- Genetically Encoded Fluorescent RNA Sensor for Ratiometric Imaging of MicroRNA in Living Tumor Cells
-
Light-up RNA aptamers are valuable tools for fluorescence imaging of RNA in living cells and thus for elucidating RNA functions and dynamics. However, no light-up RNA sensor has been reported for imaging of microRNAs (miRs) in mammalian cells. We report a novel genetically encoded RNA sensor for fluorescent imaging of miRs in living tumor cells using a light-up RNA aptamer that binds to sulforhodamine and separates it from a conjugated contact quencher. On the basis of the structural switching mechanism for molecular beacon, we show that the RNA sensor activates high-contrast fluorescence from the sulforhodamine-quencher conjugate when its stem-loop responsive motif hybridizes with target miR. The RNA sensor can be stably expressed within a designed tRNA scaffold in tumor cells and deliver light-up response to miR target. We also realize the RNA sensor for dual-emission, ratiometric imaging by coexpression of RNA sensor with GFP, enabling quantitative studies of target miR in living cells. Our design may provide a new paradigm for developing robust, sensitive light-up RNA sensors for RNA imaging applications.
- Ying, Zhan-Ming,Wu, Zhan,Tu, Bin,Tan, Weihong,Jiang, Jian-Hui
-
-
Read Online
- Genetically Encoded Ratiometric RNA-Based Sensors for Quantitative Imaging of Small Molecules in Living Cells
-
Precisely determining the intracellular concentrations of metabolites and signaling molecules is critical in studying cell biology. Fluorogenic RNA-based sensors have emerged to detect various targets in living cells. However, it is still challenging to apply these genetically encoded sensors to quantify the cellular concentrations and distributions of targets. Herein, using a pair of orthogonal fluorogenic RNA aptamers, DNB and Broccoli, we engineered a modular sensor system to apply the DNB-to-Broccoli fluorescence ratio to quantify the cell-to-cell variations of target concentrations. These ratiometric sensors can be broadly applied for live-cell imaging and quantification of metabolites, signaling molecules, and other synthetic compounds.
- Wu, Rigumula,Karunanayake Mudiyanselage, Aruni P. K. K.,Shafiei, Fatemeh,Zhao, Bin,Bagheri, Yousef,Yu, Qikun,McAuliffe, Kathleen,Ren, Kewei,You, Mingxu
-
-
Read Online
- A Color-Shifting Near-Infrared Fluorescent Aptamer–Fluorophore Module for Live-Cell RNA Imaging
-
Fluorescent light-up RNA aptamers (FLAPs) have become promising tools for visualizing RNAs in living cells. Specific binding of FLAPs to their non-fluorescent cognate ligands results in a dramatic fluorescence increase, thereby allowing RNA imaging. Here, we present a color-shifting aptamer-fluorophore system, where the free dye is cyan fluorescent and the aptamer-dye complex is near-infrared (NIR) fluorescent. Unlike other reported FLAPs, this system enables ratiometric RNA imaging. To design the color-shifting system, we synthesized a series of environmentally sensitive benzopyrylium-coumarin hybrid fluorophores which exist in equilibrium between a cyan fluorescent spirocyclic form and a NIR fluorescent zwitterionic form. As an RNA tag, we evolved a 38-nucleotide aptamer that selectively binds the zwitterionic forms with nanomolar affinity. We used this system as a light-up RNA marker to image mRNAs in the NIR region and demonstrated its utility in ratiometric analysis of target RNAs expressed at different levels in single cells.
- J?schke, Andres,Sunbul, Murat,Wang, Lu,Zhang, Jingye
-
supporting information
p. 21441 - 21448
(2021/08/23)
-
- Efficient Innate Immune Killing of Cancer Cells Triggered by Cell-Surface Anchoring of Multivalent Antibody-Recruiting Polymers
-
Binding of monoclonal antibodies (mAbs) onto a cell surface triggers antibody-mediated effector killing by innate immune cells through complement activation. As an alternative to mAbs, synthetic systems that can recruit endogenous antibodies from the blood stream to a cancer cell surface could be of great relevance. Herein, we explore antibody-recruiting polymers (ARPs) as a novel class of immunotherapy. ARPs consist of a cell-binding motif linked to a polymer that contains multiple small molecule antibody-binding motifs along its backbone. As a proof of concept, we employ a lipid anchor that inserts into the phospholipid cell membrane and make use of a polymeric activated ester scaffold onto which we substitute dinitrophenol as an antibody-binding motif. We demonstrate that ARPs allow for high avidity antibody binding and drive antibody recruitment to treated cells for several days. Furthermore, we show that ARP-treated cancer cells are prone to antibody-mediated killing through phagocytosis by macrophages.
- Uvyn, Annemiek,De Coen, Ruben,Gruijs, Mandy,Tuk, Cees W.,De Vrieze, Jana,van Egmond, Marjolein,De Geest, Bruno G.
-
supporting information
p. 12988 - 12993
(2019/08/01)
-
- Cell surface clicking of antibody-recruiting polymers to metabolically azide-labeled cancer cells
-
Triggering antibody-mediated innate immune mechanisms to kill cancer cells is an attractive therapeutic avenue. In this context, recruitment of endogenous antibodies to the cancer cell surface could be a viable alternative to the use of monoclonal antibodies. We report on antibody-recruiting polymers containing multiple antibody-binding hapten motifs and cyclooctynes that can covalently conjugate to azides introduced onto the glycocalyx of cancer cells by metabolic labeling with azido sugars.
- Uvyn, Annemiek,De Coen, Ruben,De Wever, Olivier,Deswarte, Kim,Lambrecht, Bart N.,De Geest, Bruno G.
-
supporting information
p. 10952 - 10955
(2019/09/18)
-
- SiRA: A Silicon Rhodamine-Binding Aptamer for Live-Cell Super-Resolution RNA Imaging
-
Although genetically encoded light-up RNA aptamers have become promising tools for visualizing and tracking RNAs in living cells, aptamer/ligand pairs that emit in the far-red and near-infrared (NIR) regions are still rare. In this work, we developed a li
- Wirth, Regina,Gao, Peng,Nienhaus, G. Ulrich,Sunbul, Murat,J?schke, Andres
-
supporting information
p. 7562 - 7571
(2019/05/16)
-
- Synthesis and application of poly(phenylene ethynylene)s for bioconjugation: A conjugated polymer-based fluorogenic probe for proteases
-
A set of carboxylate-functionalized poly(phenylene ethynylene)s (PPEs) has been synthesized in which the carboxylic acid groups are separated from the polymer backbone by oligo(ethylene glycol) spacer units. These polymers are soluble in water and organic
- Wosnick, Jordan H.,Mello, Charlene M.,Swager, Timothy M.
-
p. 3400 - 3405
(2007/10/03)
-