Browsing by Author "Ding, Yuzhe"

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Cross-Binding of Four Adenosine/ATP Aptamers to Caffeine, Theophylline, and Other Methylxanthines
(2023-07-11) Ding, Yuzhe; Xie, Yachen; Li, Albert Zehan; Huang, Po-Jung Jimmy; Liu, Juewen
The classical DNA aptamer for adenosine and ATP was selected twice using ATP as the target in 1995 and 2005, respectively. In 2022, this motif appeared four more times from selections using adenosine, ATP, theophylline, and caffeine as targets, suggesting that this aptamer can also bind methylxanthines. In this work, using thioflavin T fluorescence spectroscopy, this classical DNA aptamer showed Kd values for adenosine, theophylline, and caffeine of 9.5, 101, and 131 μM, respectively, and similar Kd values were obtained using isothermal titration calorimetry. Binding to the methylxanthines was also observed for the newly selected Ade1301 aptamer but not for the Ade1304 aptamer. The RNA aptamer for ATP also had no binding to the methylxanthines. Molecular dynamics simulations were performed using the classical DNA and RNA aptamers based on their NMR structures, and the simulation results were consistent with the experimental observations, explaining the selectivity profiles. This study suggests that a broader range of target analogues need to be tested for aptamers. For the detection of adenosine and ATP, the Ade1304 aptamer is a better choice due to its better selectivity.
Fundamental studies for small molecule aptamer selection using capture-SELEX
(University of Waterloo, 2025-08-11) Ding, Yuzhe
DNA aptamers for small molecules hold transformative promise in biosensing, diagnostics, and therapeutics, yet their in vitro evolution has been hampered by incomplete knowledge of the parameters that drive efficient enrichment. In recent years, the development of library-immobilization based method, so called capture-SELEX, has generated over 100 high-quality DNA aptamers for various types of small molecules. Importantly, capture-SELEX allows systematic investigation of fundamental problems in the selection of aptamers. This thesis studies the capture-SELEX platform by dissecting thermodynamic, kinetic, and methodological variables to accelerate the discovery of high-affinity DNA aptamers. Using adenosine/ATP as targets for selection has repeatedly produced the same guanine-rich aptamer motif that was first reported by the Szostak group in 1995. This aptamer has been considered as the adenosine/ATP aptamer by the field. First, by gradually increasing the selection stringency on classical targets (adenosine and ATP), we selected two new aptamers with Kd ≈ 230 nM, 35-fold tighter than that of the classical aptamer sequence. This was achieved through gradual reduction of target concentration from 5 mM to the low-micromolar range. The evolution of the sequence abundance cross different rounds was traced by deep sequencing, and the reason for the previous repeated report of the classical sequence was attributed to its short 12-nucleotide conserved binding regions, whereas the two new aptamers have approximately 16 conserved nucleotides. This study highlights the importance of using low target concentration in order to enrich high affinity aptamers. During aptamer selection, using a lower target concentration tends to favor the enrichment of higher affinity binders, raising the question of whether a practical lower limit exists. Next, we performed three capture-SELEX campaigns using 5 µM, 500 nM, and 50 nM guanine as the target, respectively, to investigate it. Both the 5 µM and 500 nM selections successfully enriched the same guanine aptamer-requiring eight rounds at 5 µM guanine versus 17 rounds at 500 nM guanine. However, the 50 nM selection failed to yield any aptamers. The highest affinity and most enriched aptamer from these selections displayed a Kd of 200 nM, indicating that if the target concentration is much lower than Kd can lead to failed selections. Mutation analysis further revealed a critical cytosine in the guanine binding pocket: substituting this cytosine with a thymine switched selectivity from guanine to adenine. A similar specificity switching was previously seen in the natural guanine riboswitches. These findings define a lower limit for target concentration in capture-SELEX and offer a practical guidance for selecting target levels to isolate high-affinity aptamers. Selection of high-affinity aptamers underpins all downstream applications, yet most protocols emphasize thermodynamic factors-such as target concentration-while overlooking binding kinetics. Third, we performed a library-immobilization selection against ampicillin to dissect these influences. Under typical gravity-flow conditions (1-2 min interaction), a low-affinity aptamer (Kd = 12.7 µM) dominated the enriched pool. In contrast, extending the incubation time to 10 min enriched a higher affinity sequence (Kd = 1.8 µM), differing by only three nucleotides from the weaker Kd aptamer. Systematic comparison of library immobilization efficiency, release fraction, and release kinetics confirmed that dissociation rate from the capture duplex was the primary determinant of the selection outcome. We observed the same kinetic bias in parallel adenosine selections, demonstrating the generality of this effect. Based on these findings, we recommend combining low target concentrations with extended incubation time to favor the enrichment of high-affinity aptamers. This study not only yields a robust, high affinity and selective ampicillin aptamer but also highlights a critical interplay between thermodynamics and kinetics during in vitro aptamer selection. Since 1990, numerous aptamer-selection techniques have been developed, yet quantitative comparisons of their enrichment efficiencies remain scarce. Finally, we evaluated three library‐immobilization SELEX methods, capture‐SELEX, GO‐SELEX, and gold‐SELEX, using a spiked library containing DNA aptamers with varying affinities for adenosine. Using 100 µM adenosine as target, all three methods showed that <1 % of the library was released by adenosine as revealed by qPCR, with gold‐SELEX showing virtually no DNA elution. Deep sequencing of three model aptamers (Ade1301, Ade1304, and the classical adenosine aptamer) revealed 30-50‐fold enrichment in capture-SELEX, whereas GO‐SELEX and gold‐SELEX both yielded enrichment factors below 1, indicating a lack of aptamer enrichment. Blocking the primer‐binding regions improved GO‐SELEX enrichment to ~14 % but still fell far short of capture‐SELEX’s performance. Finally, we compared nonspecific versus target‐induced release and elucidated why capture‐SELEX’s structural-switching mechanism offers superior aptamer enrichment. Overall, capture‐SELEX is a markedly more efficient strategy for isolating high‐affinity aptamers. Collectively, this work establishes a quantitative framework for capture-SELEX-balancing target concentration, kinetic control, and partitioning strategy-to reliably isolate nanomolar-class DNA aptamers for small molecules.
Gold Nanoparticles Synthesized Using Various Reducing Agents and the Effect of Aging for DNA Sensing
(American Chemical Society, 2022-12-28) Ding, Yuzhe; Huang, Po-Jung Jimmy; Zandieh, Mohamad; Wang, Jinghan; Liu, Juewen
Gold nanoparticles (AuNPs) are one of the most commonly used reagents in colloidal science and biosensor technology. In this work, we first compared AuNPs prepared using four different reducing agents including citrate, glucose, ascorbate, and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). At the same absorbance at the surface plasmon peak of 520–530 nm, citrate-AuNPs and glucose-AuNPs adsorbed more DNA and achieved higher affinity to the adsorbed DNA. In addition, citrate-AuNPs had better sensitivity than glucose-AuNPs for label-free DNA detection. Then, using citrate-AuNPs, the effect of aging was studied by incubation of the AuNPs at 22 °C (room temperature) and at 4 °C for up to 6 months. During aging, the colloidal stability and DNA adsorption efficiency gradually decreased. In addition, the DNA sensing sensitivity using a label-free method also dropped around 4-fold after 6 months. Heating at boiling temperature of the aged citrate-AuNPs could not rejuvenate the sensing performance. This study shows that while citrate-AuNPs are initially better than the other three AuNPs in their colloid properties and sensing properties, this edge in performance might gradually decrease due to constantly changing surface properties caused from the aging effect.
Homogeneous assays for aptamer-based ethanolamine sensing: no indication of target binding
(Royal Society of Chemistry, 2022-02-11) Ding, Yuzhe; Liu, Xun; Huang, Po-Jung Jimmy; Liu, Juewen
Ethanolamine is an important analyte for environmental chemistry and biological sciences. A few DNA aptamers were previously reported for binding ethanolamine with a dissociation constant (Kd) as low as 9.6 nM. However, most of the previous binding assays and sensing work used either immobilized ethanolamine or immobilized aptamers. In this work, we studied three previously reported DNA sequences, two of which were supposed to bind ethanolamine while the other could not bind. Isothermal titration calorimetry revealed no binding for any of these sequences. In addition, due to their guanine-rich sequences, thioflavin T was used as a probe. Little fluorescence change was observed with up to 1 μM ethanolamine. Responses within the millimolar range of ethanolamine were attributed to the general fluorescence quenching effect of ethanolamine instead of aptamer binding. Finally, after studying the adsorption of ethanolamine to gold nanoparticles (AuNPs), we confirmed the feasibility of using AuNPs as a probe when the concentration of ethanolamine was below 0.1 mM. However, no indication of specific aptamer binding was observed by comparing the three DNA sequences for their color changing trends. This work articulates the importance of careful homogeneous binding assays using free target molecules.
Machine Learning Directed Aptamer Search from Conserved Primary Sequences and Secondary Structures
(American Chemical Society, 2023-01-03) Tobia, Javier Perez; Huang, Po-Jung Jimmy; Ding, Yuzhe; Saran Narayan, Runjhun; Narayan, Apurva; Liu, Juewen
Computer-aided prediction of aptamer sequences has been focused on primary sequence alignment and motif comparison. We observed that many aptamers have a conserved hairpin, yet the sequence of the hairpin can be highly variable. Taking such secondary structure information into consideration, a new algorithm combining conserved primary sequences and secondary structures is developed, which combines three scores based on sequence abundance, stability, and structure, respectively. This algorithm was used in the prediction of aptamers from the caffeine and theophylline selections. In the late rounds of the selections, when the libraries were converged, the predicted sequences matched well with the most abundant sequences. When the libraries were far from convergence and the sequences were deemed challenging for traditional analysis methods, this algorithm still predicted aptamer sequences that were experimentally verified by isothermal titration calorimetry. This algorithm paves a new way to look for patterns in aptamer selection libraries and mimics the sequence evolution process. It will help shorten the aptamer selection time and promote the biosensor and chemical biology applications of aptamers.
Pushing Adenosine and ATP SELEX for DNA Aptamers with Nanomolar Affinity
(American Chemical Society, 2023-03-22) Ding, Yuzhe; Liu, Juewen
The classical DNA aptamer for adenosine and ATP has been the most used small molecule binding aptamer for biosensing, imaging, and DNA nanotechnology. This sequence has recurred multiple times in previous aptamer selections, and all previous selections used a high concentration of ATP as the target. Herein, two separate selections were performed using adenosine and ATP as targets. By pushing the target concentrations down to the low micromolar range, two new aptamers with Kd as low as 230 nM were obtained, showing around 30-fold higher affinity compared to the classical aptamer. The classical aptamer sequence still dominated the library in the early rounds of the selections, but it was suppressed in the later rounds. The new aptamers bind to one target molecule instead of two. Mutation studies confirmed their secondary structures and specific binding. Using the deep sequencing data from the selections, long-standing questions such as the existence of one-site aptamers and mutation distribution in the classical aptamer were addressed. Comparisons were made with previously reported DNA aptamers for ATP. Finally, a strand-displacement biosensor was tested showing selectivity for adenosine and its nucleotides.
Quantitative Comparison of Capture-SELEX, GO-SELEX, and Gold-SELEX for Enrichment of Aptamers
(American Chemical Society, 2023-09-18) Ding, Yuzhe; Liu, Juewen
Since 1990, numerous methods for aptamer selection have been developed, although a quantitative comparison of their sequence enrichment is lacking. In this study, we compared the enrichment factors of three library-immobilization SELEX methods (capture-SELEX, GO-SELEX, and gold-SELEX). We used a spiked library that contained multiple DNA aptamers with different affinities for adenosine. The aptamer separation efficiency was measured using qPCR, and all of the three methods showed a very low DNA release (<1%) in the presence of 100 μM adenosine. Among these, barely any DNA was released from the gold nanoparticles. Deep sequencing was used to compare the enrichment of three aptamers: Ade1301, Ade1304, and the classical aptamer. Enrichment up to 30 to 50-fold was observed only for the capture-SELEX method, whereas the other two methods showed enrichment factors below 1. By blocking the primer-binding regions of the library, GO-SELEX reached up to 14% enrichment. Finally, the enrichment of aptamers based on nonspecific release and target-induced release was discussed, and the advantages of capture-SELEX were rationalized. Taken together, these results indicate that capture-SELEX is a much more efficient method for enriching aptamers.
Stabilization of Gold Nanoparticles by Hairpin DNA and Implications for Label-Free Colorimetric Biosensors
(2022-04-21) Liu, Xun; Zhao, Yu; Ding, Yuzhe; Wang, Jianhua; Liu, Juewen
With extremely high extinction coefficients and other unique optical properties, gold nanoparticles (AuNPs) have received growing interest in developing biosensors. DNA hairpin structures are very popular probes for the detection of not only complementary DNA or RNA but also aptamer targets. This work aims to understand the effect of the structure and sequence of hairpin DNA for the stabilization of AuNPs and its implications in AuNP-based label-free colorimetric biosensors. A series of hairpin DNA with various loop sizes from 4 to 26 bases and sequences (random sequences, poly-A and poly-T) were tested, but they showed similar abilities to protect AuNPs from aggregation. Using hairpin DNA with a tail under the same conditions, optimal protection was achieved with a six-base or longer tail. DNA hairpins are likely adsorbed via their tail regions or with their terminal bases if no tail is present. Molecular dynamics simulations showed that the rigidity of the hairpin loop region disfavored its adsorption to AuNPs, while the flexible tail region is favored. Finally, a DNA sensing assay was conducted using different structured DNA, where hairpin DNA with a tail doubled the sensitivity compared to the tail-free hairpin.