SYPRO Orange Research Articles

A novel simple fluorometric protease assay for monitoring hydrolysis of proteins in real time

Measuring the activity of proteases is essential for investigating both the physiological functions and commercial applications of these enzymes. In contrast to the numerous protease assays that are based on chromogenic or fluorogenic peptide substrates, there is a lack of approaches to monitor degradation of proteins in real time. Here we report a protease assay where SYPRO Orange is employed as a fluorogenic probe to follow proteolysis. The functionality of the assay was demonstrated with the two subtilases of varying thermostability, using four different protein substrates. The assay is compatible with a real-time PCR instrument which allows continuous fluorescence measurements in low-volume samples even at high temperatures. This makes the assay especially suitable for high-throughput characterization of thermostable proteases.

N α -acetyl-L-ornithine deacetylase from Escherichia coli and a ninhydrin-based assay to enable inhibitor identification.

Bacteria are becoming increasingly resistant to antibiotics, therefore there is an urgent need for new classes of antibiotics to fight antibiotic resistance. Mammals do not express N ɑ -acetyl-L-ornithine deacetylase (ArgE), an enzyme that is critical for bacterial survival and growth, thus ArgE represents a promising new antibiotic drug target, as inhibitors would not suffer from mechanism-based toxicity. A new ninhydrin-based assay was designed and validated that included the synthesis of the substrate analog N 5, N 5-di-methyl N α-acetyl-L-ornithine (kcat/Km = 7.32 ± 0.94 × 104M-1s-1). This new assay enabled the screening of potential inhibitors that absorb in the UV region, and thus is superior to the established 214nm assay. Using this new ninhydrin-based assay, captopril was confirmed as an ArgE inhibitor (IC50 = 58.7μM; Ki = 37.1 ± 0.85μM), and a number of phenylboronic acid derivatives were identified as inhibitors, including 4-(diethylamino)phenylboronic acid (IC50 = 50.1μM). Selected inhibitors were also tested in a thermal shift assay with ArgE using SYPRO Orange dye against Escherichia coli ArgE to observe the stability of the enzyme in the presence of inhibitors (captopril Ki = 35.9 ± 5.1μM). The active site structure of di-Zn EcArgE was confirmed using X-ray absorption spectroscopy, and we reported two X-ray crystal structures of E. coli ArgE. In summary, we describe the development of a new ninhydrin-based assay for ArgE, the identification of captopril and phenylboronic acids as ArgE inhibitors, thermal shift studies with ArgE + captopril, and the first two published crystal structures of ArgE (mono-Zn and di-Zn).

Library screening identifies commercial drugs as potential structure correctors of abnormal apolipoprotein A-I

AapoA-I, the main protein of high-density lipoprotein, plays a key role in the biogenesis and atheroprotective properties of high-density lipoprotein. We showed previously that a naturally occurring apoA-I mutation, L178P, induces major defects in protein’s structural integrity and functions that may underlie the increased cardiovascular risk observed in carriers of the mutation. Here, a library of marketed drugs (956 compounds) was screened against apoA-I[L178P] to identify molecules that can stabilize the normal conformation of apoA-I. Screening was performed by the thermal shift assay in the presence of fluorescent dye SYPRO Orange. As an orthogonal assay, we monitored the change in fluorescence intensity of 8-anilinonaphthalene-1-sulfonic acid upon binding on hydrophobic sites on apoA-I. Screening identified four potential structure correctors. Subsequent analysis of the concentration-dependent effect of these compounds on secondary structure and thermodynamic stability of WT apoA-I and apoA-I[L178P] (assessed by thermal shift assay and circular dichroism spectroscopy), as well as on macrophage viability, narrowed the potential structure correctors to two, the drugs atorvastatin and bexarotene. Functional analysis showed that these two compounds can restore the defective capacity of apoA-I[L178P] to promote cholesterol removal from macrophages, an important step for atheroprotection. Computational docking suggested that both drugs target a positively charged cavity in apoA-I, formed between helix 1/2 and helix 5, and make extensive interactions that could underlie thermodynamic stabilization. Overall, our findings indicate that small molecules can correct defective apoA-I structure and function and may lead to novel therapeutic approaches for apoA-I–related dyslipidemias and increased cardiovascular risk.

A high-throughput differential scanning fluorimetry method for rapid detection of thermal stability and iron saturation in lactoferrin

Thermal stability and iron saturation of lactoferrin (LF) are of great significance not only for the evaluation of the biological activities of LF but also for the optimization of the isolation and drying process parameters. Differential scanning calorimetry (DSC) is a well-established and efficient method for thermal stability and iron saturation detection in LF. However, multiple DSC measurements are typically performed sequentially, thus time-consuming and low throughput. Herein, we introduced the differential scanning fluorimetry (DSF) approach to overcome such limitations. The DSF can monitor LF thermal unfolding with a commonly available real-time PCR instrument and a fluorescent dye (SYPRO orange or Glomelt), and the measured melting temperature of LF is consistent with that determined by DSC. On the basis of that, a new quantification method was established for determination of iron saturation levels using the linear correlation of the degree of ion saturation of LF with DSF measurements. Such DSF method is simple, inexpensive, rapid (<15 min), and high throughput (>96 samples per experiment), and provides a valuable alternative tool for thermal stability detection of LF and other whey proteins.

Fluorescence correlation spectroscopy measurements on amyloid fibril reveal at least two binding modes for fluorescent sensors

Interaction of fluorophore with amyloid structure is central to the design and development of fluorescent sensors. In literature ensemble spectroscopy results reveal at least two distinct binding modes for most of the torsional probes like Thioflavin-T, SYPRO Orange, etc. though single molecule localization microscopy and spectroscopy results consider only one binding mode. To address this discrepancy we employed fluorescence correlation spectroscopy (FCS) to measure the binding kinetics on immobilized fibril structures with single molecule sensitivity. Our results indicate that there are at least two possible biding configurations with differing relaxation time constants indicating possible weaker and stronger binding configurations depending on the local environment on the fibril structure. Critical evaluation of on-time distributions from the recorded wide-field image stack for single molecule localization microscopy image, further highlight the presence of two burst times. Such bi-modal binding behavior is attributed to the differing binding configurations on fibril architecture.

Fluorescence-based thermal stability screening is concentration-dependent and varies with protein size

Proteins are used in many areas including diagnostic and therapeutical applications. Screening protein stability is an essential step for production, pharmacokinetic/pharmacodynamic studies, and determination of storage conditions. Instability of proteins can cause serious problems such as activity loss and unexpected adverse effects, so determination of sensitive and reliable methods for protein stability measurement is crucial. There are several “gold-standard” protein stability tests such as differential scanning calorimetry (DSC), but they are usually not suitable for high-throughput settings and consume large amounts of proteins. Instead, more high-throughput methods such as fluorescent-based assays can be used and validated to make stability screening process more straight-forward, easier, and lower-cost. Here, two methods were systemically compared to see whether their measurements depended on protein sizes. DSC and Sypro Orange dye-based fluorescent assay were compared for various proteins with different sizes and quaternary structures. This is the first systemic comparison of these two methods for thermal stability testing for different ranges of proteins in the literature. It was shown that protein melting temperature (Tm) measured by fluorescent assay highly depends on protein concentration and protein size. Larger proteins with multi-domain structures such as monoclonal antibodies gave more deviated and lower than expected Tms compared to small proteins. It has been concluded that fluorescent-based thermal stability assays are more suitable for smaller proteins, but protein concentrations used are still needed to be optimized in their settings for more reliable results.

Ferguson plot analysis of multiple intermediate species of thermally unfolded bovine serum albumin

Ferguson plot was used to characterize the multiple intermediate species of bovine serum albumin (BSA) upon thermal unfolding. Differential scanning calorimetry showed an irreversible melting of BSA in Tris-HCl and phosphate buffers with a mid-transition temperature, Tm, of ∼68 °C. Thermally unfolded BSA was analyzed by agarose native gel electrophoresis stained by Coomassie blue and SYPRO Orange staining as a function of pH or protein concentration. SYPRO Orange was used to stain unfolded proteins. BSA heated at 70 and 80 °C, i.e., above the Tm, formed multiple intermediate species, which depended on the pH between 7.0 and 8.0, protein concentration and which buffer was used. These intermediate species were analyzed by Ferguson plot, which showed that BSA heated at 60 °C had a similar size to the native BSA, indicating that they are either native or native-like state consistent with no SYPRO Orange staining. The intermediate species observed at higher temperatures with the mobility less than that of the native BSA showed a steeper Ferguson plot and were stained by SYPRO Orange, indicating that these species had a larger hydrodynamic size than the native BSA and were unfolded.

Contrasting complexation behaviour of zwitterionic amyloid probe, SYPRO orange with β-cyclodextrin and captisol

The differential complexation studies with cyclodextrins and their charged derivatives have been well studied using cationic, anionic, as well as neutral guest molecules. However, supramolecular complexation with zwitterionic guest molecules is quite limited. Zwitterions are overall neutral molecules with localized charges that can offer additional site-specific electrostatic interaction with cyclodextrins as compared to neutral guest molecules. There is no information on how the different intra- and intermolecular interactions can modulate the binding of a zwitterion with a macrocyclic host. Herein, the supramolecular interactions of biologically important zwitterionic amyloid probe, SYPRO Orange (SO) with β-cyclodextrin (β-CD) and sulfobutylether β-cyclodextrin (SBE-β-CD, Captisol®) have been evaluated and compared using different spectroscopic methods. SO forms inclusion complexes with both β-CD and SBE-β-CD which led to the enhanced fluorescence along with a hypsochromic shift in its emission spectra. It has been shown that the presence of segmental rotation in a guest molecule with charge transfer property favours its association with the macromolecules. The fluorescence enhancement and the binding affinities show contrasting behavior in these two types of host–guest complexes. Time-resolved emission measurements could disentangle the contribution from different inclusion complexes. Ionic strength and temperature variation in the photophysical properties helped to understand the molecular interactions involved in such contrasting behavior.

The extracellular chaperone clusterin prevents primary and secondary nucleation of an amyloidogenic variant of β2-microglobulin

Amyloid fibril formation by the extracellular protein β2-microglobulin (β2m) and its subsequent accumulation in periarticular tissues have been linked to dialysis-related amyloidosis. A natural variant of human β2m responsible for aggressive systemic amyloidosis contains an aspartate to asparagine mutation at residue 76 (i.e. D76N β2m), which readily forms amyloid fibrils in vitro under physiological conditions. In this study, we examined the role of the extracellular molecular chaperone clusterin in modulating D76N β2m fibril formation in vitro under physiological conditions. The presence of extrinsic charged amino acids modulated D76N β2m fibril formation, implying that electrostatic interactions are involved in the protein’s aggregation. Thioflavin T (ThT) and 1-anilinonaphthalene-8-sulfonate fluorescence assays indicated that clusterin interacts via hydrophobic and electrostatic forces with the monomeric, prefibrillar and fibrillar species of D76N β2m. As a result, clusterin was incorporated into D76N β2m aggregates during the latter’s fibril formation, as indicated by SDS-PAGE of depolymerised fibrils. SYPRO Orange and ThT fluorescence assays suggested that, compared to pure D76N β2m fibrils, those formed in the presence of clusterin are chemically more stable with a reduced ability to act as nucleation seeds. Detailed 15N NMR relaxation studies of mixtures of 15N-labelled β2m with clusterin confirmed that the chaperone interacts transiently and non-specifically with monomeric β2m. Clusterin inhibits both primary and secondary nucleation of D76N β2m fibril formation. In doing so, clusterin binds to D76N β2m fibrils and stabilises them to prevent possible fragmentation. In vivo, the multifaceted chaperone action of clusterin may delay, if not prevent, β2m amyloid proliferation and deposition in tissues.

Analysis of bovine serum albumin unfolding in the absence and presence of ATP by SYPRO Orange staining of agarose native gel electrophoresis

An attempt was made to specifically stain unfolded proteins on agarose native gels. SYPRO Orange is routinely used to detect unfolded protein in differential scanning fluorimetry, which is based on the enhanced fluorescence intensity upon binding to the unfolded protein. We demonstrated that this dye barely bound to the native proteins, resulting in no or faint staining of the native bands, but bound to and stained the unfolded proteins, on agarose native gels. Using bovine serum albumin (BSA), it was shown that staining did not depend on whether BSA was thermally unfolded in the presence of SYPRO Orange or stained after electrophoresis. On the contrary, SYPRO Orange dye stained protein bands in the presence of sodium dodecylsulfate (SDS) due to incorporation of the dye into SDS micelles that bound to the unfolded proteins. This staining resulted in detection of new, intermediately unfolded structure of BSA during thermal unfolding. Such intermediate structure occurred at higher temperature in the presence of ATP.

Critical evaluation of fluorescent dyes to evaluate the stability and ligand binding properties of an anti-cocaine mAb, h2E2

Monoclonal antibodies have become a mainstay of modern drug development. However, unlike small molecule drugs, mAbs are large proteins that need to be characterized for their stability, heterogeneity, and tendency to aggregate. Many different extrinsic fluorescent dyes have been used to monitor the thermal stability, aggregation, and ligand binding characteristics of many different proteins. Some of these dyes change their fluorescence when bound to proteins due to changes in the hydrophobicity of their microenvironment (solvatochromic dyes such as Sypro Orange), while others respond to differences in rotational mobilities (rotor dyes such as DASPMI), and others have been used to detect fibrils and amyloid-like protein aggregation (amyloid dyes such as Thioflavin T). Previously, we used DASPMI dye and differential scanning fluorimetry to quantitate the binding of cocaine and cocaine metabolites to a humanized anti-cocaine h2E2 mAb under development for the treatment of cocaine use disorders. In the present study, we evaluated six dyes in these three classes for their ability to monitor domain denaturation and cocaine binding of the h2E2 mAb, both in its clinical formulation buffer and in PBS buffer. We noted that the Thioflavin T dye commonly used to assess amyloid formation was also capable of monitoring h2E2 mAb thermal denaturation and ligand binding using differential scanning calorimetry. However, unlike the DASPMI dye, the Thioflavin T dye caused a dose-dependent stabilization of the unliganded (apo) mAb, and when using the methodology developed with the DASPMI dye, decreased the apparent affinity of the mAb for cocaine as a function of dye concentration. Thus, although Thioflavin T differential fluorimetry data appears to be suitable for measuring cocaine affinity for this h2E2 mAb, the apparent mAb Kd values for cocaine are dependent on Thioflavin T dye concentration, reinforcing and extending the unique use of the DASPMI dye for this purpose.

Thermal Shift Assay for Small GTPase Stability Screening: Evaluation and Suitability.

Thermal unfolding methods are commonly used as a predictive technique by tracking the protein’s physical properties. Inherent protein thermal stability and unfolding profiles of biotherapeutics can help to screen or study potential drugs and to find stabilizing or destabilizing conditions. Differential scanning calorimetry (DSC) is a ‘Gold Standard’ for thermal stability assays (TSA), but there are also a multitude of other methodologies, such as differential scanning fluorimetry (DSF). The use of an external probe increases the assay throughput, making it more suitable for screening studies, but the current methodologies suffer from relatively low sensitivity. While DSF is an effective tool for screening, interpretation and comparison of the results is often complicated. To overcome these challenges, we compared three thermal stability probes in small GTPase stability studies: SYPRO Orange, 8-anilino-1-naphthalenesulfonic acid (ANS), and the Protein-Probe. We studied mainly KRAS, as a proof of principle to obtain biochemical knowledge through TSA profiles. We showed that the Protein-Probe can work at lower concentration than the other dyes, and its sensitivity enables effective studies with non-covalent and covalent drugs at the nanomolar level. Using examples, we describe the parameters, which must be taken into account when characterizing the effect of drug candidates, of both small molecules and Designed Ankyrin Repeat Proteins.

The stretch region within the TTD domain of UHRF2 is a disordered region

UHRF1 and UHRF2 are multiple‐domain epigenetic proteins that share a high degree of sequence similarity. These proteins play a very important role in histone modification and DNA methylation. Although both proteins contain a TTD and a PHD domain, the TTD domain of UHRF2 contains a unique ~35 amino acid long region that is highly basic called the “stretch”. This region was analyzed for the possibility of being a disordered region using the Sypro Orange Thermofluor assay. We found that UHRF2 TTD‐PHD has a lower melting temperature than UHRF2 TTD‐PHD without the stretch. It was also found that at higher salt concentrations, the melting temperature of UHRF2 TTD‐PHD increases, likely due to the disordered “stretch” region becoming stabilized. This data provides support towards the model that the stretch region is disordered and regulates protein stability.

Analysis of proteins by agarose native gel electrophoresis in the presence of solvent additives.

Solvent additives, including NaCl, arginine hydrochloride (ArgHCl), glycine and sucrose, are used to enhance protein stability or reduce protein aggregation. Here, we studied the effects of these additives on proteins using agarose native gel electrophoresis. Since these additives are used at relatively high concentration, we first confirmed that they do not interfere with the performance of the native gel electrophoresis. Agarose native gel electrophoresis showed that aggregation of bovine serum albumin (BSA) induced by heating was slightly reduced by NaCl and ArgHCl. On the contrary, glycine and sucrose had marginal effects. ArgHCl and NaCl promoted heat aggregation of monoclonal antibody (mAb), while glycine and sucrose stabilized the native mAb. Arginine methyl ester inhibited heat aggregation of lysozyme and, to a much lesser extent, BSA. These results show that agarose native gel electrophoresis can be used to analyze the effects of solvent additives on proteins subjected to heat stresses. SYPRO Orange that stains only unfolded proteins confirmed unfolded structures of soluble aggregates.

A low-cost 3D-printable differential scanning fluorometer for protein and RNA melting experiments

Differential scanning fluorimetry (DSF) is a widely used biophysical technique with applications to drug discovery and protein biochemistry. DSF experiments are commonly performed in commercial real-time polymerase chain reaction (qPCR) thermal cyclers or nanoDSF instruments. Here, we report the construction, validation, and example applications of an open-source DSF system for 176€, which, in addition to protein-DSF experiments, also proved to be a versatile biophysical instrument for less conventional RNA-DSF experiments. Using 3D-printed parts made of polyoxymethylene, we were able to fabricate a thermostable machine chassis for protein-melting experiments. The combination of blue high-power LEDs as the light source and stage light foil as filter components was proven to be a reliable and affordable alternative to conventional optics equipment for the detection of SYPRO Orange or Sybr Gold fluorescence. The ESP32 microcontroller is the core piece of this openDSF instrument, while the in-built I2S interface was found to be a powerful analog-to-digital converter for fast acquisition of fluorescence and temperature data. Airflow heating and inline temperature control by thermistors enabled high-accuracy temperature management in PCR tubes (±0.1 °C) allowing us to perform high-resolution thermal shift assays (TSA) from exemplary biological applications.

Cocaine binding to the Fab fragment of a humanized anti-cocaine mAb quantitated by dye absorption and fluorescence spectroscopy

In this work, we establish that cocaine binding to the Fab fragment of a recombinant humanized anti-cocaine mAb (h2E2) can be directly and easily quantitated using simple and inexpensive absorption and fluorescence measurements, employing dyes typically used for differential scanning fluorimetry, DASPMI and SYPRO Orange. For concentrated samples of the Fab fragment, absorbance spectroscopy employing these dyes reveals the number of cocaine sites present, using either DASPMI (by measuring the increase in dye absorbance) or SYPRO Orange (by measuring the change in dye maximal absorbance wavelength). Interestingly, we observed that cocaine binding to the Fab fragment had a much different effect on the SYPRO Orange dye absorbance than previously reported for the intact h2E2 mAb, resulting in a large decrease in the total dye absorbance for the Fab fragment, in contrast to previous results with the intact h2E2 mAb. For dilute samples of Fab fragment, a dye fluorescence emission spectroscopy assay was developed to quantitate the number of cocaine (and other high affinity cocaine metabolites) binding sites via the ligand-induced decrease in fluorescence emission of both of these extrinsic dyes. The difference in the cocaine titrations for the high affinity (Kd < 30 nM) ligands, cocaine, cocaethylene and benzoylecgonine and the low affinity (Kd > 30 μM) ligands, norcocaine, ecgonine methyl ester, and ecgonine were obvious using this assay. These simple, direct, and inexpensive techniques should prove useful for evaluation of other small molecule antigen binding Fab fragments, enabling quantitation and rapid biochemical assessments necessary for determining Fab fragment suitability for in vivo uses and other assays and experiments.

Understanding the amyloid sensing mechanism of SYPRO Orange

Amyloid fibrils, an aggregated form of proteins, are responsible for different types of neurological diseases. A constant effort is there for the search of a sensitive and selective probe for amyloid fibrils. Recently, it has been shown that a zwitterionic merocyanine dye, SYPRO Orange, can detect the amyloid fibrils with much higher sensitivity and with much preferred spectroscopic properties than present gold standard amyloid probe, Thioflavin T. In the present work, we have investigated the detailed photophysical properties of SYPRO Orange in different molecular solvents and solvent mixtures with wide range of properties to disentangle the fundamental processes that are responsible for its amyloid sensing activity. It has been shown that SYPRO Orange forms aggregates in water and results in extremely low emission quantum yield. The amyloid induced de-aggregation of SYPRO Orange is primarily responsible for enormous increase in its emission intensity upon binding with amyloid fibrils. Photophysical studies using steady state and time-resolved emission spectroscopy further enlighten us on the role of solvent polarity, solute-solvent H-bonding and solvent viscosity on its sensitivity towards amyloid fibrils. Thus, present results will be quite useful for the use of SYPRO Orange in different other spectroscopic applications.

Fluorescent thermal shift-based method for detection of NF-\u03baB binding to double-stranded DNA

The nuclear factor kappa B (NF-κB) family of dimeric transcription factors regulates a wide range of genes by binding to their specific DNA regulatory sequences. NF-κB is an important therapeutic target linked to a number of cancers as well as autoimmune and inflammatory diseases. Therefore, effective high-throughput methods for the detection of NF-κB DNA binding are essential for studying its transcriptional activity and for inhibitory drug screening. We describe here a novel fluorescence-based assay for quantitative detection of κB consensus double-stranded (ds) DNA binding by measuring the thermal stability of the NF-κB proteins. Specifically, DNA binding proficient NF-κB probes, consisting of the N-terminal p65/RelA (aa 1–306) and p50 (aa 1–367) regions, were designed using bioinformatic analysis of protein hydrophobicity, folding and sequence similarities. By measuring the SYPRO Orange fluorescence during thermal denaturation of the probes, we detected and quantified a shift in the melting temperatures (ΔTm) of p65/RelA and p50 produced by the dsDNA binding. The increase in Tm was proportional to the concentration of dsDNA with apparent dissociation constants (KD) of 2.228 × 10–6 M and 0.794 × 10–6 M, respectively. The use of withaferin A (WFA), dimethyl fumarate (DMF) and p-xyleneselenocyanate (p-XSC) verified the suitability of this assay for measuring dose-dependent antagonistic effects on DNA binding. In addition, the assay can be used to analyse the direct binding of inhibitors and their effects on structural stability of the protein probe. This may facilitate the identification and rational design of new drug candidates interfering with NF-κB functions.

Ligand binding to a humanized anti-cocaine mAb measured by dye absorption spectroscopy

Here we demonstrate that the antigen binding function of a humanized anti-cocaine mAb (h2E2) can be directly and easily determined using simple and inexpensive absorption spectroscopy and dyes commonly used for differential scanning fluorimetry, such as DASPMI and SYPRO Orange. Therapeutic monoclonal antibodies are commonly formulated in buffers which can interfere with necessary functional assays, containing additives and excipients such as mild detergents. Using the undiluted therapeutic product h2E2 mAb in its formulation buffer containing 0.01% polysorbate 80, the number of antigen/cocaine binding sites can be determined by the increase in absorbance (for DASPMI dye) or by the decrease in absorbance maximum wavelength (for SYPRO Orange dye), confirming proper function of the therapeutic mAb product. This ligand-induced visible dye absorption change can also be used to qualitatively evaluate the relative affinities of various metabolites of cocaine. These results are confirmed and extended by binding data obtained in the same formulation buffer using intrinsic tyrosine and tryptophan fluorescence quenching by cocaine, as well as by differential scanning fluorimetry. Interestingly, the binding of the cocaine metabolite norcocaine was demonstrated to be differentially affected by the pH 6 formulation buffer used for this mAb, presumably due to the differential ionizability of the demethylated norcocaine tropane ring nitrogen. This simple, direct, and inexpensive technique should prove useful for evaluation of other small molecule binding mAbs directly in their formulation buffers containing detergent, allowing rapid functional assessment of the produced therapeutic proteins.

NanoDSF: In vitro Label-Free Method to Monitor Picornavirus Uncoating and Test Compounds Affecting Particle Stability.

Thermal shift assays measure the stability of macromolecules and macromolecular assemblies as a function of temperature. The Particle Stability Thermal Release Assay (PaSTRy) of picornaviruses is based on probes becoming strongly fluorescent upon binding to hydrophobic patches of the protein capsid (e.g., SYPRO Orange) or to the viral RNA genome (e.g., SYTO-82) that become exposed upon heating virus particles. PaSTRy has been exploited for studying the stability of viral mutants, viral uncoating, and the effect of capsid-stabilizing compounds. While the results were usually robust, the thermal shift assay with SYPRO Orange is sensitive to surfactants and EDTA and failed at least to correctly report the effect of excipients on an inactivated poliovirus 3 vaccine. Furthermore, interactions between the probe and capsid-binding antivirals as well as mutual competition for binding sites cannot be excluded. To overcome these caveats, we assessed differential scanning fluorimetry with a nanoDSF device as a label-free alternative. NanoDSF monitors the changes in the intrinsic tryptophan fluorescence (ITF) resulting from alterations of the 3D-structure of proteins as a function of the temperature. Using rhinovirus A2 as a model, we demonstrate that nanoDFS is well suited for recording the temperature-dependence of conformational changes associated with viral uncoating with minute amounts of sample. We compare it with orthogonal methods and correlate the increase in viral RNA exposure with PaSTRy measurements. Importantly, nanoDSF correctly identified the thermal stabilization of RV-A2 by pleconaril, a prototypic pocket-binding antiviral compound. NanoDFS is thus a label-free, high throughput-customizable, attractive alternative for the discovery of capsid-binding compounds impacting on viral stability.