Direct Screening for Cytometric Bead Assays for Adenosine Triphosphate

Qu, Hao; Wang, Lu; Liu, Jian; Zheng, Lei

doi:10.1021/acssensors.8b00224

Cited by 18 publications

(16 citation statements)

References 36 publications

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“…The only other comparable small molecular target is probably estradiol (selected at least eight times) . Aside from what already been discussed, at least two additional DNA aptamer selections were performed, each using a different selection method. , We aligned their sequences with our new aptamers and the classical aptamer, and none of the conserved regions aligned (Figure S3). Their binding properties are compared in Table S4.…”

Section: Resultsmentioning

confidence: 99%

Pushing Adenosine and ATP SELEX for DNA Aptamers with Nanomolar Affinity

Ding

Liu

2023

J. Am. Chem. Soc.

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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 K d 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.

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Section: Resultsmentioning

confidence: 99%

Pushing Adenosine and ATP SELEX for DNA Aptamers with Nanomolar Affinity

Ding

Liu

2023

J. Am. Chem. Soc.

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“… 27 Most recently, Zheng et al reported an ATP aptamer that can be used in cytometric bead assays. 28 Although these SELEX strategies are powerful for guiding the selectivity, still no aptamers are available for highly selective binding of adenosine.…”

Section: Introductionmentioning

confidence: 99%

Engineering base-excised aptamers for highly specific recognition of adenosine

Liu

Huang

et al. 2020

Chem. Sci.

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The DNA aptamer for adenosine and ATP has been used as a model system for developing analytical biosensors. For practical reasons, it is important to distinguish adenosine from ATP, although this has yet to be achieved despite extensive efforts made on selection of new aptamers. We herein report a strategy of excising an adenine nucleotide from the backbone of a one-site adenosine aptamer, and the adenineexcised aptamer allowed highly specific binding of adenosine. Cognate analytes including AMP, ATP, guanosine, cytidine, uridine, and theophylline all failed to bind to the engineered aptamer according to the SYBR Green I (SGI) fluorescence spectroscopy and isothermal titration calorimetry (ITC) results. Our A-excised aptamer has two binding sites: the original aptamer binding site in the loop and the newly created one due to base excision from the DNA backbone. ITC demonstrated that the A-excised aptamer strand can bind to two adenosine molecules, with a K d of 14.8 AE 2.1 mM at 10 C and entropydriven binding. Since the wild-type aptamer cannot discriminate adenosine from AMP and ATP, we attributed this improved specificity to the excised site. Further study showed that these two sites worked cooperatively. Finally, the A-excised aptamer was tested in diluted fetal bovine serum and showed a limit of detection of 46.7 mM adenosine. This work provides a facile, cost-effective, and non-SELEX method to engineer existing aptamers for new features and better applications.

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“…Among the available candidates, optical recognition is the commonly effective manner because of its unique features relating to speed, intuitiveness, and controllability. Most recently, many studies have incorporated optical supports and presented their commendable abilities in bioassays. − …”

mentioning

confidence: 99%

Breaking Through Bead-Supported Assay: Integration of Optical Tweezers Assisted Fluorescence Imaging and Luminescence Confined Upconversion Nanoparticles Triggered Luminescent Resonance Energy Transfer (LRET)

Kang

et al. 2019

Anal. Chem.

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Herein, a conceptual approach for significantly enhancing a bead-supported assay is proposed. For the fluorescence imaging technology, optical tweezers are introduced to overcome the fluid viscosity interference and immobilize a single tested bead at the laser focus to guarantee a fairly precise imaging condition. For the selection of fluorescent materials and the signal acquisition means, a type of innovative luminescence confined upconversion nanoparticle with a unique sandwich structure is specially designed to act as an efficient energy donor to trigger the luminescent resonance energy transfer (LRET) process. By further combining the double breakthrough with a molecular beacon model, the newly developed detection strategy allows for achieving a pretty high LRET ratio (≈ 88%) to FAM molecules and offering sound assay performance toward miRNA analysis with a detection limit as low as the sub-fM level, and is capable of well identifying single-base mismatching. Besides, this approach not only is able to accurately qualify the low-abundance targets from as few as 30 cancer cells but also can be employed as a valid cancer early warning tool for performing liquid biopsy.

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Direct Screening for Cytometric Bead Assays for Adenosine Triphosphate

Cited by 18 publications

References 36 publications

Pushing Adenosine and ATP SELEX for DNA Aptamers with Nanomolar Affinity

Pushing Adenosine and ATP SELEX for DNA Aptamers with Nanomolar Affinity

Engineering base-excised aptamers for highly specific recognition of adenosine

Breaking Through Bead-Supported Assay: Integration of Optical Tweezers Assisted Fluorescence Imaging and Luminescence Confined Upconversion Nanoparticles Triggered Luminescent Resonance Energy Transfer (LRET)

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