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1.
J Am Chem Soc ; 144(12): 5471-5477, 2022 03 30.
Article in English | MEDLINE | ID: mdl-35294188

ABSTRACT

DNA and RNA can spontaneously self-assemble into various structures, including aggregates, complexes, and ordered structures. The self-assembly reactions cannot be genetically encoded to occur in living mammalian cells since the double-stranded nucleic acids generated by current self-assembly approaches are unstable and activate innate RNA immunity pathways. Here, we show that recently described dimeric aptamers can be used to create RNAs that self-assemble and create RNA and RNA-protein assemblies in cells. We find that incorporation of five copies of Corn, a dimeric fluorogenic RNA aptamer, into an RNA causes the RNA to form large clusters in cells, reflecting multivalent RNA-RNA interactions enabled by these RNAs. Here, we also describe a second dimeric fluorogenic aptamer, Beetroot, which shows partial sequence similarity to Corn. Both Corn and Beetroot form homodimers with themselves but do not form Corn-Beetroot heterodimers. We thus use Corn and Beetroot to encode distinct RNA-protein assemblies in the same cells. Overall, these studies provide an approach for inducing RNA self-assembly, enable multiplexing of distinct RNA assemblies in cells, and demonstrate that proteins can be recruited to RNA assemblies to genetically encode intracellular RNA-protein assemblies.


Subject(s)
Aptamers, Nucleotide , Nucleic Acids , Animals , Aptamers, Nucleotide/genetics , DNA/chemistry , Mammals/genetics , Mammals/metabolism , RNA/chemistry , Zea mays
2.
Nat Methods ; 16(9): 862-865, 2019 09.
Article in English | MEDLINE | ID: mdl-31471614

ABSTRACT

Fluorogenic RNA aptamers bind and activate the fluorescence of otherwise nonfluorescent dyes. However, fluorogenic aptamers are limited by the small number of fluorogenic dyes suitable for use in live cells. In this communication, fluorogenic proteins whose fluorescence is activated by RNA aptamers are described. Fluorogenic proteins are highly unstable until they bind RNA aptamers inserted into messenger RNAs, resulting in fluorescent RNA-protein complexes that enable live imaging of mRNA in living cells.


Subject(s)
Aptamers, Nucleotide/metabolism , Fluorescence , Fluorescent Dyes/chemistry , Microscopy, Fluorescence/methods , Molecular Imaging/methods , RNA, Messenger/analysis , Aptamers, Nucleotide/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , HEK293 Cells , Humans , Luminescent Proteins/genetics , Luminescent Proteins/metabolism
3.
Angew Chem Int Ed Engl ; 59(11): 4511-4518, 2020 03 09.
Article in English | MEDLINE | ID: mdl-31850609

ABSTRACT

Spinach and Broccoli are fluorogenic RNA aptamers that bind DFHBI, a mimic of the chromophore in green fluorescent protein, and activate its fluorescence. Spinach/Broccoli-DFHBI complexes exhibit high fluorescence in vitro, but they exhibit lower fluorescence in mammalian cells. Here, computational screening was used to identify BI, a DFHBI derivative that binds Broccoli with higher affinity and leads to markedly higher fluorescence in cells compared to previous ligands. BI prevents thermal unfolding of Broccoli at 37 °C, leading to more folded Broccoli and thus more fluorescent Broccoli-BI complexes in cells. Broccoli-BI complexes are more photostable owing to impaired photoisomerization and rapid unbinding of photoisomerized cis-BI. These properties enable single mRNA containing 24 Broccoli aptamers to be imaged in live mammalian cells treated with BI. Small molecule ligands can thus promote RNA folding in cells, and thus allow single mRNA imaging with fluorogenic aptamers.


Subject(s)
Aptamers, Nucleotide/chemistry , Benzyl Compounds/chemistry , Brassica/genetics , Fluorescent Dyes/chemistry , Imidazolines/chemistry , RNA, Messenger/chemistry , Aptamers, Nucleotide/metabolism , Benzyl Compounds/metabolism , Gene Expression Regulation , HEK293 Cells , Humans , Imidazolines/metabolism , Isomerism , Optical Imaging , Photochemical Processes , RNA Folding , Single Molecule Imaging , Transition Temperature
4.
Nat Chem Biol ; 13(11): 1187-1194, 2017 Nov.
Article in English | MEDLINE | ID: mdl-28945233

ABSTRACT

Quantitative measurement of transcription rates in live cells is important for revealing mechanisms of transcriptional regulation. This is particularly challenging when measuring the activity of RNA polymerase III (Pol III), which transcribes growth-promoting small RNAs. To address this issue, we developed Corn, a genetically encoded fluorescent RNA reporter suitable for quantifying RNA transcription in cells. Corn binds and induces fluorescence of 3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime, which resembles the fluorophore found in red fluorescent protein (RFP). Notably, Corn shows high photostability, enabling quantitative fluorescence imaging of mTOR-dependent Pol III transcription. We found that, unlike actinomycin D, mTOR inhibitors resulted in heterogeneous transcription suppression in individual cells. Quantitative imaging of Corn-tagged Pol III transcript levels revealed distinct Pol III transcription 'trajectories' elicited by mTOR inhibition. Together, these studies provide an approach for quantitative measurement of Pol III transcription by direct imaging of Pol III transcripts containing a photostable RNA-fluorophore complex.


Subject(s)
Aptamers, Nucleotide/genetics , Chromophore-Assisted Light Inactivation , Fluorescent Dyes/metabolism , Optical Imaging/methods , RNA Polymerase III/analysis , Transcription, Genetic , Aptamers, Nucleotide/metabolism , Base Pairing , Base Sequence , Gene Expression Regulation , HEK293 Cells , Humans , Luminescent Proteins/metabolism , Nucleic Acid Conformation , RNA Polymerase III/genetics , Sirolimus/analogs & derivatives , Sirolimus/pharmacology , TOR Serine-Threonine Kinases/antagonists & inhibitors , Red Fluorescent Protein
5.
Nano Lett ; 15(5): 2938-44, 2015 May 13.
Article in English | MEDLINE | ID: mdl-25806671

ABSTRACT

Natural membrane vesicles (MVs) derived from various types of cells play an essential role in transporting biological materials between cells. Here, we show that exogenous compounds are packaged in the MVs by engineering the parental cells via liposomes, and the MVs mediate autonomous intercellular migration of the compounds through multiple cancer cell layers. Hydrophobic compounds delivered selectively to the plasma membrane of cancer cells using synthetic membrane fusogenic liposomes were efficiently incorporated into the membrane of MVs secreted from the cells and then transferred to neighboring cells via the MVs. This liposome-mediated MV engineering strategy allowed hydrophobic photosensitizers to significantly penetrate both spheroids and in vivo tumors, thereby enhancing the therapeutic efficacy. These results suggest that innate biological transport systems can be in situ engineered via synthetic liposomes to guide the penetration of chemotherapeutics across challenging tissue barriers in solid tumors.


Subject(s)
Cell Engineering , Liposomes/chemistry , Neoplasms/drug therapy , Biological Transport , Cell Membrane/chemistry , Humans , Hydrophobic and Hydrophilic Interactions , Liposomes/ultrastructure , Neoplasms/chemistry , Tumor Cells, Cultured
6.
Cell Chem Biol ; 28(11): 1569-1580.e4, 2021 11 18.
Article in English | MEDLINE | ID: mdl-34010626

ABSTRACT

Small molecules can be imaged in living cells using biosensors composed of RNA. However, RNA-based devices are difficult to design. Here, we describe a versatile platform for designing RNA-based fluorescent small-molecule sensors using naturally occurring highly stable three-way junction RNAs. We show that ligand-binding aptamers and fluorogenic aptamers can be inserted into three-way junctions and connected in a way that enables the three-way junction to function as a small-molecule-regulated fluorescent sensor in vitro and in cells. The sensors are designed so that the interhelical stabilizing interactions in the three-way junction are only induced upon ligand binding. We use these RNA-based devices to measure the dynamics of S-adenosylmethionine levels in mammalian cells in real time. We show that this strategy is compatible with diverse metabolite-binding RNA aptamers, fluorogenic aptamers, and three-way junctions. Overall, these data demonstrate a versatile method for readily generating RNA devices that function in living cells.


Subject(s)
Aptamers, Nucleotide/chemistry , Biosensing Techniques , Fluorescent Dyes/chemistry , RNA/genetics , Small Molecule Libraries/chemistry , Aptamers, Nucleotide/metabolism , Female , Fluorescent Dyes/metabolism , HEK293 Cells , HeLa Cells , Humans , Ligands , Small Molecule Libraries/chemical synthesis , Small Molecule Libraries/metabolism
7.
Cell Chem Biol ; 26(12): 1725-1731.e6, 2019 12 19.
Article in English | MEDLINE | ID: mdl-31631009

ABSTRACT

Corn is a fluorogenic RNA aptamer that forms a high-affinity quasi-symmetric homodimer. The Corn dimer interface binds DFHO, resulting in highly photostable yellow fluorescence. Because of its photostability, Corn would be useful in RNA-based small-molecule biosensors, where quantitative accuracy would be affected by photobleaching. Here we describe a strategy for converting the constitutive Corn dimer into a small-molecule-regulated fluorescent biosensor that detects S-adenosylmethionine (SAM) in vitro and in living cells. We fused the Corn aptamer into a helical stem that was engineered by circularly permuting the SAM aptamer from the SAM-III riboswitch. In the absence of SAM, the Corn portion of this fusion RNA is unable to dimerize. However, upon binding SAM, the RNA dimerizes and binds DFHO. This RNA-based biosensor enables detection of SAM dynamics in living mammalian cells. Together, these data describe a class of RNA-based biosensor based on small-molecule-regulated dimerization of Corn.


Subject(s)
Aptamers, Nucleotide/metabolism , Biosensing Techniques/methods , RNA/metabolism , Aptamers, Nucleotide/chemistry , Dimerization , Fluorescent Dyes/chemistry , Fluorescent Dyes/metabolism , HEK293 Cells , Humans , Kinetics , Nucleic Acid Conformation , RNA/chemistry , Riboswitch , S-Adenosylmethionine/chemistry
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