The ability of non-invasive monitoring of deep-tissue developmental, metabolic, and pathogenic processes will advance modern biotechnology. Imaging of live mammals using fluorescent probes is more feasible within a “near-infrared optical window” (NIRW)1. Here we report a phytochrome-based near infra-red fluorescent protein (iRFP) with the excitation/emission maxima at 690/713 nm. Bright fluorescence in a living mouse proved iRFP to be a superior probe for non-invasive imaging of internal mammalian tissues. Its high intracellular stability, low cytotoxicity, and lack of the requirement to add external biliverdin-chromophore makes iRFP as easy to use as conventional GFP-like proteins. Compared to earlier phytochrome-derived fluorescent probes, the iRFP protein has better in vitro characteristics and performs well in cells and in vivo, having greater effective brightness and photostability. Compared to the far-red GFP-like proteins, iRFP has substantially higher signal to background ratio in a mouse model owing to its infra-red shifted spectra.

Genetically encoded fluorescent ribonucleic acids (RNAs) have diverse applications, including imaging RNA trafficking and as a component of RNA-based sensors that exhibit fluorescence upon binding small molecules in live cells. These RNAs include the Spinach and Spinach2 aptamers, which bind and activate the fluorescence of fluorophores similar to that found in green fluorescent protein. Although additional highly fluorescent RNA–fluorophore complexes would extend the utility of this technology, the identification of novel RNA–fluorophore complexes is difficult. Current approaches select aptamers on the basis of their ability to bind fluorophores, even though fluorophore binding alone is not sufficient to activate fluorescence. Additionally, aptamers require extensive mutagenesis to efficiently fold and exhibit fluorescence in living cells. Here we describe a platform for rapid generation of highly fluorescent RNA–fluorophore complexes that are optimized for function in cells. This procedure involves selection of aptamers on the basis of their binding to fluorophores, coupled with fluorescence-activated cell sorting (FACS) of millions of aptamers expressed in Escherichia coli. Promising aptamers are then further optimized using a FACS-based directed evolution approach. Using this approach, we identified several novel aptamers, including a 49-nt aptamer, Broccoli. Broccoli binds and activates the fluorescence of (Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one. Broccoli shows robust folding and green fluorescence in cells, and increased fluorescence relative to Spinach2. This reflects, in part, improved folding in the presence of low cytosolic magnesium concentrations. Thus, this novel fluorescence-based selection approach simplifies the generation of aptamers that are optimized for expression and performance in living cells.

Quantitative measurement of transcription rates in live cells is important for revealing mechanisms of transcriptional regulation. This is particularly challenging for measuring the activity of RNA polymerase III (Pol III), which transcribes growth-promoting small RNAs. To address this, 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. Unlike actinomycin D, we found that 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 measurements of Pol III transcription by direct imaging of Pol III transcripts containing a photostable RNA-fluorophore complex.

SUMMARY 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.

Corn, a 28-nucleotide RNA, induces yellow fluorescence of its cognate ligand (3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime, DFHO) by >1000-fold. It was selected in vitro to overcome limitations of other fluorogenic RNAs, particularly rapid photobleaching. We now report the Corn-DFHO co-crystal structure, discovering that the functional species is a quasisymmetric homodimer. Unusually, the dimer interface, where six unpaired adenosines break overall 2-fold symmetry, lacks any intermolecular base pairs. The homodimer encapsulates one DFHO at its inter-protomer interface, sandwiching it with a G-quadruplex from each protomer. Corn and the green-fluorescent Spinach RNA are structurally unrelated. Their convergent use of G-quadruplexes underscores the usefulness of this motif for RNA-induced small-molecule fluorescence. The asymmetric dimer interface of Corn could form the basis for the development of mutants that only fluoresce as heterodimers. Such variants would be analogous to Split GFP, and may be useful in analyzing RNA co-expression or association, or in designing self-assembling RNA nanostructures.

We report the discovery of a class of abundant circular noncoding RNAs that are produced during metazoan tRNA splicing. These transcripts, termed tRNA intronic circular (tric)RNAs, are conserved features of animal transcriptomes. Biogenesis of tricRNAs requires anciently conserved tRNA sequence motifs and processing enzymes, and their expression is regulated in an agedependent and tissue-specific manner. Furthermore, we exploited this biogenesis pathway to develop an in vivo expression system for generating "designer" circular RNAs in human cells. Reporter constructs expressing RNA aptamers such as Spinach and Broccoli can be used to follow the transcription and subcellular localization of tricRNAs in living cells. Owing to the superior stability of circular vs. linear RNA isoforms, this expression system has a wide range of potential applications, from basic research to pharmaceutical science.

Photoactivatable fluorescent proteins (PAFPs) are required for super-resolution imaging of live cells. Recently, the first red PAFP, PAmCherry1, was reported, which complements the photoactivatable GFP by providing a red super-resolution color. PAmCherry1 is originally ''dark'' but exhibits red fluorescence after UV-violet light irradiation. To define the structural basis of PAmCherry1 photoactivation, we determined its crystal structure in the dark and red fluorescent states at 1.50 Å and 1.65 Å, respectively. The non-coplanar structure of the chromophore in the dark PAmChery1 suggests the presence of an N-acylimine functionality and a single non-oxidized C ␣ -C ␤ bond in the Tyr-67 side chain in the cyclized Met-66-Tyr-67-Gly-68 tripeptide. MS data of the chromophore-bearing peptide indicates the loss of 20 Da upon maturation, whereas tandem MS reveals the C ␣ -N bond in Met-66 is oxidized. These data indicate that PAmCherry1 in the dark state possesses the chromophore N-[(E)-(5-hydroxy-1H-imidazol-2-yl)methylidene]acetamide, which, to our knowledge, has not been previously observed in PAFPs. The photoactivated PAmCherry1 exhibits a non-coplanar anionic DsRed-like chromophore but in the trans configuration. Based on the crystallographic analysis, MS data, and biochemical analysis of the PAmCherry1 mutants, we propose the detailed photoactivation mechanism. In this mechanism, the excited-state PAmCherry1 chromophore acts as the oxidant to release CO2 molecule from Glu-215 via a Koble-like radical reaction. The Glu-215 decarboxylation directs the carbanion formation resulting in the oxidation of the Tyr-67 C ␣ -C ␤ bond. The double bond extends the -conjugation between the phenolic ring of Tyr-67, the imidazolone, and the N-acylimine, resulting in the red fluorescent chromophore.chromophore ͉ localization microscopy ͉ photoconversion S uper-resolution imaging approaches such as photoactivation localization microscopy provide the ability to observe details of cellular and even macromolecular structure that were not previously discernible with less than 40 nm resolution (1). There is significant demand for a broader and more diverse range of photoactivatable fluorescent probes (2), in particular irreversibly photoactivatable fluorescent proteins (PAFPs). To develop PAFPs with new spectral properties, it is essential to gain understanding of the underlying mechanisms of photoactivation.X-ray structures have been reported for a number of irreversible PAFPs, including those that change their fluorescence from green to red upon irradiation with violet light such as EosFP (3), its derivative IrisFP (4), Kaede (5), KikGR (6), and Dendra2 (7). These PAFPs share the same His-Tyr-Gly chromophore-forming tripeptide and the same mechanism of photoactivation. This mechanism is associated with a ␤-elimination reaction, which results in the cleavage of the peptide bond between the amide nitrogen and ␣-carbon of the His residue in the chromophore tripeptide (8).Another group of irreversible PAFPs includes photoactivatable GFP (P...

Skin‐deep: The combination of a near‐infrared fluorescent protein (iRFP) and deep‐tissue photoacoustic tomography clearly demonstrates the superiority of iRFP over other genetically encoded probes. Impressive resolution (280 μm lateral and 75 μm axial) was obtained at a depth of 4 mm in a live animal, and volumetric images of a tumor were produced (see image), thus allowing the spatially resolved monitoring of its development.