In Vitro Selection of Structure‐Switching Signaling Aptamers

Nutiu, Razvan; Li, Yingfu

doi:10.1002/anie.200461848

Cited by 378 publications

(292 citation statements)

References 27 publications

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“…That consideration made it likely that this simplest internal loop would be recovered from all selections, so that the ease of isolating it could be compared at all randomized lengths. Publications showing the recurrence of a solution for a variety of targets (Hanczyc and Dorit 2000;Salehi-Ashtiani and Szostak 2001;Cruz et al 2004;Nutiu and Li 2005) supported the idea that the recurrence of the simplest isoleucine motif is highly probable in our case. Our experiments show that the probability of this motif, judged by the apparent initial abundance, does not increase monotonically as randomized RNA tracts are lengthened (as calculation suggested).…”

Section: Introductionsupporting

confidence: 80%

Size, constant sequences, and optimal selection

Łęgiewicz

Lozupone²,

Knight³

et al. 2005

RNA

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Because the abundance of functional molecules in RNA sequence space has many unexplored aspects, we compared the outcome of 11 independent selections, performed using the same affinity selection protocol and contiguous randomized regions of 16, 22, 26, 50, 70, and 90 nucleotides. All affinity selections targeted the simplest isoleucine aptamer, an asymmetric internal loop. This loop should be abundant in all selections, so that it can be compared across all experiments. In some cases, two primer sets intended to favor selection of different structures have also been compared. The simplest isoleucine aptamer dominates all selections except with the shortest tract, 16 contiguous randomized nucleotides. Here the isoleucine aptamer cannot be accommodated and no other motif can be selected. Our results suggest an optimum length for selection; surprisingly, both the shortest and the longest randomized tracts make it more difficult to recover the motif. Estimated apparent initial abundances suggest that the simplest isoleucine motif was 20-to 40-fold more frequent in selection with 50-or 70-nucleotide randomized regions than with any other length. Considering primer sets, a pre-formed stable stem within fixed flanking sequences had a fiveto 10-fold negative effect on apparent motif abundance at all lengths. Differing random tract lengths also determined the probable motif permutation and the most abundant helix lengths. These data support a significant but lesser role for primer sequences in the outcome of selections.

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Section: Introductionsupporting

confidence: 80%

Size, constant sequences, and optimal selection

Łęgiewicz

Lozupone²,

Knight³

et al. 2005

RNA

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“…33 Avoiding target immobilization, capture-SELEX selection has so far been indirectly evaluated by comparing the amount of eluted ssDNA or RNA at each cycle with a blank control elution. [10][11][12][13]15,34 We therefore developed a direct protocol that allows for the first time in-stream direct quality control of the selected cycle-libraries of ssDNA via SPR. Briefly, an aliquot of each selected ssDNA cycle-library (even cycles) and of the initial library are used as template for a PCR with a thiolated 5′ primer and a phosphorylated 3′.…”

Section: ■ Resultsmentioning

confidence: 99%

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

2015

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To address limitations in the production of DNA aptamers against small molecules, we introduce a DNA-based capture-SELEX (systematic evolution of ligands by exponential enrichment) protocol with long and continuous randomized library for more flexibility, coupled with in-stream direct-specificity monitoring via SPR and high throughput sequencing (HTS). Applying this capture-SELEX on tobramycin shows that target-specificity arises at cycle number 8, which is confirmed by sequence convergence in HTS analysis. Interestingly, HTS also shows that the most enriched sequences are already visible after only two capture-SELEX cycles. The best aptamers displayed K D of approximately 200 nM, similar to RNA and DNA-based aptamers previously selected for tobramycin. The lowest concentration of tobramycin detected on label-free SPR experiments with the selected aptamers is 20-fold smaller than the clinical range limit, demonstrating suitability for smalldrug biosensing.

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“…A simple yet general approach of engineering any aptamer into structure-switching aptamer for real-time signaling applications has also been reported (11,27). Finally, with a combination of the fast turnaround of automatic SELEX techniques (29) and novel selection approaches (30,31), virtually any protein target can have at least one conformation-changing aptamer sequence. Thus, numerous excimer-signaling probes may be created quickly for proteomics with a similar approach reported here.…”

Section: Discussionmentioning

confidence: 99%

Light-switching excimer probes for rapid protein monitoring in complex biological fluids

Yang

Jockusch²,

Vicens³

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

332

265

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Quantitative protein bioanalysis in complex biological fluids presents considerable challenges in biological studies and disease diagnosis. The major obstacles are the background signals from both the probe and the biological fluids where the proteins reside. We have molecularly engineered light-switching excimer aptamer probes for rapid and sensitive detection of a biomarker protein, platelet-derived growth factor (PDGF). Labeled with one pyrene at each end, the aptamer switches its fluorescence emission from Ϸ400 nm (pyrene monomer) to 485 nm (pyrene excimer) upon PDGF binding. This fluorescence wavelength change from monomer to excimer emission is a result of aptamer conformation rearrangement induced by target binding. The excimer probe is able to effectively detect picomolar PDGF in homogeneous solutions. Because the excimer has a much longer fluorescence lifetime (Ϸ40 ns) than that of the background (Ϸ5 ns), time-resolved measurements were used to eliminate the biological background. We thus were able to detect PDGF in a cell sample quantitatively without any sample pretreatment. This molecular engineering strategy can be used to develop other aptamer probes for protein monitoring. Combined with lifetime-based measurements and molecular engineering, light-switching excimer aptamer probes hold great potential in protein analysis for biomedical studies.aptamer ͉ biomarker ͉ platelet-derived growth factor ͉ pyrene ͉ time-resolved fluorescence

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In Vitro Selection of Structure‐Switching Signaling Aptamers

Cited by 378 publications

References 27 publications

Size, constant sequences, and optimal selection

Size, constant sequences, and optimal selection

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

Light-switching excimer probes for rapid protein monitoring in complex biological fluids

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