Selection of DNA Aptamers for Sensing Uric Acid in Simulated Tears

Zheng

et al. 2022

ChemBioChem

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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 K d of 15.0 mM glucose, which was comparable with the 10 mM K d previously reported using the strand displacement assay. The binding required two Na + ions or one Mg 2 + 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 glucosecontaining disaccharides, namely trehalose and sucrose, reached a similar fluorescence level as glucose although with over 10-fold higher K d values. Detection limits in both the selection buffer (0.9 mM) and in artificial interstitial fluids (6.0 mM) were measured.

Section: Magnesium-dependent Glucose Bindingmentioning

confidence: 99%

2‐Aminopurine Fluorescence Spectroscopy for Probing a Glucose Binding Aptamer

Zheng

et al. 2022

ChemBioChem

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“…The RNA aptamer was obtained by immobilization of theophylline . While this method has yielded a high-quality aptamer, we were more interested in using the library immobilization method because the aptamer can access all parts of the target molecule, resulting in potentially better binding performance. − In this work, we report our isolation of a high-quality DNA aptamer for theophylline with submicromolar affinity and even better specificity than the RNA aptamer. The use of this aptamer-based biosensor for the detection of theophylline in serum was also demonstrated.…”

Section: Introductionmentioning

confidence: 99%

A DNA Aptamer for Theophylline with Ultrahigh Selectivity Reminiscent of the Classic RNA Aptamer

Liu

2022

ACS Chem. Biol.

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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 (K d ) around 0.5 μM theophylline. The Theo2201 aptamer can be truncated down to 23-mer while still has a K d of 9.8 μM. The selectivity for theophylline over caffeine is around 250,000-fold based on a stranddisplacement 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.

“…An example is shown in Figure 1A for two aptamers that can bind uric acid. 28 Aside from the 6-mer conserved motifs in blue, the middle part can form a hairpin (the stem region marked in red). In this example, the nucleotides inside the hairpin differed a lot, but the secondary structure was conserved (Figure 1B).…”

Section: Resultsmentioning

confidence: 99%

“…The fact that they switched sides in some sequences (see the sequence alignment in Figure 5B) indicated that these are likely to be real aptamers. 28,32 Since they were not present in the top 70 sequences in round 20, we did not test them in our previous studies. Seq8 is the same as the 9th most abundant sequence in round 20, and its binding to caffeine was previously confirmed.…”

Section: Prediction Of Caffeine Aptamersmentioning

confidence: 99%

Machine Learning Directed Aptamer Search from Conserved Primary Sequence and Secondary Structure

Tobia

Narayan

et al. 2022

Preprint

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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 a secondary structure information into consideration, a new algorithm combining conserved primary sequences and secondary structures is developed, that combines three scores based on sequence abundance, stability, and structure, respectively. This algorithm was used in the prediction of aptamers from caffeine and theophylline selections. In the late rounds of the selection, when the library was converged, the predicted sequences matched well with the most abundant sequences. When the library was far from convergence and the sequences were deemed impossible for traditional analysis methods, the 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 application of aptamers.