Using dual exonucleases to finely distinguish structural adjustment of aptamers for small-molecule detection

The binding of small molecules to their DNA aptamers can modulate their susceptibility to digestion by exonucleases, however, absolute differentiation between digestion and inhibition has never been reported. Here, we show that the digestion of aptamers by T7 exonuclease can be completely inhibited upon binding of small-molecule targets and exploit this finding for the first time to achieve sensitive, label-free small-molecule detection. We use a quinine-binding aptamer to show that target binding entirely halts T7 exonuclease digestion, leaving behind an intact double-stranded product that retains strong target affinity. On the contrary, digestion of nontarget-bound aptamer produces a single-stranded product incapable of target binding. Exonuclease I efficiently eliminates these single-stranded products but is unable to digest the target-bound double-stranded product. The remaining products can be fluorescently quantified with SYBR Gold to determine target concentrations, giving a limit of detection of 100 nM with the linear range from 0 to 8 μM. We demonstrate the first example of a dual-exonuclease-mediated approach capable of producing a concentration-dependent response in terms of aptamer digestion modules, therefore improving performance of the current aptamer-based assay for small-molecule detection.

Dual exonucleases to finely distinguish structural adjustment of aptamers to produce absolute differentiation between digestion and inhibition.     

Selection of aptamers for AMACR detection from DNA libraries with different primers

In this work, we investigated aptamer selection against alpha-methylacyl-CoA racemase (AMACR) with three DNA libraries bearing different primers. Because of increased selection diversity, aptamers with varied structural properties, such as pre-folded, induced-folding, high and low primer-dependent conformations, were discovered. From the selection results, a dimeric aptamer was constructed and capable of detecting over-expression of AMACR in prostate cancer cell lines. In summary, this study demonstrates a library-based approach to obtain aptamers with different binding properties and provides distinct aptamers for flexible design of AMACR detection.

This study demonstrates a library-based approach to obtain aptamers with different binding properties for flexible design of AMACR detection.     

Enhanced antitumor activity of carbendazim on HeLa cervical cancer cells by aptamer mediated controlled release

Carbendazim, is a broad-spectrum fungicide and also a promising experimental antitumor drug as reproduction and developmental toxicant, which is currently under phase II preclinical trials. In this study, an approach based on controlled and targeted release with aptamers and mesoporous silica nanoparticles was investigated to improve the antitumor activity of carbendazim. To this end, we synthesized aptamer conjugated silica nanoparticles for testing cytotoxicity properties in vitro with human cervical adenocarcinoma (HeLa) cultured cells. Nucleolin (AS1411) binding aptamers were used to entrap carbendazim molecules inside nanopores of MCM-41 type silica nanoparticles to obtain a stimuli-dependent release system. The effect of carbendazim loaded aptamer silica complex was tested and compared to free carbendazim treatment on HeLa cells, demonstrating 3.3 fold increase of toxicity on targeted cells with our delivery system. In addition, cytotoxicity of the complex was determined to be mostly due to increased apoptosis and to a less extend necrosis related pathways.

Carbendazim doped and aptamer-gate functionalized mesoporous silica nanoparticles targeted nucleolin on HeLa cell surface for specific delivery. This delivery system improved antitumor activity of carbendazim by about 3 folds increase of EC₅₀ values.     

Proximity hybridization triggered strand displacement and DNAzyme assisted strand recycling for ATP fluorescence detection in vitro and imaging in living cells

We developed a novel strategy for ATP detection in vitro and imaging in living cells based on integrating proximity hybridization-induced strand displacement and metal ion-dependent DNAzyme recycling amplification. Four DNA oligonucleotides were used in the sensing system including two aptamer probes, enzymatic sequences and FAM-linked substrate strands. Upon the addition of ATP, the proximity binding of two aptamers to ATP led to the release of the enzymatic sequences, which hybridized with the FAM-linked substrate strand on the graphene oxide (GO) surface to form the ion-dependent DNAzyme. Subsequent catalytic cleavage of the DNAzyme by the corresponding metal ions results in recycling of the enzymatic sequences and cyclic cleavage of the substrate strand, liberating many short FAM-linked oligonuleotide fragments separated from the GO surface, which results in fluorescence enhancement due to the weak affinity of the short FAM-linked oligonuleotide fragment to GO. The amount of produced short FAM-linked oligonuleotide fragments is positively related to the concentration of ATP. This means that one target binding could result in cleaving multiplex fluorophore labelled substrate strands, which provided effective signal amplification. The vivo studies suggested that the nanoprobe was efficiently delivered into living cells and worked for specific, high-contrast imaging of target ATP. More importantly, this target-responsive nanoscissor model is an important approach for intracellular amplified detection and imaging of various analytes by selecting appropriate affinity ligands.

A novel strategy for ATP imaging was developed based on proximity binding-induced strand displacement and metal ion-dependent DNAzyme recycling.     

A proof of concept application of aptachain: ligand-induced self-assembly of a DNA aptamer

A challenge for the use of aptamers as biosensors is how to signal the occurrence of their ligand binding event into a signal that can be exploited in a detection scheme. Here, we present the concept of “aptachain” formation, where an aptamer is split into two overlapping or staggered strands and assembles into an extended oligomer upon ligand binding. This assembly of aptamers can then be used as a way to detect ligand binding by the aptamer. As an example of this concept, we employed the cocaine-binding aptamer as a model system, used its ability to tightly bind quinine and demonstrated its capability in a gold nanoparticle-based biosensing application. We used isothermal titration calorimetry to demonstrate that, when split into two overlapping DNA strands, the aptamer remains functional. Size-exclusion chromatography showed that the quinine-bound oligos form a larger assembly of aptamer units than in the absence of ligand. Finally, we used the oligomer forming ability of the aptachain oligos in a biosensor application for quinine that brings gold nanoparticles closer together resulting in a shift in their plasmonic resonance to a longer wavelength and an observed colour shift. We propose that splitting aptamers into overlapping strands that form oligomers in the presence of a ligand, aptachain formation, will be generally applicable to aptamers and prove useful in a variety of biotechnology applications.

We present the concept of aptachain. An aptamer is split into two overlapping strands that form an oligomer when it binds its target. Aptachain formation can be used to detect ligand binding and may be beneficial in other biotechnology applications.     

Selection and characterization of toxic Aspergillus spore-specific DNA aptamer using spore-SELEX

As airborne spores of toxic Aspergillus species cause mild symptoms to invasive fungal infections, their indoor concentration should be controlled through real-time management. Aptamer-based biosensors could provide economical and simple solutions for point-of-care. In this study, we isolated aptamers binding to the spores of three representative toxic Aspergillus species (A. fumigatus, A. flavus, and A. niger) for the first time, using cell-SELEX (systematic evolution of ligands through exponential enrichment). Among the aptamer candidates, Asp-3 showed a broad and high binding affinity for the Aspergillus spores. Considering the low binding affinity with proteinase-treated spores, we speculated that the Asp-3 binding sites could be possibly associated with cell surface proteins. The high Asp-3 specificity was confirmed by comparing the binding affinity between the Aspergillus target species and other common indoor fungal species. Moreover, we also established quantitative linear relationships between Asp-3 and the spore concentration of each Aspergillus species. Therefore, the selected Asp-3 aptamer, conjugated with detection sensors, could be an effective biorecognition element for the spores of three toxic Aspergillus species.

Spore-SELEX was performed for the isolation of an Aspergillus spore-specific aptamer.     

Adsorption–desorption nano-aptasensors: fluorescent screening assays for ochratoxin A

In this study, a FRET-based fluorescent aptasensor for the detection of ochratoxin A (OTA) was optimized based on the quenching efficiency of single-walled carbon nanotubes (SWCNTs) and the binding affinity of aptamers. OTA aptamers were conjugated with quantum dots and adsorbed to the surface of both acid-modified and unmodified SWCNTs. The maximum fluorescence quenching efficiency of the SWCNTs were compared. Acid-modified SWCNTs (amSWCNTs) have moderate quenching efficiency, providing an optimal sensitivity for qualitative fluorescence-enhancement biosensor assays. The binding parameters of the QD-modified OTA aptamers (1.12.2 and A08min) on the surface of amSWCNTs were compared. Based on our results, the A08min aptamer is a better candidate for OTA detection. Using the A08min aptamer, the SWCNT method had a limit of detection (LOD) of 40 nM. The amSWCNT method had a significantly lower LOD of 14 nM. Turn-on fluorescent nano-aptasensors are emerging as an effective diagnostic tool for simple detection of mycotoxins. Nanocomplexes designed for the detection of mycotoxins in solution and paper-based tests have proven to be useful.

A fluorescent-enhancement biosensor was developed for the mycotoxin ochratoxin A using aptamer-modified quantum dots noncovalently immobilized on carbon nanotubes.     

Smartphone-based kanamycin sensing with ratiometric FRET

Smartphone-based fluorescence detection is a promising avenue for biosensing that can aid on-site analysis. However, quantitative detection with fluorescence in the field has been limited due to challenges with robust excitation and calibration requirements. Here, we show that ratiometric analysis with Förster resonance energy transfer (FRET) between dye pairs on DNA aptamers can enable rapid and sensitive kanamycin detection. Since our detection scheme relies on ligand binding-induced changes in the aptamer tertiary structure, it is limited only by the kinetics of ligand binding to the aptamer. Our FRET-based kanamycin binding aptamer (KBA) sensor displays two linear ranges of 0.05–5 nM (detection limit of 0.18 nM) and 50–900 nM of kanamycin. The aptamer displays high specificity even in the presence of the ‘natural’ background from milk. By immobilizing the aptamer in the flow cell, our KBA sensor design is also suitable for repeated kanamycin detection. Finally, we show that the ratiometric FRET-based analysis can be implemented on a cheap custom-built smartphone setup. This smartphone-based FRET aptamer scheme detects kanamycin in a linear range of 50–500 nM with a limit of detection (LOD) of 28 nM.

FRET aptamer based kanamycin detection enables reusable and smartphone sensing.     

Selection of potential aptamers for specific growth stage detection of Yersinia enterocolitica

Yersinia enterocolitica remains a threat to public health, and a sensitive detection method is a prerequisite due to its complicated diagnosis associated with slow growth. Recently, aptamer-based detection systems have played a vital role in the development of simple, rapid, sensitive, and specific detection methods. Herein, highly specific ssDNA aptamers were screened against Y. enterocolitica at the different growth stages by whole cell-SELEX. Cells at different growth stages were harvested and incubated with an ssDNA library to get an enriched pool of specific aptamer candidates. After the 10^th round of SELEX, the enriched pool was sequenced and grouped into seven families based on homology and similarity of the secondary structure. Flow cytometry analysis revealed that the aptamers M1, M5, and M7 with K_d values of 37.93 ± 7.88 nM, 74.96 ± 21.34 nM, and 73.02 ± 18.76 nM had the highest affinity and specificity to the target, respectively. The selected aptamers showed binding to the different growth stages of Y. enterocolitica with a significant increase in the gated fluorescence. Our aptamer selection strategy is convenient, and the developed aptamer can be useful for an accurate and reliable detection system.

Yersinia enterocolitica remains a threat to public health, and a sensitive detection method is a prerequisite due to its complicated diagnosis associated with slow growth.     

The selection of highly specific and selective aptamers using modified SELEX and their use in process analytical techniques for Lucentis bioproduction

Aptamers for Lucentis were selected using 10 rounds of a modified and highly stringent SELEX process. Affinity column chromatography was used for the binding, partitioning, and elution steps, and the regeneration of ssDNA was performed via asymmetric PCR in the SELEX process. The interaction of aptamers with Lucentis was studied by means of the HADDOCK web server docking program. In addition, the secondary structures of aptamers were interrogated using the mfold web server to check common regions responsible for better affinity towards Lucentis. The two best aptamers for Lucentis (aptamers 1 and 25) were found to have dissociation constant (K_d) values between 23 and 35 nM by means of thermofluorimetric and non-faradaic impedance spectroscopy (NFIS) analysis. The low dissociation constants in the nanomolar range showed the high specificities of the aptamers for Lucentis. Selectivity tests were also performed using both aptamers with different proteins in which negligible responses were obtained from interfering proteins with respect to Lucentis. Although neither of the two aptamers showed prominent responses to the interfering proteins, slightly better selectivity was shown by aptamer 1. The same aptamers were tested for their application in the detection of Lucentis in spiked and real media broth samples. For this detection test, interdigitated (IDT) gold electrodes on a glass substrate were fabricated using standard photolithography and thermal deposition techniques. NFIS measurements were used for the label-free detection of Lucentis in samples. The linear ranges of detection for aptamers 1 and 25 were found to be 22–100 nM and 40–100 nM, respectively. The LODs for aptamers 1 and 25 were calculated to be 22 nM and 40 nM, respectively, which were significantly better than the values from a HPLC-based detection method (about 240 nM). The real sample analysis results were cross-checked via a standard HPLC method, and better correlation was found between the HPLC and aptamer 1 results than the aptamer 25 results; hence, aptamer 1 can be further analyzed and tested for use in affinity column chromatography and detection-kit/chip-based PAT for Lucentis bioproduction.

Highly specific and selective aptamers for Lucentis were selected using 10 rounds of a modified and highly stringent SELEX process.     

Aptamer fluorescence anisotropy assays for detection of aflatoxin B1 and adenosine triphosphate using antibody to amplify signal change

Fluorescence polarization/anisotropy (FP/FA) is an attractive technology for determining small molecules in homogeneous solution based on rotation changes of a fluorescent reporter. Binding induced conformation change is a specific property of aptamers. This property has been integrated into aptamer based FA assays for small molecules. In this work, we reported aptamer FA assays for aflatoxin B1 (AFB1) and adenosine triphosphate (ATP) by using antibody conjugated complementary DNA at the 3′ end and a fluorescein (FAM)-labeled aptamer at the 5′ end. The hybridization of aptamer and cDNA induced a FAM label close to the large-sized antibody, which restricts the local rotation of FAM and gives high FA signal. With the addition of target, the aptamer probe binds with the target, and the aptamer–cDNA duplex is inhibited, causing FA signal decreases. This method achieved detection of 25 pM AFB1 and 1 μM ATP, respectively. The assay is promising for application.

Aptamer fluorescence anisotropy assays for small molecules (aflatoxin B1 and ATP) using antibody to amplify signal change.     

Enzyme-free ultrasensitive fluorescence detection of epithelial cell adhesion molecules based on a toehold-aided DNA recycling amplification strategy

Epithelial cell adhesion molecules (EpCAMs) play a significant role in tumorigenesis and tumor development. EpCAMs are considered to be tumor signaling molecules for cancer diagnosis, prognosis and therapy. Herein, an enzyme-free and highly sensitive fluorescent biosensor, with a combined aptamer-based EpCAM recognition and toehold-aided DNA recycling amplification strategy, was developed for sensitive and specific fluorescence detection of EpCAMs. Due to highly specific binding between EpCAMs and corresponding aptamers, strand a, which is released from the complex of aptamer/strand a in the presence of EpCAMs which is bound to the corresponding aptamer, triggered the toehold-mediated strand displacement process. An amplified fluorescent signal was achieved by recycling strand a for ultrasensitive EpCAM detection with a detection limit as low as 0.1 ng mL⁻¹, which was comparable or superior to that of reported immunoassays and biosensor strategies. In addition, high selectivity towards EpCAMs was exhibited when other proteins were selected as control proteins. Finally, this strategy was successfully used for the ultrasensitive fluorescence detection of EpCAMs in human serum samples with satisfactory results. Importantly, the present strategy may be also expanded for the detection of other targets using the corresponding aptamers.

A fluorescent biosensor with a combined aptamer-based EpCAM recognition and toehold-aided DNA recycling amplification strategy was developed.     

In silico post-SELEX screening and experimental characterizations for acquisition of high affinity DNA aptamers against carcinoembryonic antigen

DNA aptamers against carcinoembryonic antigen (CEA) have been identified through the systematic evolution of ligands by exponential enrichment (SELEX) technique, but their affinity needs to be improved. In this study, an in silico approach was firstly used to screen the mutation sequences of a reported DNA aptamer (the parent aptamer, denoted as P) against CEA. The affinities of several high-score DNA mutants were determined by the biolayer interferometry technique. Finally, the newly obtained aptamers were verified in an aptasensor application. For the in silico approach, Mfold and RNA Composer were combined to generate the 3D RNA structures of the DNA mutants. The RNA structures were then modified to 3D DNA structures with the Write program. The docking model and binding ability of the 3D DNA structures with CEA were simulated and predicted with the ZDOCK program. Two mutation sequences (P-ATG and GAC-P) exhibited significantly higher ZDOCK scores than P. The dissociation constant of P-ATG and GAC-P to CEA was determined to be 4.62 and 3.93 nM respectively, obviously superior to that of P (6.95 nM). The detection limit of the P-ATG and GAC-P based aptasensors was 1.5 and 1.2 ng mL⁻¹, respectively, markedly better than that based on P (3.4 ng mL⁻¹). The consistency between the in silico and the experimental results indicates that the developed in silico post-SELEX screening approach is feasible for improving DNA aptamers. The P-ATG and GAC-P aptamers found in this study could be used for future CEA aptasensor design and fabrication, promisingly applicable for highly sensitive CEA detection and early cancer diagnosis.

High affinity DNA aptamers against carcinoembryonic antigen were selected and verified by using an in silico approach and experimental characterizations.     

Selection and preliminary application of a single stranded DNA aptamer targeting colorectal cancer serum

Colorectal cancer is one of the common causes of malignant tumors in recent years, thus the discovery of potential compounds that detect the occurrence of colorectal cancer by efficient approaches is necessary. In this study, the method of systematic evolution of ligands by exponential enrichment (SELEX) was used for recognizing serum from colorectal cancer patients by a single-stranded DNA library of aptamers assisted by single-walled carbon nanotubes (SWCNTs) to remove single-stranded DNA with low affinity. Ten rounds of selection were applied using colorectal cancer serum as a target with the serum of healthy individuals as a control. As the result, we have successfully identified four candidate aptamers after high-throughput genome sequencing analysis, comparison analysis and secondary structure prediction. Among them, aptamer Seq-2 exhibited the highest affinity and the strongest selectivity with an equilibrium dissociation constant (K_d) of 11.31 ± 3.25 nM and a C_t difference value of 4.25 ± 0.38 between the colorectal cancer group and the healthy group. Moreover, with fifty negative control serum samples, the positive detection rate of fifty positive serum samples tested by aptamer Seq-2 was over 90%. In particular, aptamer Seq-2 can strongly bind the colorectal cancer serum, less strongly bind the non-colon cancer serum and hardly bind the healthy serum. Therefore, aptamer Seq-2 presents enormous potential for exploring as a tumor diagnostic kit and detecting unknown tumor markers in serum to reflect colorectal cancer.

Aptamer Seq-2 with high affinity and selectivity was screened against colorectal cancer serum directly for clinical application.     

DNA aptamers from whole-serum SELEX as new diagnostic agents against gastric cancer

Gastric cancer is still among the leading causes of cancer deaths worldwide. Despite the improvements in diagnostic methods, the status of early detection has not been achieved so far. Early diagnosis of gastric cancer may significantly improve the cure rate of patients. Therefore, a new diagnostic method is needed. In this study, subtractive SELEX was performed to screen gastric cancer serum-specific DNA aptamers by using gastric cancer serum and normal serum as the target and negative serum, respectively. Four highly specific aptamers generated for gastric cancer serum, Seq-3, Seq-6, Seq-19 and Seq-54, were developed using whole-serum subtractive SELEX technology with K_d of 128 ± 26.3 nM, 149 ± 23.6 nM, 232 ± 44.2 nM, 202 ± 25.6 nM, respectively. These generated aptamers showed higher specificities toward their target serum by differentiating normal serum but closely related other cancer serums. The selected four high affinity DNA aptamers were further applied to the development based on qPCR method for the early detection of gastric cancer. In addition, we performed MALDI-TOF MS followed by secondary peptide sequencing MS analysis for the identification of the aptamer binding proteins. Among these potential biomarkers, APOA1, APOA4, PARD3, Importin subunit alpha-1 showed a relatively high score probability. Therefore, these four ssDNA aptamers generated in our study could be a promising molecular probe for gastric cancer diagnosis.

Gastric cancer is still among the leading causes of cancer deaths worldwide.     

Site-specific anchoring aptamer C2NP on DNA origami nanostructures for cancer treatment

Because of the remarkable features, including biocompatibility and biodegradability, DNA origami nanostructures have drawn much attention as ideal carriers for drug delivery. However, the cellular uptake of DNA origami nanostructures was a passive targeting process, resulting in limited therapeutic effect. To address this problem, we anchored the aptamer C2NP (Apt) on rectangular DNA origami nanostructures (RE) to enhance the tumor targeting properties and anticancer effects of doxorubicin (DOX). Apt was anchored onto RE with low or high density (RE-4Apt, RE-16Apt), followed by incubation with DOX to obtain DOX@RE-4Apt and DOX@RE-16Apt. The results showed that DOX@RE-4Apt and DOX@RE-16Apt exhibited excellent biocompatibility and targeting ability, as well as a synergic biological effect with chemotherapy on cancer therapy. More importantly, after conjugation with RE, the bioactivity of Apt was significantly increased. These results revealed that Apt anchored DNA nanostructures not only are potential carriers for precise therapy, but also supply a strategy to enhance the bioactivity of aptamers.

Aptamer anchored DNA nanostructures not only can enhance the anticancer activity of DOX, but also exhibit synergic biological effect with chemotherapy on cancer therapy.     

A label-free RTP sensor based on aptamer/quantum dot nanocomposites for cytochrome c detection

Given the outstanding room-temperature phosphorescence (RTP) of Mn–ZnS quantum dots (QDs) and the specific recognition performance of the aptamer, we built phosphorescent composites from aptamers conjugated with polyethyleneimine quantum dots (PEI-QDs) and applied them to cytochrome c (Cyt c) detection. Specifically, QDs/CBA composites were generated from the electrostatic interaction between the positively-charged PEI-QDs and the negatively-charged Cyt c binding aptamer (CBA). With the presence of Cyt c, the Cyt c can specifically bind with the QDs/CBA composites, and quench the RTP of QDs through photoinduced electron-transfer (PIET). Thereby, an optical biosensor for Cyt c detection was built, which had a detection range of 0.166–9.96 μM and a detection limit of 0.084 μM. This aptamer-mediated phosphorescent sensor with high specificity and operational simplicity can effectively avoid the interference of scattering light from complex substrates. Our findings offer a new clue for building biosensors based on QDs and aptamers.

In this study, the nanocomposites from polyethyleneimine-capped Mn-doped ZnS QDs (PEI-QDs) and Cyt c binding aptamer (CBA) were prepared and used as Cyt c RTP sensors..     

A nanoporous gold-based electrochemical aptasensor for sensitive detection of cocaine

The increasing application of aptamers in bioassays has triggered a lot of research interest for development of highly sensitive and selective sensing platforms. Herein, we report on the design of a sensitive cocaine biosensor by immobilizing the 5′-disulfide-functionalized end of an aptamer sequence on a nanoporous gold (NPG) electrode followed by the conjugation of its 3′-amino-functionalized end to 2,5-dihydroxybenzoic acid (DHBA) as the redox probe. In the presence of cocaine, the aptamer undergoes a conformational change from an open unfolded state to a closed conformation, which reduces the distance between DHBA and the electrode surface, resulting in the enhanced electron-transfer efficiency. Using square wave voltammetric method and under the optimal conditions, the cocaine aptasensor presented two linear responses in the concentration ranges between 0.05–1 and 1–35 μM, with an excellent detection limit of 21 nM. The proposed aptasensor provides a simple and low-cost method for cocaine detection with good reproducibility and accuracy. Furthermore, it could be regarded as a general model to investigate the unique function of aptamer-functionalized nanostructured electrodes to stablish highly advanced electrochemical biosensors for various target analytes of diagnostic importance.

The increasing application of aptamers in bioassays has triggered a lot of research interest for development of highly sensitive and selective sensing platforms.     

Considering both small and large scale motions of vascular endothelial growth factor (VEGF) is crucial for reliably predicting its binding affinities to DNA aptamers

Vascular endothelial growth factor 165 (VEGF₁₆₅), a predominant isoform of VEGF signal proteins, is an ideal target for developing drugs against various diseases. It is composed of a heparin binding domain (HBD) and a receptor binding domain (RBD), which are connected by a flexible linker. Among the two domains, RBD is utilized in binding with the signal transduction protein, the VEGF receptor (VEGFR). None the less for its pharmaceutical importance, structure-based studies for developing drugs has been severely hindered by the lack of its whole structure determination, mainly owing to the existence of the flexible linker. Fortunately, the utilization of computer simulation methods can offer a possibility to circumvent this difficult issue. Here, we employ ensemble docking in combination with the anisotropic network model analysis to examine the interactions between DNA aptamers and VEGF₁₆₅. We model three-dimensional structures of aptamer variants based on their sequence information and perform docking calculations with the whole VEGF₁₆₅ structure. Indeed, we show that we can closely reproduce the experimental binding affinity order among different DNA aptamer variants by inclusively considering the flexible nature of VEGF. In addition, we address how DNA aptamer that binds to HBD of VEGF₁₆₅ impedes the interaction between VEGFR and VEGF₁₆₅ through RBD, even though HBD and RBD are rather distant. The present study illustrates that the flexible docking scheme employed here can be applied to tricky cases that involve flexible proteins with undetermined structures, toward effectively predicting ligand binding affinities to such proteins.

Considering both small and large scale motions of VEGF is crucial to predict its relative binding affinities to DNA aptamer variants with docking.