More DNA–Aptamers for Small Drugs: A Capture–SELEX Coupled with Surface Plasmon Resonance and High-Throughput Sequencing

Spiga, Fabio Mario; Maietta, Paolo; Guiducci, Carlotta

doi:10.1021/acscombsci.5b00023

Cited by 91 publications

(68 citation statements)

References 41 publications

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“…The measured concentrations of tobramycin in patients' blood range between 0.5 and 80 μM, 30 and the minimum is less than 1 order of magnitude below the theoretical LOD reported in this work. Interestingly, the DNA aptamer used in this work showed excellent selectivity in propagating SPR toward molecules belonging to the class of coadministered antibiotics (carbenicillin, penicillin-like) and good selectivity against a similar aminoglycoside antibiotic (kanamycin), 25 therefore confirming its potential in clinical analysis.…”

Section: Analytical Chemistrysupporting

confidence: 64%

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Label-Free Detection of Tobramycin in Serum by Transmission-Localized Surface Plasmon Resonance

et al. 2015

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In order to improve the efficacy and safety of treatments, drug dosage needs to be adjusted to the actual needs of each patient in a truly personalized medicine approach. Key for widespread dosage adjustment is the availability of point-of-care devices able to measure plasma drug concentration in a simple, automated, and costeffective fashion. In the present work, we introduce and test a portable, palm-sized transmission-localized surface plasmon resonance (T-LSPR) setup, comprised of offthe-shelf components and coupled with DNA-based aptamers specific to the antibiotic tobramycin (467 Da). The core of the T-LSPR setup are aptamer-functionalized gold nanoislands (NIs) deposited on a glass slide covered with fluorine-doped tin oxide (FTO), which acts as a biosensor. The gold NIs exhibit localized plasmon resonance in the visible range matching the sensitivity of the complementary metal oxide semiconductor (CMOS) image sensor employed as a light detector. The combination of gold NIs on the FTO substrate, causing NIs size and pattern irregularity, might reduce the overall sensitivity but confers extremely high stability in high-ionic solutions, allowing it to withstand numerous regeneration cycles without sensing losses. With this rather simple T-LSPR setup, we show real-time label-free detection of tobramycin in buffer, measuring concentrations down to 0.5 μM. We determined an affinity constant of the aptamer−tobramycin pair consistent with the value obtained using a commercial propagating-wave based SPR. Moreover, our label-free system can detect tobramycin in filtered undiluted blood serum, measuring concentrations down to 10 μM with a theoretical detection limit of 3.4 μM. While the association signal of tobramycin onto the aptamer is masked by the serum injection, the quantification of the captured tobramycin is possible during the dissociation phase and leads to a linear calibration curve for the concentrations over the tested range (10−80 μM). The plasmon shift following surface binding is calculated in terms of both plasmon peak location and hue, with the latter allowing faster data elaboration and real-time display of the results. The presented T-LSPR system shows for the first time label-free direct detection and quantification of a small molecule in the complex matrix of filtered undiluted blood serum. Its uncomplicated construction and compact size, together with the remarkable performances, represent a leap forward toward effective point-of-care devices for therapeutic drug concentration monitoring.

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Section: Analytical Chemistrysupporting

confidence: 64%

“…467 Da) on propagating SPR (Biacore X100, GE), in a label-free assay, down to 50 nM in buffer solution. 25 We therefore deployed the same aptamer in order to observe the signal generated by the small molecule tobramycin on a nonconnected pattern of plasmonic gold NIs in a transmission localized SPR configuration.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Label-Free Detection of Tobramycin in Serum by Transmission-Localized Surface Plasmon Resonance

et al. 2015

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“…Various approaches for monitoring the SELEX process have been reported [42,[57][58][59][60][61][62][63][64][65][66][67][68][69][70][71][72][73][74][75]. The most straightforward monitoring method involves the assessment of sequence enrichment in RNA pools, a technique made possible by the advent of HTS technology [42,[57][58][59][60][61]. However, HTS is expensive, and sample preparation for HTS is time-consuming.…”

Section: Monitoring the Enrichment Of The Dynamic Libraries In Selex mentioning

confidence: 99%

“…However, HTS is expensive, and sample preparation for HTS is time-consuming. Another effective way of monitoring is to assess the average affinity of the RNA pools to the target molecule [42,[64][65][66][67][68][69][70][71][72][73][74]. Several such assessment methods are available, including SPR, EMSA, and the filter-binding assay.…”

Section: Monitoring the Enrichment Of The Dynamic Libraries In Selex mentioning

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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“…However, HTS is expensive, and sample preparation for HTS is time-consuming. Another effective way of monitoring is to assess the average affinity of the RNA pools to the target molecule 16, 23–33 . Several such assessment methods are available, including surface plasmon resonance (SPR), the electrophoretic mobility shift assay (EMSA), and the filter-binding assay.…”

Section: Introductionmentioning

confidence: 99%

NMR monitoring of the SELEX process to confirm enrichment of structured RNA

Amano

Aoki²,

Miyakawa³

et al. 2017

Sci Rep

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RNA aptamers are RNA molecules that bind to a target molecule with high affinity and specificity using uniquely-folded tertiary structures. RNA aptamers are selected from an RNA pool typically comprising up to 1015 different sequences generated by iterative steps of selection and amplification known as Systematic Evolution of Ligands by EXponential enrichment (SELEX). Over several rounds of SELEX, the diversity of the RNA pool decreases and the aptamers are enriched. Hence, monitoring of the enrichment of these RNA pools is critical for the successful selection of aptamers, and several methods for monitoring them have been developed. In this study, we measured one-dimensional imino proton NMR spectra of RNA pools during SELEX. The spectrum of the initial RNA pool indicates that the RNAs adopt tertiary structures. The structural diversity of the RNA pools was shown to depend highly on the design of the primer-binding sequence. Furthermore, we demonstrate that enrichment of RNA aptamers can be monitored using NMR. The RNA pools can be recovered from the NMR tube after measurement of NMR spectra. We also can monitor target binding in the NMR tubes. Thus, we propose using NMR to monitor the enrichment of structured aptamers during the SELEX process.

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More DNA–Aptamers for Small Drugs: A Capture–SELEX Coupled with Surface Plasmon Resonance and High-Throughput Sequencing

Cited by 91 publications

References 41 publications

Label-Free Detection of Tobramycin in Serum by Transmission-Localized Surface Plasmon Resonance

Label-Free Detection of Tobramycin in Serum by Transmission-Localized Surface Plasmon Resonance

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

NMR monitoring of the SELEX process to confirm enrichment of structured RNA

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