNA and also a high affinity (2.7 nM) for RNA—RNA hybrids. We used the microscale thermophoresis method allowing surface independent interaction studies and electromobility shift assays to evaluate additional RNA-DNA hybrid sequences and to quantify the binding affinities of the S9.6 antibody with respect to distinct sequences and their GC-content. Our results confirm high affinity binding to previously analysed sequences, but reveals that binding affinities are highly sequence specific. Our study presents R-loop sequences that independent of GC-content and in different sequence variations exhibit either no binding, binding affinities in the micromolar range and as well high affinity binding in the nanomolar range. Our study questions the usefulness of the S9.6 antibody in the quantitative analysis of R-loop sequences in vivo.

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Section: Resultsmentioning

confidence: 99%

The monoclonal S9.6 antibody exhibits highly variable binding affinities towards different R-loop sequences

König¹,

Schubert²,

Längst³

2017

PLoS ONE

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“…While highly sensitive techniques employing radioactivity and SPR can measure K d up to sub-picomolar range; Fluorescence and ITC can measure K d limited to nanomolar and spectrophotometry techniques can measure K d in micromolar range. Recently, MicroScale Thermophoresis (MST), a novel low cost highly sensitive technique has been described by numerous aptamer—research groups that can estimate apparent dissociation constant in pico to nanomolar range, with high accuracy in limited microliter scale solutions (Stoltenburg et al, 2015 ; Breitsprecher et al, 2016 ; Entzian and Schubert, 2016 ; Jauset Rubio et al, 2016 ). In addition to less volume and unlike several techniques, MST also offers the advantage, that it can estimate the aptamer K d independent of the target size in wide range of buffers as well as in complex biological samples, in pico to nanomolar range (Entzian and Schubert, 2016 ).…”

Section: Enhancing Aptamer Evaluation Parametersmentioning

confidence: 99%

Simple Methods and Rational Design for Enhancing Aptamer Sensitivity and Specificity

Kalra

Dhiman

Cho

et al. 2018

Front. Mol. Biosci.

122

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Aptamers are structured nucleic acid molecules that can bind to their targets with high affinity and specificity. However, conventional SELEX (Systematic Evolution of Ligands by EXponential enrichment) methods may not necessarily produce aptamers of desired affinity and specificity. Thus, to address these questions, this perspective is intended to suggest some approaches and tips along with novel selection methods to enhance evolution of aptamers. This perspective covers latest novel innovations as well as a broad range of well-established approaches to improve the individual binding parameters (aptamer affinity, avidity, specificity and/or selectivity) of aptamers during and/or post-SELEX. The advantages and limitations of individual aptamer selection methods and post-SELEX optimizations, along with rational approaches to overcome these limitations are elucidated in each case. Further the impact of chosen selection milieus, linker-systems, aptamer cocktails and detection modules utilized in conjunction with target-specific aptamers, on the overall assay performance are discussed in detail, each with its own advantages and limitations. The simple variations suggested are easily available for facile implementation during and/or post-SELEX to develop ultrasensitive and specific assays. Finally, success studies of established aptamer-based assays are discussed, highlighting how they utilized some of the suggested methodologies to develop commercially successful point-of-care diagnostic assays.

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“…Therefore, the thermophoresis changes as a function of ligand concentration allow calculating dissociation constants ( K d ) [40]. Several MST applications for aptamer research have been reported (see [42] for detailed methods). Here, we label the aptamer with a fluorescent probe (probe FAM ) and use it at a constant concentration and increased the concentrations of HMG-box Pf , ranging from 0.1 nM to 2 μM.…”

Section: Resultsmentioning

confidence: 99%

DNA aptamers for the recognition of HMGB1 from Plasmodium falciparum

Joseph

Nakamoto

Ruiz

et al. 2019

Preprint

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21 Rapid Diagnostic Tests (RDTs) for malaria are restricted to a few biomarkers and antibody-22 mediated detection. However, the expression of commonly used biomarkers varies 23 geographically and the sensibility of immunodetection can be affected by batch-to-batch 24 differences or limited thermal stability. In this study we aimed to overcome these limitations 25 by identifying a potential biomarker and by developing molecular sensors based on aptamer 26 technology. Using gene expression databases, ribosome profiling analysis, and structural 27 modeling, we find that the High Mobility Group Box 1 protein (HMGB1) of Plasmodium 28 falciparum is highly expressed, structurally stable and steadily present along all blood-stages 29 of P. falciparum infection. To develop biosensors, we used in vitro evolution techniques to 30 produce DNA aptamers for the recombinantly expressed HMG-box, the conserved domain 31 of HMGB1. An evolutionary approach for evaluating the dynamics of aptamer populations 32 suggested three predominant aptamer motifs. Representatives of the aptamer families were 33 tested for binding parameters to the HMG-box domain using microscale thermophoresis and 34 rapid kinetics. Dissociation constants of the aptamers varied over two orders of magnitude 35 between nano-and micromolar ranges while the aptamer-HMG-box interaction occurred in 36 less than 30 seconds. The specificity of aptamer binding to the HMG-box of P. falciparum 37 compared to its human homolog depended on pH conditions. Altogether, our study proposes 38 HMGB1 as a potential biomarker and a set of sensing aptamers that can be further developed 39 into rapid diagnostic tests for P. falciparum detection. 40 41 42 3 43 44 45 46 47 Malaria is an infectious disease that affects animals and humans, caused by protozoans of the 48 genus Plasmodium. Malaria remains the cause of 435,000 deaths worldwide, with 219 49 million cases reported during 2017 [1]. Accurate treatment requires the identification of the 50 parasite of the genus Plasmodium, in addition to the species causing the disease. Currently, 51 there are several methods to diagnose malaria, such as PCR-based, Giemsa microscopy, and 52 Rapid Diagnostic Tests (RDTs). Among these options, the last two appear to be suitable for 53 low-income and mostly affected countries; nonetheless, they also present certain limitations 54 [2]. 55 Giemsa microscopy is inexpensive to perform, can differentiate malaria species and stages, 56 and can quantify the parasites [2]. However, well-trained personnel is required and, 57 frequently unavailable in low-income countries [2]. RDTs, on the other hand, 58 detect Plasmodium spp. biomarker antigens in a small amount of blood using immobilized 59 antibodies. The two most used biomarkers are histidine-rich protein 2 (HRP-2) and lactate 60 dehydrogenase (LDH), which have been shown of high sensitivity and specificity for 61 detecting P. falciparum [3]. However, P. falciparum strains lacking the HRP-2 and HRP-3 62 genes have appeared, increasing false negative re...

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Aptamer Binding Studies Using MicroScale Thermophoresis

Cited by 29 publications

References 10 publications

The monoclonal S9.6 antibody exhibits highly variable binding affinities towards different R-loop sequences

The monoclonal S9.6 antibody exhibits highly variable binding affinities towards different R-loop sequences

Simple Methods and Rational Design for Enhancing Aptamer Sensitivity and Specificity

DNA aptamers for the recognition of HMGB1 from Plasmodium falciparum

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