Surfactant-Assisted Label-Free Fluorescent Aptamer Biosensors and Binding Assays

Zhang, Hanxiao; Li, Albert Zehan; Liu, Juewen

doi:10.3390/bios13040434

Cited by 9 publications

(9 citation statements)

References 38 publications

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“…The guanine-rich conserved regions allowed us to use ThT as a probe to study aptamer binding. − Free ThT is almost nonfluorescent, but its fluorescence is enhanced upon binding to guanine-rich DNA. Aptamer binding to its target may displace ThT, leading to decreased fluorescence (Figure A).…”

Section: Resultsmentioning

confidence: 99%

Cross-Binding of Four Adenosine/ATP Aptamers to Caffeine, Theophylline, and Other Methylxanthines

Ding

Xie

et al. 2023

Biochemistry

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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 K d values for adenosine, theophylline, and caffeine of 9.5, 101, and 131 μM, respectively, and similar K d 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.

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Section: Resultsmentioning

confidence: 99%

Cross-Binding of Four Adenosine/ATP Aptamers to Caffeine, Theophylline, and Other Methylxanthines

Ding

Xie

et al. 2023

Biochemistry

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“…Nucleic acid-binding dyes can be used as efficient indicators of aptamer-target interactions . Different fluorescent dyes, such as SGI and thioflavin T (ThT), have been used to develop label-free biosensors and verify aptamer-target binding. – It was hypothesized that the interaction between an aptamer and its target can either foster dye binding or displace the dye, leading to either an increase or decrease in fluorescence, respectively.…”

Section: Resultsmentioning

confidence: 99%

Electrostatic-Mediated Binding of DNA to Lysozymes: Evaluation of Aptamer-Based Assays for Highly Positively Charged Targets

Shi,

Jin,

Liu

2023

Langmuir

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Lysozymes are a highly popular protein target for the development of aptamer-based biosensors. Because a lysozyme is a polycation and DNA is a polyanion, it is essential to separate the contribution of nonspecific electrostatic interactions from specific aptamer binding. In this study, various factors affecting the binding of DNA and lysozymes, including the DNA sequence, DNA length, pH, and salt concentration, were explored using fluorescence polarization. We concluded that direct fluorescence polarization and fluorescence intensity changes are unlikely to be directly applicable for aptamer-based biosensors to detect lysozymes because all of the tested DNA sequences showed binding. These fundamental studies confirm the dominant role of electrostatic binding. We further evaluated three other methods, including label-free fluorescent detection using a DNA staining dye, label-free colorimetric detection using gold nanoparticles, and a fluorescent sensor based on the strand displacement reaction. In each case, we focused on a random DNA sequence that is not expected to bind to the lysozyme as an aptamer. Of all the methods, only the strand displacement strategy can be potentially used to evaluate aptamer binding, as the other methods all responded to nonaptamer sequences. This study provides valuable insights for assaying aptamer binding to cationic proteins that can exhibit a nonspecific attraction to DNA.

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“…The sample was temperature controlled using a water bath at 22 °C, and the titration started only after a stable background fluorescence reading was achieved. 49 Note that guanine solutions need to be diluted freshly each time.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

“…The fluorescence intensity at 490 nm was measured using a Horiba Fluormax-4 instrument with 420 nm excitation. The sample was temperature controlled using a water bath at 22 °C, and the titration started only after a stable background fluorescence reading was achieved . Note that guanine solutions need to be diluted freshly each time.…”

Section: Methodsmentioning

confidence: 99%

Affinity-Guided Coevolution of Aptamers for Guanine, Xanthine, Hypoxanthine, and Adenine

Ding,

Gu,

Wang

et al. 2024

ACS Chem. Biol.

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The simultaneous evolution of multiple aptamers can drastically increase the speed of aptamer discovery. Most previous studies used the same concentration for different targets, leading to the dominance of the libraries by one or a few aptamers and a low success rate. To foster the best aptamers to grow independently in the sequence space, it is important to (1) use low target concentrations close to their dissociation constants and (2) stop at an early round before any sequence starts to dominate. In this study, we demonstrate this affinity-guided selection concept using the capture-SELEX method to isolate aptamers for four important purines: guanine (5 μM), xanthine (50 μM), hypoxanthine (10 μM), and adenine (10 μM). The round 9 library was split, and in round 10, the four targets were individually used to elute the binding sequences. Using thioflavin T fluorescence spectroscopy and isothermal titration calorimetry, we confirmed highly selective aptamers for xanthine, guanine, and adenine. These aptamers have K d values below 1 μM and around 100-fold selectivity against most competing analytes, and they compare favorably with existing RNA aptamers and riboswitches. A separate selection was performed using hypoxanthine alone, and no selective aptamer was achieved, even with negative selection, explaining the lack of its aptamer in our mixed selection. This affinity-guided multiplex SELEX study offers fundamental insights into aptamer selection and provides high-quality aptamers for three important purines.

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Surfactant-Assisted Label-Free Fluorescent Aptamer Biosensors and Binding Assays

Cited by 9 publications

References 38 publications

Cross-Binding of Four Adenosine/ATP Aptamers to Caffeine, Theophylline, and Other Methylxanthines

Cross-Binding of Four Adenosine/ATP Aptamers to Caffeine, Theophylline, and Other Methylxanthines

Electrostatic-Mediated Binding of DNA to Lysozymes: Evaluation of Aptamer-Based Assays for Highly Positively Charged Targets

Affinity-Guided Coevolution of Aptamers for Guanine, Xanthine, Hypoxanthine, and Adenine

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