Engineering base-excised aptamers for highly specific recognition of adenosine

Li, Yuqing; Liu, Biwu; Huang, Zhicheng; Liu, Juewen

doi:10.1039/d0sc00086h

Cited by 27 publications

(24 citation statements)

References 54 publications

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“…This implies that ATP-33 can bind to all adenosine analogs but not nucleotide trisphosphates in general, which reflects the binding profile of this aptamer as reported by originally by Huizenga and Szostak ( 35 ). Moreover, aptamer digestion in the presence of each ligand resulted in a different amount of retained product and the level of enzymatic inhibition for each analog coincided with their previously reported cross-reactivity ( 17 , 36 , 37 ). For example, more 28-nt product (as well as undigested aptamer) was retained for ADP and adenosine relative to ATP and AMP (Figure 1D ), which implies that the aptamer binds more strongly to the former pair.…”

Section: Resultssupporting

confidence: 85%

Label-free profiling of DNA aptamer-small molecule binding using T5 exonuclease

Alkhamis

Yang

Farhana

et al. 2020

Nucleic Acids Research

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In vitro aptamer isolation methods can yield hundreds of potential candidates, but selecting the optimal aptamer for a given application is challenging and laborious. Existing aptamer characterization methods either entail low-throughput analysis with sophisticated instrumentation, or offer the potential for higher throughput at the cost of providing a relatively increased risk of false-positive or -negative results. Here, we describe a novel method for accurately and sensitively evaluating the binding between DNA aptamers and small-molecule ligands in a high-throughput format without any aptamer engineering or labeling requirements. This approach is based on our new finding that ligand binding inhibits aptamer digestion by T5 exonuclease, where the extent of this inhibition correlates closely with the strength of aptamer-ligand binding. Our assay enables accurate and efficient screening of the ligand-binding profiles of individual aptamers, as well as the identification of the best target binders from a batch of aptamer candidates, independent of the ligands in question or the aptamer sequence and structure. We demonstrate the general applicability of this assay with a total of 106 aptamer-ligand pairs and validate these results with a gold-standard method. We expect that our assay can be readily expanded to characterize small-molecule-binding aptamers in an automated, high-throughput fashion.

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Section: Resultssupporting

confidence: 85%

Label-free profiling of DNA aptamer-small molecule binding using T5 exonuclease

Alkhamis

Yang

Farhana

et al. 2020

Nucleic Acids Research

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“…It is known that G8, G9, G18, G19, and A10 are all highly conserved, and any mutation to them can abolish aptamer binding [8a] . In our previous study we found that the A10‐excised aptamer can specifically recognize adenosine [12] . Taking together the observations of the G19‐excised aptamer here, and the two Na + aptamers ΔG16 and ΔA10, highly conserved purines are more likely to be optimal excision sites than nonconserved purines, although not all conserved sites work.…”

Section: Resultsmentioning

confidence: 88%

“…In general, an aptamer should bind its target with a well‐defined three‐dimensional structure and contain guanines that are involved in target binding. We previously excised an adenine from a duplex region, but this did not allow the rebinding of adenosine [12] . Therefore, not all DNA structures can be used for targeted binding of purines.…”

Section: Resultsmentioning

confidence: 99%

“…This break generates new 3′ and 5′ termini, and we anticipated that this cleaved site would be rejoined by binding a nucleoside or nucleotide. After excising A10 in the adenosine aptamer (along with a few other changes to the structure to stabilize the engineered aptamer, Figure 1 B), the sequence was able to specifically bind adenosine, [12] whereas the original aptamer could barely differentiate adenosine from AMP and ATP [8a] …”

Section: Resultsmentioning

confidence: 99%

“…For example, a vacancy‐bearing DNA scaffold (such as a duplex or G‐quadruplex) can act as an aptamer to bind free purine nucleosides [11] . Furthermore, we reported a DNA sequence (32‐mer) with excellent specificity for adenosine using an adenosine/adenosine triphosphate (ATP) aptamer as scaffold [12] . We called it a base‐excised aptamer because an adenine nucleotide close to the aptamer binding site was completely removed.…”

Section: Introductionmentioning

confidence: 99%

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Highly Specific Recognition of Guanosine Using Engineered Base‐Excised Aptamers

Liu

2020

Chemistry A European J

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Purines and their derivatives are highly important molecules in biology for nucleic acid synthesis, energy storage, and signaling. Although many DNA aptamers have been obtained for binding adenine derivatives such as adenosine, adenosine monophosphate, and adenosine triphosphate, success for the specific binding of guanosine has been limited. Instead of performing new aptamer selections, we report herein a base‐excision strategy to engineer existing aptamers to bind guanosine. Both a Na+‐binding aptamer and the classical adenosine aptamer have been manipulated as base‐excising scaffolds. A total of seven guanosine aptamers were designed, of which the G16‐deleted Na+ aptamer showed the highest bindng specificity and affinity for guanosine with an apparent dissociation constant of 0.78 mm. Single monophosphate difference in the target molecule was also recognizable. The generality of both the aptamer scaffold and excised site were systematically studied. Overall, this work provides a few guanosine binding aptamers by using a non‐SELEX method. It also provides deeper insights into the engineering of aptamers for molecular recognition.

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Advances and Challenges in Small‐Molecule DNA Aptamer Isolation, Characterization, and Sensor Development

Alkhamis

Canoura

et al. 2021

Angewandte Chemie

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Aptamers are short oligonucleotides isolated in vitro from randomizedlibraries that can bind to specific molecules with high affinity,and offer an umber of advantages relative to antibodies as biorecognition elements in biosensors.H owever,i tremains difficult and labor-intensive to develop aptamer-based sensors for small-molecule detection. Here,w er eview the challenges and advances in the isolation and characterization of small-molecule-binding DNAa ptamers and their use in sensors.First, we discuss in vitro methodologies for the isolation of aptamers,and provide guidance on selecting the appropriate strategy for generating aptamers with optimal binding properties for agiven application. We next examine techniques for characterizing aptamer-target binding and structure.A fterwards,w ediscuss various small-molecule sensing platforms based on original or engineered aptamers,and their detection applications.F inally,w econclude with ageneral workflow to develop aptamer-based small-molecule sensors for real-world applications. Aus dem Inhalt 1. Introduction 16939 2. Challenges and Advances in Aptamer Isolation 16942 3. Challenges and Advances in Aptamer Characterization 16949 4. Challenges and Advances in Developing Aptamer-Based Small-Molecule Sensors 16953

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Engineering base-excised aptamers for highly specific recognition of adenosine

Cited by 27 publications

References 54 publications

Label-free profiling of DNA aptamer-small molecule binding using T5 exonuclease

Label-free profiling of DNA aptamer-small molecule binding using T5 exonuclease

Highly Specific Recognition of Guanosine Using Engineered Base‐Excised Aptamers

Advances and Challenges in Small‐Molecule DNA Aptamer Isolation, Characterization, and Sensor Development

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