GFP and its derivatives revolutionized the study of proteins. Spinach is a recently reported in v... more GFP and its derivatives revolutionized the study of proteins. Spinach is a recently reported in vitro-evolved RNA mimic of GFP, which as genetically encoded fusions makes possible live-cell, real-time imaging of biological RNAs without resorting to large RNA-binding protein-GFP fusions. To elucidate the molecular basis of Spinach fluorescence, we solved the cocrystal structure of Spinach bound to its cognate exogenous chromophore, showing that Spinach activates the small molecule by immobilizing it between a base triple, a G-quadruplex and an unpaired G. Mutational and NMR analyses indicate that the G-quadruplex is essential for Spinach fluorescence, is also present in other fluorogenic RNAs and may represent a general strategy for RNAs to induce fluorescence of chromophores. The structure guided the design of a miniaturized 'Baby Spinach', and it provides a foundation for structure-driven design and tuning of fluorescent RNAs.
Genetically encoded fluorescent sensors can be valuable tools for studying the abundance and flux... more Genetically encoded fluorescent sensors can be valuable tools for studying the abundance and flux of molecules in living cells. We recently developed a novel class of sensors composed of RNAs that can be used to detect diverse small molecules and untagged proteins. These sensors are based on Spinach, an RNA mimic of GFP, and they have successfully been used to image several metabolites and proteins in living bacteria. Here we discuss the generation and optimization of these Spinach-based sensors, which, unlike most currently available genetically encoded reporters, can be readily generated to any target of interest. We also provide a detailed protocol for imaging ADP dynamics in living Escherichia coli after a change from glucose-containing medium to other carbon sources. The entire procedure typically takes ∼4 d including bacteria transformation and image analysis. The majority of this protocol is applicable to sensing other metabolites and proteins in living bacteria.
RNA aptamers can be expressed in cells to influence and image cellular processes. Aptamer folding... more RNA aptamers can be expressed in cells to influence and image cellular processes. Aptamer folding is maintained by inserting the aptamers into highly structured RNA scaffolds. Here, we show that commonly used RNA scaffolds exhibit unexpected instability and cleavage in bacterial and mammalian cells. Using an in-gel staining approach for rapid and simple detection of Spinach- or Broccoli-tagged RNAs in cells, we monitored the processing of RNAs tagged with scaffolded aptamers, revealing endonucleolytic cleavage, RNA instability, and poor expression. We reengineered a natural three-way junction structure to generate an alternative scaffold that enables stable aptamer expression in cells. This scaffold was used to create cassettes containing up to four Broccoli units, markedly enhancing the brightness of mammalian cells expressing cassette-tagged RNAs. These experiments describe methods for screening RNA cleavage events in cells and identify cell-compatible scaffolds that enable efficient tagging of RNAs with aptamers for cellular expression.
GFP and its derivatives revolutionized the study of proteins. Spinach is a recently reported in v... more GFP and its derivatives revolutionized the study of proteins. Spinach is a recently reported in vitro-evolved RNA mimic of GFP, which as genetically encoded fusions makes possible live-cell, real-time imaging of biological RNAs without resorting to large RNA-binding protein-GFP fusions. To elucidate the molecular basis of Spinach fluorescence, we solved the cocrystal structure of Spinach bound to its cognate exogenous chromophore, showing that Spinach activates the small molecule by immobilizing it between a base triple, a G-quadruplex and an unpaired G. Mutational and NMR analyses indicate that the G-quadruplex is essential for Spinach fluorescence, is also present in other fluorogenic RNAs and may represent a general strategy for RNAs to induce fluorescence of chromophores. The structure guided the design of a miniaturized 'Baby Spinach', and it provides a foundation for structure-driven design and tuning of fluorescent RNAs.
Several long-wavelength (365 nm) photoactivatable diaryltetrazoles were discovered by screening a... more Several long-wavelength (365 nm) photoactivatable diaryltetrazoles were discovered by screening a series of substituted diaryltetrazoles and subsequently showed excellent reactivity in the photoactivated 1,3-dipolar cycloaddition reactions toward electron-deficient and conjugated alkenes in organic solvents as well as an alkene-containing protein in the aqueous buffer.
Genetically encoded fluorescent sensors can be valuable tools for studying the abundance and flux... more Genetically encoded fluorescent sensors can be valuable tools for studying the abundance and flux of molecules in living cells. We recently developed a novel class of sensors composed of RNAs that can be used to detect diverse small molecules and untagged proteins. These sensors are based on Spinach, an RNA mimic of GFP, and they have successfully been used to image several metabolites and proteins in living bacteria. Here we discuss the generation and optimization of these Spinach-based sensors, which, unlike most currently available genetically encoded reporters, can be readily generated to any target of interest. We also provide a detailed protocol for imaging ADP dynamics in living Escherichia coli after a change from glucose-containing medium to other carbon sources. The entire procedure typically takes ∼4 d including bacteria transformation and image analysis. The majority of this protocol is applicable to sensing other metabolites and proteins in living bacteria.
GFP and its derivatives revolutionized the study of proteins. Spinach is a recently reported in v... more GFP and its derivatives revolutionized the study of proteins. Spinach is a recently reported in vitro-evolved RNA mimic of GFP, which as genetically encoded fusions makes possible live-cell, real-time imaging of biological RNAs without resorting to large RNA-binding protein-GFP fusions. To elucidate the molecular basis of Spinach fluorescence, we solved the cocrystal structure of Spinach bound to its cognate exogenous chromophore, showing that Spinach activates the small molecule by immobilizing it between a base triple, a G-quadruplex and an unpaired G. Mutational and NMR analyses indicate that the G-quadruplex is essential for Spinach fluorescence, is also present in other fluorogenic RNAs and may represent a general strategy for RNAs to induce fluorescence of chromophores. The structure guided the design of a miniaturized 'Baby Spinach', and it provides a foundation for structure-driven design and tuning of fluorescent RNAs.
Genetically encoded fluorescent sensors can be valuable tools for studying the abundance and flux... more Genetically encoded fluorescent sensors can be valuable tools for studying the abundance and flux of molecules in living cells. We recently developed a novel class of sensors composed of RNAs that can be used to detect diverse small molecules and untagged proteins. These sensors are based on Spinach, an RNA mimic of GFP, and they have successfully been used to image several metabolites and proteins in living bacteria. Here we discuss the generation and optimization of these Spinach-based sensors, which, unlike most currently available genetically encoded reporters, can be readily generated to any target of interest. We also provide a detailed protocol for imaging ADP dynamics in living Escherichia coli after a change from glucose-containing medium to other carbon sources. The entire procedure typically takes ∼4 d including bacteria transformation and image analysis. The majority of this protocol is applicable to sensing other metabolites and proteins in living bacteria.
RNA aptamers can be expressed in cells to influence and image cellular processes. Aptamer folding... more RNA aptamers can be expressed in cells to influence and image cellular processes. Aptamer folding is maintained by inserting the aptamers into highly structured RNA scaffolds. Here, we show that commonly used RNA scaffolds exhibit unexpected instability and cleavage in bacterial and mammalian cells. Using an in-gel staining approach for rapid and simple detection of Spinach- or Broccoli-tagged RNAs in cells, we monitored the processing of RNAs tagged with scaffolded aptamers, revealing endonucleolytic cleavage, RNA instability, and poor expression. We reengineered a natural three-way junction structure to generate an alternative scaffold that enables stable aptamer expression in cells. This scaffold was used to create cassettes containing up to four Broccoli units, markedly enhancing the brightness of mammalian cells expressing cassette-tagged RNAs. These experiments describe methods for screening RNA cleavage events in cells and identify cell-compatible scaffolds that enable efficient tagging of RNAs with aptamers for cellular expression.
GFP and its derivatives revolutionized the study of proteins. Spinach is a recently reported in v... more GFP and its derivatives revolutionized the study of proteins. Spinach is a recently reported in vitro-evolved RNA mimic of GFP, which as genetically encoded fusions makes possible live-cell, real-time imaging of biological RNAs without resorting to large RNA-binding protein-GFP fusions. To elucidate the molecular basis of Spinach fluorescence, we solved the cocrystal structure of Spinach bound to its cognate exogenous chromophore, showing that Spinach activates the small molecule by immobilizing it between a base triple, a G-quadruplex and an unpaired G. Mutational and NMR analyses indicate that the G-quadruplex is essential for Spinach fluorescence, is also present in other fluorogenic RNAs and may represent a general strategy for RNAs to induce fluorescence of chromophores. The structure guided the design of a miniaturized 'Baby Spinach', and it provides a foundation for structure-driven design and tuning of fluorescent RNAs.
Several long-wavelength (365 nm) photoactivatable diaryltetrazoles were discovered by screening a... more Several long-wavelength (365 nm) photoactivatable diaryltetrazoles were discovered by screening a series of substituted diaryltetrazoles and subsequently showed excellent reactivity in the photoactivated 1,3-dipolar cycloaddition reactions toward electron-deficient and conjugated alkenes in organic solvents as well as an alkene-containing protein in the aqueous buffer.
Genetically encoded fluorescent sensors can be valuable tools for studying the abundance and flux... more Genetically encoded fluorescent sensors can be valuable tools for studying the abundance and flux of molecules in living cells. We recently developed a novel class of sensors composed of RNAs that can be used to detect diverse small molecules and untagged proteins. These sensors are based on Spinach, an RNA mimic of GFP, and they have successfully been used to image several metabolites and proteins in living bacteria. Here we discuss the generation and optimization of these Spinach-based sensors, which, unlike most currently available genetically encoded reporters, can be readily generated to any target of interest. We also provide a detailed protocol for imaging ADP dynamics in living Escherichia coli after a change from glucose-containing medium to other carbon sources. The entire procedure typically takes ∼4 d including bacteria transformation and image analysis. The majority of this protocol is applicable to sensing other metabolites and proteins in living bacteria.
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Papers by Wenjiao Song