Multistep Fluorescence Resonance Energy Transfer Research Articles

Multi-step FRET systems based on discrete supramolecular assemblies.

Fluorescence resonance energy transfer (FRET) from the excited state of the donor to the ground state of the acceptor is one of the most important fluorescence mechanisms and has wide applications in light-harvesting systems, light-mediated therapy, bioimaging, optoelectronic devices, and information security fields. The phenomenon of sequential energy transfer in natural photosynthetic systems provides great inspiration for scientists to make full use of light energy. In recent years, discrete supramolecular assemblies (DSAs) have been successively constructed to incorporate donor and multiple acceptors, and to achieve multi-step FRET between them. This perspective describes recent advances in the fabrication and application of DSAs with multi-step FRET. These DSAs are categorized based on the non-covalent scaffolds, such as amphiphilic nanoparticles, host-guest assemblies, metal-coordination scaffolds, and biomolecular scaffolds. This perspective will also outline opportunities and future challenges in this research area.

Construction of an Artificial Light-Harvesting System with Efficient Photocatalytic Activity in an Aqueous Solution Based on a FRET-Featuring Metallacage.

Over the past few decades, the design and construction of high-efficiency artificial light-harvesting systems (LHSs) involving multistep fluorescence-resonance energy transfer (FRET) processes have gradually received considerable attention within wide fields ranging from supramolecular chemistry to chemical biology and even materials science. Herein, through coordination-driven self-assembly, a novel tetragonal prismatic metallacage featuring a FRET process using tetraphenylethene (TPE) units as donors and BODIPY units as acceptors has been conveniently synthesized. Subsequently, taking advantage of supramolecular hydrophobic interactions, a promising artificial LHS involving two-step FRET processes from TPE to BODIPY and then to Nile Red (NiR) has been successfully fabricated in an aqueous solution using the FRET-featuring metallacage, NiR, and an amphiphilic polymer (mPEG-DSPE). Notably, this obtained aqueous LHS exhibits highly efficient photocatalytic activity in the dehalogenation of a bromoacetophenone derivate. This study provides a unique strategy for fabricating artificial LHSs in aqueous solutions with multistep FRET processes and further promotes the future development of mimicking the photosynthesis process.

Orthogonal Self-Assembly of a Two-Step Fluorescence-Resonance Energy Transfer System with Improved Photosensitization Efficiency and Photooxidation Activity.

During the past few decades, fabrication of multistep fluorescence-resonance energy transfer (FRET) systems has become one of the most attractive topics within supramolecular chemistry, chemical biology, and materials science. However, it is challenging to efficiently prepare multistep FRET systems with precise control of the distances between locations and the numbers of fluorophores. Herein we present the successful fabrication of a two-step FRET system bearing specific numbers of anthracene, coumarin, and BODIPY moieties at precise distances and locations through an efficient and controllable orthogonal self-assembly approach based on metal-ligand coordination and host-guest interactions. Notably, the photosensitization efficiency and photooxidation activity of the two-step FRET system gradually increased with the number of energy transfer steps. For example, the two-step FRET system exhibited 1.5-fold higher 1O2 generation efficiency and 1.2-fold higher photooxidation activity than that of its corresponding one-step FRET system. This research not only provides the first successful example of the efficient preparation of multistep FRET systems through orthogonal self-assembly involving coordination and host-guest interactions but also pushes multistep FRET systems toward the application of photosensitized oxidation of a sulfur mustard simulant.

A multifunctional DNA nanostructure based on multicolor FRET for nuclease activity assay.

Nucleases play a crucial role in DNA replication, recombination and repair which are associated with cancers. Herein, we develop a four-color fluorescent probe for ratiometric detection of multiple nucleases. This four-color fluorescent probe consists of four fluorescent dyes connected by a DNA tetrahedral nanostructure with the involvement of multistep fluorescence resonance energy transfer (FRET). Based on the principle of self-assembly, the four-color fluorescent probe is constructed by integrating one acceptor with three spatially and spectrally distinct acceptors. A DNA tetrahedral nanostructure functions as a scaffold to link the acceptor dyes (i.e., diethylaminocoumarin (DEA), carboxyfluorescein (FAM), Texas Red, and Cy5). The fluorescence emissions of DEA, FAM, Texas Red and Cy5 can be observed through efficient multi-step energy transfer. This four-color fluorescent probe enables single excitation/four emissions, and it can be used for ratiometric detection of nucleases (i.e., XhoI, HindIII and KpnI) and the screening of nuclease inhibitors. Importantly, this four-color fluorescent probe can be further applied to discriminate multiple biomolecule targets by simply integrating the recognition sites of various biomolecules into the DNA tetrahedral nanostructure.

Multi-step fluorescence resonance energy transfer between the fluorophores via cosolubilization in cationic, anionic and non-ionic micelles

The interaction of three different fluorescent molecules viz, 2,5-dimethoxybenzaldehyde (DMB), fluorescein (Flu) and rhodamine B (RhB) with the surfactant systems of varied nature has been explored using steady state spectrofluorometry. Cosolubilization studies have been carried out to understand the micellar effects on the spectral properties of flourophores in their mixed state within the micelles and accordingly evaluated for their energy transfer processes. FRET studies have been carried out between DMB-Flu and Flu-RhB and thus two new FRET pairs have been established using micellar systems as scaffolds. It has been found that the nature of surfactant has a significant effect on the FRET efficiency of both the pairs. Herein, we also demonstrate the three step FRET taking place simultaneously from DMB to RhB via an intermediate Flu which otherwise does not occur due to the lack of spectral overlap between DMB and RhB and it is operative in all the micellar systems studied. This three component FRET so obtained can have vast applications in biological systems.

Multi step energy transfer between three Si_LTL and SiGe_LTL zeolite-loaded dyes

We report a novel potential light-harvesting antenna material constructed with three dyes loaded on two types of zeolite LTL (SiGe_LTL and Si_LTL). Fluorescence resonance energy transfer (FRET) occurs from acridine hydrochloride (Ac) to Thionine acetate (Th) via Acriflavine hydrochloride (AF), which acts as an antenna to receive and transfer energy from Ac to Th. We compared multistep FRET systems based on dye-loaded SiGe_LTL paired with an Si_LTL-based system. Our results show that in both cases, FRET efficiency increases with Th loading and decreases with increasing AF loading. Moreover, the zeolite LTL microenvironment causes a red shift in the fluorescence spectra of the three SiGe_LTL-loaded-dyes compared to those of the Si_LTL-based dyes.

Visual Detection of Multiplex MicroRNAs Using Cationic Conjugated Polymer Materials.

A simple, visual, and specific method for simultaneous detection of multiplex microRNAs (miRNAs) has been developed by integrating duplex-specific nuclease (DSN)-induced amplification with cationic conjugated polymer (CCP) materials. The probe DNA with a complementary sequence to target miRNA is labeled with fluorescein dye (FAM). Without target miRNA, the single-strand DNA probe cannot be digested by DSN. Upon adding CCPs, efficient fluorescence resonance energy transfer (FRET) from CCP to FAM occurs owing to strong electrostatic interactions between CCP and the DNA probe. In the presence of target miRNA, the DNA probe hybridizes with target miRNA followed by digestion to small nucleotide fragments by DSN; meanwhile, the miRNA is released and subsequently interacts again with the probe, resulting in the cycled digestion of the DNA probe. In this case, weak electrostatic interactions between oligonucleotide fragments and CCP lead to inefficient FRET from CCP to FAM. Thus, by triggering the FRET signal from CCP to FAM, miRNA can be specially detected, and the fluorescence color change based on FRET can be visualized directly with the naked eye under an UV lamp. Furthermore, an energy transfer cascade can be designed using CCP and DNA probes labeled at the 5'-terminus with FAM and Cy3 dyes, and the multistep FRET processes offer the ability of simultaneous detection of multiplex miRNAs.

Multi step FRET among three laser dyes Pyrene, Acriflavine and Rhodamine B

Fluorescence Resonance Energy Transfer (FRET) system using three dyes has been demonstrated. It has been observed that multi step energy transfer occurred from Pyrene to Rhodamine B via Acriflavine. Here Acriflavine acts as an antenna to receive energy from Pyrene and transfer the same to Rhodamine B. This multi step FRET system is advantageous compared to the conventional FRET as this can be used to study molecular level interaction beyond conventional FRET distance (1–10nm) as well as studying multi-branched macromolecules. The introduction of clay enhances the FRET efficiencies among the dye pair, which is an advantage to make the multi step system more useful. Similar approach can be used for increasing FRET efficiencies by using other dyes.

Borromean three-body FRET in frozen Rydberg gases.

Controlling the interactions between ultracold atoms is crucial for quantum simulation and computation purposes. Highly excited Rydberg atoms are considered in this prospect for their strong and controllable interactions known in the dipole-dipole case to induce non-radiative energy transfers between atom pairs, similarly to fluorescence resonance energy transfer (FRET) in biological systems. Here we predict few-body FRET processes in Rydberg atoms and observe the first three-body resonance energy transfer in cold Rydberg atoms using cold caesium atoms. In these resonances, additional relay atoms carry away an energy excess preventing the two-body resonance, leading thus to a Borromean type of energy transfer. These few-body processes present strong similarities with multistep FRET between chromophores sometimes called donor-bridge-acceptor or superexchange. Most importantly, they generalize to any Rydberg atom and could lead to new implementations of few-body quantum gates or entanglement.

Multiplex detection of KRAS and BRAF mutations using cationic conjugated polymers

The mutation detections of KRAS and BRAF genes are of significant importance to predict the responses to anti-cancer therapy and develop new drugs. In this paper, we developed a multi-step fluorescence resonance energy transfer (FRET) assay for multiplex detection of KRAS and BRAF mutations using cationic conjugated polymers (CCP). The newly established detection system could detect as low as 2% mutant DNAs in DNA admixtures. By triggering the emission intensity change of CCP and the dyes labeled in the DNA, four possible statuses (three mutations and one wildtype) can be differentiated in one extension reaction. The detection efficiency of this new method in clinical molecular diagnosis was validated by determining KRAS and BRAF mutations of 51 formalin-fixed paraffin-embedded (FFPE) ovary tissue samples. Furthermore, the result of the CCP-based multi-step FRET assay can be directly visualized under UV light so that no expensive instruments and technical expertise are needed. Thus, the assay provides a sensitive, reliable, cost-effective and simple method for the detection of disease-related gene mutations.

Fluorescence Logic‐Signal‐Based Multiplex Detection of Nucleases with the Assembly of a Cationic Conjugated Polymer and Branched DNA

An energy-transfer cascade is generated from a cationic conjugated polymer (PFP) and negatively charged, Y-shaped DNA labeled with three dyes at its termini (fluorescein (Fl), Tex Red, and Cy5). Multistep fluorescence resonance energy transfer regulates the fluorescence intensities of PFP and the dyes. Different types of logic gates can be operated by observing the emission wavelengths of different dyes with multiplex nucleases as inputs.

Self-Assembled DNA Photonic Wire for Long-Range Energy Transfer

DNA is a promising material for use in nanotechnology; the persistence length of double stranded DNA gives it a rigid structure in the several-nanometer regime, and its four letter alphabet enables addressability. We present the construction of a self-assembled DNA-based photonic wire capable of transporting excitation energy over a distance of more than 20 nm. The wire utilizes DNA as a scaffold for a chromophore with overlapping absorption and emission bands enabling fluorescence resonance energy transfer (FRET) between pairs of chromophores leading to sequential transfer of the excitation energy along the wire. This allows for the creation of a self-assembled photonic wire using straightforward construction and, in addition, allows for a large span in wire lengths without changing the basic components. The intercalating chromophore, YO, is chosen for its homotransfer capability enabling effective diffusive energy migration along the wire without loss in energy. In contrast to heterotransfer, i.e., multistep cascade FRET, where each step renders a photon with less energy than in the previous step, homotransfer preserves the energy in each step. By using injector and detector chromophores at opposite ends of the wire, directionality of the wire is achieved. The efficiency of the wire constructs is examined by steady-state and time-resolved fluorescence measurements and the energy transfer process is simulated using a Markov chain model. We show that it is possible to create two component DNA-based photonic wires capable of long-range energy transfer using a straightforward self-assembly approach.

Time-resolved Laser Spectroscopic Analysis of Multi-step Fluorescence Resonance Energy Transfer on Chromophore Array Constructed by Oligo-DNA Assembly

Specific sequential arrangements of three kinds of chromophores separated by regulated distances equaling approximately one pitch of the DNA duplex (34 Å) in non-covalent molecular assembly systems are constructed using chromophore/oligo-DNA conjugates. Vectorial photoenergy transmission along the DNA helix axis by fluorescence resonance energy transfer (FRET) in a sequential chromophore array is observed and analyzed by time-resolved fluorescence spectroscopy and lifetime measurements using a femtosecond pulse laser system. The results suggest a FRET occurs on a picosecond scale between the donor chromophore and the acceptor chromophore through a mediator chromophore via a multi-step FRET over the molecular assemblies (two helical pitches, 68 Å).

Multistep fluorescence resonance energy transfer in sequential chromophore array constructed on oligo-DNA assemblies.

Sequential arrays of chromophores at regulated distances were constructed on a noncovalent DNA molecular assembly system in aqueous media. Photoinduced fluorescence resonance energy transfer (FRET) behaviors were then observed. We designed a number of chromophore/oligo-DNA conjugates with varying sequences. The chromophores eosin (Eo), TexasRed (TR), and tetramethylrhodamine (Rho) were employed as the energy donor, acceptor, and mediator, respectively, based on overlapping excitation and emission spectra. The chromophores were attached via aminolinkers to the 5'-terminals of 10mer oligo-DNAs consisting of AT rich sequences. The arrangement of Eo-Rho or Rho-TR with 10-residue (1 pitch of duplex) distances was ensured by duplex formation of the conjugates with a 20mer matrix oligo-DNA composed of complementary sequences to the conjugates. Single-step FRET from Eo to Rho and from Rho to TR was confirmed on the duplex. The three chromophore conjugates were then mixed with longer matrix oligo-DNAs (30 or 40mer) consisting of complementary sequences to the conjugates, producing Eo-(Rho)(n)-TR (n = 1 or 2) arrays with 10-residue distances. Multistep FRET from Eo to TR through the Rho mediator(s) was observed on the molecular assemblies. This photoenergy transmission system offers a good model for a photoenergy transmission system mimicking photosynthetic systems.

Change in Fluorescence Spectra Depending on the Arrangement of the Chromophores in Multistep Fluorescence Resonance Energy Transfer

Multistep fluorescence resonance energy transfer in a system containing three chromophores is dependent upon the relative arrangement of the chromophore triad.