An efficient method to evaluate experimental factor influence on in vitro binding of aptamers

Diao, Donglin; Qiao, Na; Wu, Xiao; Li, Jiyuan; Lou, Xinhui

doi:10.1016/j.ab.2018.06.015

Cited by 8 publications

(7 citation statements)

References 53 publications

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“…In contrast, the binding percentages of PD-L1 from R1 to R3 by MB-SELEX were quite random, changing from 3.52% in R1 to 0.078% in R2 and 2.93% in R3 (Figure D). This phenomenon should be caused by the uncontrolled nonspecific absorption of DNAs on the magnetic beads and the low binding specificity of aptamers with the immobilized proteins on surface . Previously we reported that the binding specificity varied significantly ranging from 0.03 ± 0.03 to 14.60 ± 2.30.…”

Section: Resultsmentioning

confidence: 99%

“…However, target immobilization may result in many problems such as masking the target binding sites, changing the target natural conformation, and causing serious nonspecific absorption of the library on the solid phase matrix. These factors can cause the selection of aptamers to be inefficient or even fail . The library-immobilized methods for the selection of SSAs, or named falling-off-SELEX, avoid such problems, whereas the spontaneous dissociation of the immobilized library seriously limited the selection efficiency. , …”

Section: Introductionmentioning

confidence: 99%

“…These factors can cause the selection of aptamers to be inefficient or even fail. 23 The library- immobilized methods for the selection of SSAs, or named falling-off-SELEX, avoid such problems, whereas the spontaneous dissociation of the immobilized library seriously limited the selection efficiency. 11,12 Immuno-precipitation (IP) is a widely used method for isolating small amount of antigens or target protein from complex samples such as cell lysates, serum, and tissue homogenates.…”

Section: ■ Introductionmentioning

confidence: 99%

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Speeding up in Vitro Discovery of Structure-Switching Aptamers via Magnetic Cross-Linking Precipitation

Qiao

et al. 2019

Anal. Chem.

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We report here a modified aptamer selection method, magnetic cross-linking precipitation (MCP)-SELEX, for highly efficient library enrichment and aptamer isolation. MCP-SELEX isolates bound aptamers via highly efficient chemical cross-linking between amino groups of target proteins and activated carboxylic acid groups on magnetic beads (>90% coupling efficiency). Importantly, MCP-SELEX avoids surface interferences in conventional target-fixed methods and substantially minimizes nonspecific binding. The enrichment efficiencies of MCP-SELEX for various proteins (PD-L1, ubiquitin, thrombin, and HSA) were all greatly higher than those of the conventional target-bound magnetic bead based-SELEX (MB-SELEX). Antithrombin aptamer with KD of 33 nM was successfully isolated by four rounds of MCP-SELEX. MCP-SELEX also enabled the efficient aptamer isolation by coupling with MB-SELEX or falling-off-SELEX. We identified structure-switching aptamers (SSAs) that specifically bind to HSA with low nanomolar dissociation constant via three rounds of MCP-SELEX and 1 round of falling-off-SELEX. Our HSA SSAs also have ∼3-fold higher specificity against streptavidin relative to thrombin SSAs discovered through falling-off-SELEX only. The enriched library has ∼78-fold higher signal-to-noise ratio (the number of DNAs eluted by 50 nM HSA divided by the number of DNAs self-dissociated in blank buffer) than that obtained by 4 rounds of direct falling-off-SELEX. We finally demonstrated the application of the selected SSA in fluorescent detection of HSA in urine with diagnostic required sensitivity and dynamic range. We expect that MCP-SELEX may be coupled with other selection methods to substantially accelerate aptamer discovery.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

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Speeding up in Vitro Discovery of Structure-Switching Aptamers via Magnetic Cross-Linking Precipitation

Qiao

et al. 2019

Anal. Chem.

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“…However, immobilized targets were especially ill-suited for small molecules, where the immobilization itself is a difficult process due to the small molecular structure and the presence of fewer active sites for conjugation [ 32 ]. In other words, the aptamer screened by immobilized targets may be different from the aptamer screened by targets in the natural conformations [ 33 ]. Gu [ 34 ] and Ha [ 35 ] screened two STX aptamers through the immobilized libraries by graphene oxide SELEX (GO-SELEX), overcoming the obstacle that STX was difficult to immobilize.…”

Section: Introductionmentioning

confidence: 99%

A Novel SELEX Based on Immobilizing Libraries Enables Screening of Saxitoxin Aptamers for BLI Aptasensor Applications

Zhou¹,

Gao²,

Yang³

et al. 2022

Toxins

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Saxitoxin (STX) is one of the potent marine biotoxins that has high rate of lethality. However, there are no effective treatments at present, and the existing detection methods need to be further explored because of ethical problems or technical limitations. In this work, oligonucleotide aptamers toward STX were screened based on immobilizing libraries on Immobilized Metal-Chelate (IMC), such as Ni-NTA Sepharose, and the IMC-SELEX was conducted by the G-quadruplex library and the random library, respectively. Aptamer 45e (from the G-quadruplex library) and aptamer 75a were obtained after optimization, and aptamer 45e turned out to have a higher affinity toward STX. Furthermore, it was found that the hydrogen bonding and the van der Waals forces (VDW) played major roles in the high efficiency and specificity between STX and 45e by means of molecular docking and dynamics simulation. Based on this, aptamer 45e-1 with the Kd value of 19 nM was obtained by further optimization, which was then used to construct a simple, label-free and real-time optical BLI aptasensor for the detection of STX. This aptasensor showed good reproducibility and stability. In summary, with the advantages of screening aptamers of high efficiency and specificity toward the targets, the proposed IMC-SELEX provides a promising screening strategy for discovering aptamers, which could be used as the potential molecular recognition elements in the fields of biomedicine, food safety and environmental monitoring.

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“…Since SELEX emerged in 1990, many selection methods have been developed for isolating aptamers such as purified-protein-based SELEX, whole-cell-based SELEX, capillary electrophoresis (CE)-based SELEX, Capture-SELEX, and so on [27][28][29][30]. Due to the low successful rates for aptamer selection, several factors in selection process have still been studying [31][32][33]. Importantly, monitoring enriching progress of candidate aptamers has always been the key and restrictive step in SELEX process.…”

Section: Introductionmentioning

confidence: 99%

SELEX tool: a novel and convenient gel-based diffusion method for monitoring of aptamer-target binding

et al. 2020

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Background: Aptamers, single-stranded DNAs or RNAs, can be selected from a library containing random sequences using a method called Systematic Evolution of Ligands by EXponential Enrichment (SELEX). In SELEX, monitoring the enriching statuses of aptamer candidates during the process is a key step until today. Conformational change of an aptamer caused by target-binding in gel can be used to indicate its statuses of binding. Results: In this study, an easy-to-implement gel-based diffusion method (GBDM) was developed to monitor the interaction between enriched aptamer candidates and their targets. In order to prove the concept, characterization of aptamers targeting their targets including protein (thrombin) and non-protein molecules (acetamiprid, ATP, atrazine, profenofos and roxithromycin), respectively, were performed using mini gels. Our method has advantages over the common methods including easy performed with labor-and time-saving in experimental operation. The concept has been proven by monitoring enrichment of dynamic aptamer candidate libraries targeting a small molecule 2,2-bis(4-chlorophenyl) acetic acid (DDA) during SELEX process. A mini gel cassette was designed and fabricated by our laboratory to make mini agarose gels for diffusion with different directions. Conclusions:These results indicate that GBDM, in particular, chasing diffusion is suitable for monitoring the interaction between enriched aptamer candidates and their targets. These pioneering efforts are helpful for novel aptamer selection by breaking through the technical bottleneck of aptamer development and helpful for development of novel aptasensors.

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An efficient method to evaluate experimental factor influence on in vitro binding of aptamers

Cited by 8 publications

References 53 publications

Speeding up in Vitro Discovery of Structure-Switching Aptamers via Magnetic Cross-Linking Precipitation

Speeding up in Vitro Discovery of Structure-Switching Aptamers via Magnetic Cross-Linking Precipitation

A Novel SELEX Based on Immobilizing Libraries Enables Screening of Saxitoxin Aptamers for BLI Aptasensor Applications

SELEX tool: a novel and convenient gel-based diffusion method for monitoring of aptamer-target binding

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