Browsing by Author "Huang, Po-Jung Jimmy"

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2-Aminopurine Fluorescence Spectroscopy for Probing a Glucose Binding Aptamer
(Chemistry Europe: European Chemical Societies Publishing, 2022-04-25) Lu, Chang; Huang, Po-Jung Jimmy; Zheng, Jingkai; Liu, Juewen
Glucose is the most important analyte for biosensors. Recently a DNA aptamer was reported allowing binding-based detection. However, due to a relatively weak binding affinity, it is difficult to perform binding assays to understand the property of this aptamer. In this work, we replaced the only adenine base in the aptamer binding pocket with a 2-aminopurine (2AP) and used fluorescence spectroscopy to study glucose binding. In the selection buffer, glucose increased the 2AP fluorescence with a Kd of 15.0 mM glucose, which was comparable with the 10 mM Kd previously reported using the strand displacement assay. The binding required two Na+ ions or one Mg2+ that cannot be replaced by Li+ or K+. The binding was weaker at higher temperature and its van't Hoff plot indicated enthalpy-driven binding. While other monosaccharides failed to achieve saturated binding even at high concentrations, two glucose-containing disaccharides, namely trehalose and sucrose, reached a similar fluorescence level as glucose although with over 10-fold higher Kd values. Detection limits in both the selection buffer (0.9 mM) and in artificial interstitial fluids (6.0 mM) were measured.
A DNA Aptamer for Theophylline with Ultrahigh Selectivity Reminiscent of the Classic RNA Aptamer
(American Chemical Society, 2022-08-09) Huang, Po-Jung Jimmy; Liu, Juewen
Since the report of the RNA aptamer for theophylline, theophylline has become a key molecule in chemical biology for designing RNA switches and riboswitches. In addition, theophylline is an important drug for treating airway diseases including asthma. The classic RNA aptamer with excellent selectivity for theophylline has been used to design biosensors, although DNA aptamers are more desirable for stability and cost considerations. In this work, we selected DNA aptamers for theophylline, and all the top sequences shared the same binding motifs. Binding was confirmed using isothermal titration calorimetry and a nuclease digestion assay, showing a dissociation constant (Kd) around 0.5 μM theophylline. The Theo2201 aptamer can be truncated down to 23-mer while still has a Kd of 9.8 μM. The selectivity for theophylline over caffeine is around 250,000-fold based on a strand-displacement assay, which was more than 20-fold higher compared to the classic RNA aptamer. For other tested analogs, the DNA aptamer also showed better selectivity. Using the structure-switching aptamer sensor design method, a detection limit of 17 nM theophylline was achieved in the selection buffer, and a detection limit of 31 nM was obtained in 10% serum.
Adsorption of DNA Oligonucleotides by Self-Assembled Metalloporphyrin Nanomaterials
(American Chemical Society, 2022-03-08) Wang, Jinghan; Wang, Zhen; Huang, Po-Jung Jimmy; Bai, Feng; Liu, Juewen
Porphyrin assemblies have controllable morphology, high biocompatibility, and good optical properties and were widely used in biomedical diagnosis and treatment. With the development of DNA biotechnology, combining DNA with porphyrin assemblies can broaden the biological applications of porphyrins. Porphyrin assemblies can serve as nanocarriers for DNA, although the fundamental interactions between them are not well understood. In this work, zinc meso-tetra(4-pyridyl)porphyrin (ZnTPyP) assemblies were prepared in the presence of various surfactants and at different pH values, yielding a variety of aggregation forms. Among them, the hexagonal stacking form exposes more pyridine substituents, and the hydrogen bonding force between the substituents and the DNA bases allows the DNA to be quickly adsorbed on the surface of the assemblies. The effects of DNA sequence and length were systematically tested. In particular, the adsorption of duplex DNA was less efficient compared to the adsorption of single-stranded DNA. This fundamental study is useful for the further combination of DNA and porphyrin assemblies to prepare new functional hybrid nanomaterials.
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.
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.
Label-free and Dye-free Fluorescent Sensing of Tetracyclines Using a Capture-Selected DNA Aptamer
(American Chemical Society, 2022-07-01) Zhao, Yichen; Ong, Steven; Chen, Yijing; Huang, Po-Jung Jimmy; Liu, Juewen
Tetracyclines are a group of important antibiotics with a common four-ring scaffold. While most tetracyclines are currently used only in animals, their leaching into the environment and residues in food have caused health concerns. Aptamers are an attractive way to detect tetracyclines, and all previously reported aptamers for tetracyclines were obtained by immobilizing target molecules. In this work, we selected a few DNA aptamers by immobilizing the DNA library using oxytetracycline as the target. We obtained new aptamers with no overlapping sequences compared to the previously reported ones, and a representative sequence named OTC5 had a dissociation constant of 147 nM measured by isothermal titration calorimetry. Similar binding affinities were also observed with tetracycline and doxycycline. Because tetracyclines are fluorescent and their fluorescence intensity was enhanced by binding to the aptamers, a label-free and dye-free fluorescent biosensor was developed with a detection limit of 25 nM oxytetracycline. The sensor was able to detect targets in milk after extraction. Fluorescence polarization measurement showed that this aptamer is insensitive to sodium concentration but requires magnesium. Finally, a strand-displacement biosensor was designed, and it has a detection limit of 1.2 μM oxytetracycline.
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.
Protection of DNA by metal ions at 95 °C: from lower critical solution temperature (LCST) behavior to coordination-driven self-assembly
(Royal Society of Chemistry, 2022-09-05) Lu, Chang; Xu, Yuancong; Huang, Po-Jung Jimmy; Zandieh, Mohamad; Wang, Yihao; Zheng, Jinkai; Liu, Juewen
While polyvalent metal ions and heating can both degrade nucleic acids, we herein report that a combination of them leads to stabilization. After incubating 4 mM various metal ions and DNA oligonucleotides at 95 °C for 3 h at pH 6 or 8, metal ions were divided into four groups based on gel electrophoresis results. Mg2+ can stabilize DNA at pH 6 without forming stable nanoparticles at room temperature. Co2+, Cu2+, Cd2+, Mn2+ and Zn2+ all protected the DNA and formed nanoparticles, whereas the nanoparticles formed with Fe2+ and Ni2+ were so stable that they remained even in the presence of EDTA. At pH 8, Ce3+ and Pb2+ showed degraded DNA bands. For Mg2+, better protection was achieved with higher metal and DNA concentrations. By monitoring temperature-programmed fluorescence change, a sudden drop in fluorescence intensity attributable to the lower critical solution temperature (LCST) transition of DNA was found to be around 80 °C for Mg2+, while this transition temperature decreased with increasing Mn2+ concentration. The unexpected thermal stability of DNA enabled by metal ions is useful for extending the application of DNA at high temperatures, forming coordination-driven nanomaterials, and it might offer insights into the origin of life on the early Earth.
Selection of Aptamers for Sensing Caffeine and Discrimination of Its Three Single Demethylated Analogues
(American Chemical Society, 2022-02-10) Huang, Po-Jung Jimmy; Liu, Juewen
With the growing consumption of caffeine-containing beverages, detection of caffeine has become an important biomedical, bioanalytical, and environmental topic. We herein isolated four high-quality aptamers for caffeine with dissociation constants ranging from 2.2 to 14.6 μM as characterized using isothermal titration calorimetry. Different binding patterns were obtained for the three single demethylated analogues: theobromine, theophylline, and paraxanthine, highlighting the effect of the molecular symmetry of the arrangement of the three methyl groups in caffeine. A structure-switching fluorescent sensor was designed showing a detection limit of 1.2 μM caffeine, which reflected the labeled caffeine concentration within 6.1% difference for eight commercial beverages. In 20% human serum, a detection limit of 4.0 μM caffeine was achieved. With the four aptamer sensors forming an array, caffeine and the three analogues were well separated from nine other closely related molecules.
Signaling Kinetics of DNA and Aptamer Biosensors Revealing Graphene Oxide Surface Heterogeneity
(Springer Nature, 2021-12-16) Huang, Po-Jung Jimmy; Liu, Juewen
Adsorption of fluorescently labeled DNA and aptamer probes to graphene oxide (GO) has been one of the most popular methods for developing biosensors. In the presence of target analytes, the quenched fluorescence would recover. In this work, we followed the kinetics of the reactions and found that the fluorescence would eventually drop after an initial increase, and this was attributed to the re-adsorption of the desorbed probe DNA molecules. Both a DNA probe and an aptamer for adenosine were used. This re-adsorption was attributed to the surface heterogeneity of GO, and the DNA probes desorbed from relatively weaker binding sites were re-adsorbed on the stronger binding sites. This re-adsorption can be avoided by extensive washing the samples, and also by blocking the GO surface or by heating. This fundamental understanding is important for achieving a stable signal of such biosensors.