Expanding molecular logic capabilities in DNA-scaffolded multiFRET triads

Buckhout‐White, Susan; Brown, Carl W.; Hastman, David A.; Ancona, Mario G.; Melinger, Joseph S.; Goldman, Ellen R.; Medintz, Igor L.

doi:10.1039/c6ra23079b

Cited by 24 publications

(40 citation statements)

References 46 publications

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“…Due to a strong dependence of the energy transfer efficiency on the donor-acceptor distance, FRET is particularly useful while determining the intra-and intermolecular distances on a nanometer scale [4,5]. Although most FRET studies involve analysing a conventional one-step energy transfer, a multistep FRET (msFRET) has been attracting much attention in recent years [6][7][8][9][10], inspired by the natural photosynthetic systems containing several lightharvesting complexes that efficiently transfer the absorbed energy between a number of chromophores [11,12]. The energy transfer within multiple chromophore systems usually follows a cascade route, moving from an initial donor chromophore through the intermediate donors/acceptors onto a final acceptor chromophore [6][7][8][9][10]13].…”

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confidence: 99%

“…Although most FRET studies involve analysing a conventional one-step energy transfer, a multistep FRET (msFRET) has been attracting much attention in recent years [6][7][8][9][10], inspired by the natural photosynthetic systems containing several lightharvesting complexes that efficiently transfer the absorbed energy between a number of chromophores [11,12]. The energy transfer within multiple chromophore systems usually follows a cascade route, moving from an initial donor chromophore through the intermediate donors/acceptors onto a final acceptor chromophore [6][7][8][9][10]13]. The multistep FRET offers several advantages over the one-step FRET: i) a higher efficiency of long-range transfer [14]; ii) a larger Stokes shift [13,15]; iii) the possibility to monitor inter-and intramolecular interactions beyond the range 1-10 nm [16]; and iv) an extended excitation wavelength range for fluorescence lifetime measurements [14].…”

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confidence: 99%

“…The multistep FRET offers several advantages over the one-step FRET: i) a higher efficiency of long-range transfer [14]; ii) a larger Stokes shift [13,15]; iii) the possibility to monitor inter-and intramolecular interactions beyond the range 1-10 nm [16]; and iv) an extended excitation wavelength range for fluorescence lifetime measurements [14]. As a result, the cascade, or multistep FRET appeared to be especially useful in developing molecular photonic wires and light harvesting systems [6][7][8]17]. Most of such artificial systems are devised through synthesizing the arrays of covalently linked chromophores with a specific design to ensure large collection efficiencies, as well as fast and efficient energy migration.…”

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confidence: 99%

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Three-Step Resonance Energy Transfer in Insulin Amyloid Fibrils

Tarabara

Shchuka

Vus

et al. 2019

EEJP

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The applicability of the three-step Förster resonance energy transfer (FRET) to detection of insulin amyloid fibrils was evaluated, using the chromophore system, containing Thioflavin T (ThT), 4-dimethylaminochalcone (DMC), and two squaraine dyes, referred to here as SQ1 and SQ4. The mediator chromophore DMC was found to enhance the fluorescence intensity of the terminal acceptor, SQ1, excited at 440 nm (at the absorption maximum of the principal donor, ThT), in fibrillar insulin compared to the system without DMC, providing the evidence for the cascade energy transfer in the chain ThT→DMC→SQ4→SQ1. Furthermore, the resulting Stokes shift in the four-chromophore system was 240 nm, as compared to 45 nm for the fibril-bound ThT, suggesting that higher signal-to-noise ratio is the advantage of amyloid fibril detection by multistep FRET. The maximum efficiencies of energy transfer in the insulin fibrils estimated from the quenching of the donor fluorescence in the presence of acceptor for the donor-acceptor pairs ThT-DMC, DMC-SQ4 and SQ4-SQ1 were 40%, 60% and 30% respectively, while negligible FRET occurred in the non-fibrillized protein. The most pronounced differences between fibrillar and non-fibrillized insulin were observed in the 3D fluorescence spectra. Specifically, two intensive spots centered at the emission wavelengths ~ 650 nm (SQ4) and ~ 685 nm (SQ1) were revealed at the excitation wavelength ~ 440 nm in the 3D patterns of insulin amyloid aggregates. In contrast, in the case of the non-fibrillized protein, the barely noticeable spots centered at the same wavelengths, as well as higher fluorescence intensities at the excitation above 550 nm were observed, suggesting the predominant impact of the direct excitation of SQ1 and SQ4 on their fluorescence responses. The inter-chromophore distances calculated from the experimental values of the energy transfer efficiency assuming the isotropic rotation of the dyes, were found to be 2.4, 4.5 and 4.3 nm for the ThT-DMC, DMC-SQ4 and SQ4-SQ1 pairs, respectively, revealing the different fibril binding sites for the examined dyes. The quantum-chemical calculations and simple docking studies provided evidence for the SQ1, SQ4 and ThT, DMC binding to the wet and dry interface of the insulin amyloid protofilament, respectively. The dye-protein complexes are likely to be stabilized by the hydrophobic, van der Waals, aromatic and electrostatic interactions. In summary, the above technique based on the multistep FRET can be employed for the identification and characterization of amyloid fibrils in vitro along with the classical ThT assay, allowing the increase of the amyloid detection sensitivity and lowering the probability of the pseudo-positive result. The applicability of the multistep FRET for amyloid visualization in vivo can be also tested by the involvement of the near-infrared fluorescent dyes to the cascade.

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Three-Step Resonance Energy Transfer in Insulin Amyloid Fibrils

Tarabara

Shchuka

Vus

et al. 2019

EEJP

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“…From a purely conceptual perspective, these results are proof‐of‐principle that transducing enzymatic activity into DNA language in the form of producing unique DNA sequences is indeed feasible. This opens the door to the possibility of doing information processing with these types of assemblies . In particular, the DNA released from the input gate (see Figure ) of multiple sensor assemblies can become the logic inputs of a DNA computation.…”

Section: Discussionmentioning

confidence: 99%

Transducing Protease Activity into DNA Output for Developing Smart Bionanosensors

Bui

Brown

Buckhout‐White

et al. 2019

Small

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DNA can process information through sequence‐based reorganization but cannot typically receive input information from most biological processes and translate that into DNA compatible language. Coupling DNA to a substrate responsive to biological events can address this limitation. A two‐component sensor incorporating a chimeric peptide‐DNA substrate is evaluated here as a protease‐to‐DNA signal convertor which transduces protease activity through DNA gates that discriminate between different input proteases. Acceptor dye‐labeled peptide‐DNAs are assembled onto semiconductor quantum dot (QD) donors as the input gate. Addition of trypsin or chymotrypsin cleaves their cognate peptide sequence altering the efficiency of Förster resonance energy transfer (FRET) with the QD and frees a DNA output which interacts with a tetrahedral output gate. Downstream output gate rearrangement results in FRET sensitization of a new acceptor dye. Following characterization of component assembly and optimization of individual steps, sensor ability to discriminate between the two proteases is confirmed along with effects from joint interactions where potential for cross‐talk is highest. Processing multiple bits of information for a sensing outcome provides more confidence than relying on a single change especially for the discrimination between different targets. Coupling other substrates to DNA that respond similarly could help target other types of enzymes.

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“…From this base, the switch can be assembled with a range of different dyes and different linker lengths to produce a vast range of optical output. 36 This modularity makes it an ideal candidate to explore restriction enzyme-enabled sensing and rearrangement. Although we have explored many dye triads as well as structural modification, to date, only DNA inputs have been used to modify the optical response.…”

Section: Introductionmentioning

confidence: 99%

Restriction Enzymes as a Target for DNA-Based Sensing and Structural Rearrangement

et al. 2018

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DNA nanostructures have been shown viable for the creation of complex logic-enabled sensing motifs. To date, most of these types of devices have been limited to the interaction with strictly DNA-type inputs. Restriction endonuclease represents a class of enzyme with endogenous specificity to DNA, and we hypothesize that these can be integrated with a DNA structure for use as inputs to trigger structural transformation and structural rearrangement. In this work, we reconfigured a three-arm DNA switch, which utilizes a cyclic Förster resonance energy transfer interaction between three dyes to produce complex output for the detection of three separate input regions to respond to restriction endonucleases, and investigated the efficacy of the enzyme targets. We demonstrate the ability to use three enzymes in one switch with no nonspecific interaction between cleavage sites. Further, we show that the enzymatic digestion can be harnessed to expose an active toehold into the DNA structure, allowing for single-pot addition of a small oligo in solution.

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Expanding molecular logic capabilities in DNA-scaffolded multiFRET triads

Abstract: Dynamic rearrangement of DNA nanostructures provides a straightforward yet powerful mechanism for sequence-specific sensing and potential signaling of such interactions.

Cited by 24 publications

References 46 publications

Three-Step Resonance Energy Transfer in Insulin Amyloid Fibrils

Three-Step Resonance Energy Transfer in Insulin Amyloid Fibrils

Transducing Protease Activity into DNA Output for Developing Smart Bionanosensors

Restriction Enzymes as a Target for DNA-Based Sensing and Structural Rearrangement

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