DNA mimics of red fluorescent proteins (RFP) based on G-quadruplex-confined synthetic RFP chromophores

Abstract

Red fluorescent proteins (RFPs) have emerged as valuable biological markers for biomolecule imaging in living systems. Developing artificial fluorogenic systems that mimic RFPs remains an unmet challenge. Here, we describe the design and synthesis of six new chromophores analogous to the chromophores in RFPs. We demonstrate, for the first time, that encapsulating RFP chromophore analogues in canonical DNA G-quadruplexes (G4) can activate bright fluorescence spanning red and far-red spectral regions (Em = 583−668 nm) that nearly match the entire RFP palette. Theoretical calculations and molecular dynamics simulations reveal that DNA G4 greatly restricts radiationless deactivation of chromophores induced by a twisted intramolecular charge transfer (TICT). These DNA mimics of RFP exhibit attractive photophysical properties comparable or superior to natural RFPs, including high quantum yield, large Stokes shifts, excellent anti-photobleaching properties, and two-photon fluorescence. Moreover, these RFP chromophore analogues are a novel and distinctive type of topology-selective G4 probe specific to parallel G4 conformation. The DNA mimics of RFP have been further exploited for imaging of target proteins. Using cancer-specific cell membrane biomarkers as targets, long-term real-time monitoring in single live cell and two-photon fluorescence imaging in tissue sections have been achieved without the need for genetic coding.

A Multicolor Large Stokes Shift Fluorogen‐Activating RNA Aptamer with Cationic Chromophores

“…80 % of their maximum intensity even in the absence of Mg 2+ (Figure S6), suggesting that the positively charged side chain partially compensated for the reduced availability of divalent metal ions. Other recently reported systems, in which arylvinyl‐substituted HBI analogues were shown to be activated by parallel‐stranded DNA quadruplexes, required 50 m m Mg 2+ …”

“…Other recently reporteds ystems, in which arylvinyl-substituted HBI analogues were shown to be activated by parallel-stranded DNA quadruplexes, required 50 mm Mg 2 + . [25] The dissociation constants of the new FLAP complexes were determined by titrating each fluorophore with increasing amountso fR NA (Figure 2c,T able1). We found a K D of 570 nm for DMHBI binding to Chili RNA, similar to the original1 3-2 aptamer.…”

Inverted solvatochromic Stokes shift in GFP-like chromophores with extended conjugation

“…In test tubes, the fluorescence is re-activated when synthetic HBI analogues interact with supra-molecular hosts [6], metal-organic framework [7], aggregation-induced emission [8] and protein hosts [9]. In living cells, HBI analogues, represented by represented by 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI), have been used to visualize RNA aptamers [10], DNA quadruplex [11], and more recently the detection of protein aggregation [12].…”

“…In particular, GFP analogues recently emerge to enable fluorogenic detection of biomolecules in living cells [11,12]. Compared to commonly used HBI analogues, RFP analogues harbor an extended moiety at the C2 position of the imidazolinone group (Ib position in left panel, FIG.…”

Engineering of Nucleic Acids and Synthetic Cofactors as Holo Sensors for Probing Signaling Molecules in the Cellular Membrane Microenvironment

“…In this DNA sensor,G 4w as chose as the cofactor-binding DNAm otif because the G-tetrads in G4 afford as pecific scaffold for organic ligands with al arge p-planar structure. [10] Thes elfassembly of the cofactor and afluorophore-coupled G4 DNA motif forms the DNA-and FRET-based sensor,inwhich the fluorophore serves as the donor for the bound cofactor (Scheme 1). After the reaction with signaling molecules,t he fluorescence emission of the cofactors is altered, accompanied by aF RET ratio change.M oreover,t he DNA-based sensor can be easily used to decorate the cell surface by appending amembrane-anchored moiety during the chemical synthesis.A sar esult, the ratiometric monitoring of target signaling molecules in the plasma membrane environment with spatiotemporal resolution is achieved by mapping the changes in the FRET ratio.I ti sn oteworthy that the intact functional DNAmotif in the sensor is accessible by commercialized oligonucleotide synthesis,a voiding cumbersome design and synthesis.M ore importantly,t aking advantage of the modular design, DNAs ensors for other signaling molecules can be readily constructed by rationally tuning the cofactors and donor fluorophores,w hich provides av ersatile strategy for the construction of cell surface DNA-based sensors.…”