Fluorescent RNA Probes Research Articles

Development of a RNA-protein complex based smart drug delivery system for 9-hydroxycamptothecin

Camptothecin (CPT) is a monoterpenoid indole alkaloid with a wide spectrum of anticancer activity. However, its application is hindered by poor solubility, lack of targeting specificity, and severe side effects. Structural derivatization of CPT and the development of suitable drug delivery systems are potential strategies for addressing these issues. In this study, we discovered that the protein Cytochrome P450 Family 1 Subfamily A Member 1 (CYP1A1) from Homo sapiens catalyzes CPT to yield 9-hydroxycamptothecin (9-HCPT), which exhibits increased water solubility and cytotoxicity. We then created a RNA-protein complex based drug delivery system with enzyme and pH responsiveness and improved the targeting and stability of the nanomedicine through protein module assembly. The subcellular localization of nanoparticles can be visualized using fluorescent RNA probes. Our results not only identified the protein CYP1A1 responsible for the structural derivatization of CPT to synthesize 9-HCPT but also offered potential strategies for enhancing the utilization of silk-based drug delivery systems in tumor therapy.

Split Luciferase-Fragment Reconstitution for Unveiling RNA Localization and Dynamics in Live Cells.

The intracellular distribution and dynamics of RNAs play pivotal roles in various physiological phenomena. The ability to monitor the amount and localization of endogenous RNAs in living cells allows for elucidating the mechanisms of various intracellular events. Protein-based fluorescent RNA probes are now widely used to visualize and analyze RNAs in living cells. However, continuously monitoring the temporal changes in RNA localization and dynamics in living cells is challenging. In this study, we developed a bioluminescent probe for spatiotemporal monitoring of RNAs in living cells by using a split-luciferase reconstitution technique. The probe consists of split fragments of a bioluminescent protein, NanoLuc, connected with RNA-binding protein domains generated from a custom-made mutation of a PUM-HD. The probe showed rapid luminescence intensity changes in response to an increase or decrease in the amount of a target RNA in vitro. In live-cell imaging, temporal alteration of the intracellular distribution of endogenous β-actin mRNA was visualized in response to extracellular stimulation. Furthermore, the application of the probe to the visualization of the specific localization of β-actin mRNA in primary hippocampal neurons was conducted. These results demonstrate the capability of the bioluminescent RNA probe to monitor the changes in localization, dynamics, and the amount of target RNA in living cells.

Ethylenediamine derivatives efficiently react with oxidized RNA 3' ends providing access to mono and dually labelled RNA probes for enzymatic assays and in vivo translation.

Development of RNA-based technologies relies on the ability to detect, manipulate, and modify RNA. Efficient, selective and scalable covalent modification of long RNA molecules remains a challenge. We report a chemical method for modification of RNA 3′-end based on previously unrecognized superior reactivity of N-substituted ethylenediamines in reductive amination of periodate-oxidized RNA. Using this method, we obtained fluorescently labelled or biotinylated RNAs varying in length (from 3 to 2000 nt) and carrying different 5′ ends (including m7G cap) in high yields (70–100% by HPLC). The method is scalable (up to sub-milligrams of mRNA) and combined with label-facilitated HPLC purification yields highly homogeneous products. The combination of 3′-end labelling with 5′-end labelling by strain-promoted azide-alkyne cycloaddition (SPAAC) afforded a one-pot protocol for site-specific RNA bifunctionalization, providing access to two-colour fluorescent RNA probes. These probes exhibited fluorescence resonance energy transfer (FRET), which enabled real-time monitoring of several RNA hydrolase activities (RNase A, RNase T1, RNase R, Dcp1/2, and RNase H). Dually labelled mRNAs were efficiently translated in cultured cells and in zebrafish embryos, which combined with their detectability by fluorescent methods and scalability of the synthesis, opens new avenues for the investigation of mRNA metabolism and the fate of mRNA-based therapeutics.

Visualization of subdiffusive sites in a live single cell.

We measured anomalous diffusion in human prostate cancer cells which were transfected with the Alexa633 fluorescent RNA probe and co-transfected with enhanced green fluorescent protein-labeled argonaute2 protein by laser scanning microscopy. The image analysis arose from diffusion based on a “two-level system”. A trap was an interaction site where the diffusive motion was slowed down. Anomalous subdiffusive spreading occurred at cellular traps. The cellular traps were not immobile. We showed how the novel analysis method of imaging data resulted in new information about the number of traps in the crowded and heterogeneous environment of a single human prostate cancer cell. The imaging data were consistent with and explained by our modern ideas of anomalous diffusion of mixed origins in live cells. Our original research presented in this study is significant as we obtained a complex diffusion mechanism in live single cells.

Dynamically Long-Term Imaging of Cellular RNA by Fluorescent Carbon Dots with Surface Isoquinoline Moieties and Amines.

Cellular RNA dynamics are closely associated with a vast range of physiological processes that are mostly long-lasting. To uncover the association between RNA dynamics and these processes, fluorescent RNA probes with high specificity, photostability, and biocompatibility are compulsory. Herein, a series of fluorescent carbon dots (CDs) have been prepared by one-pot hydrothermal treatment of o-, m-, or p-phenylenediamines with triethylenetetramine. Only CDs derived from the meta precursor ( m-CDs) with excellent photostability and biocompatibility can specifically bind to cellular RNA, allowing successfully long-term (up to 3 days) monitoring of RNA dynamics during cell apoptosis, mitosis, and proliferation. This RNA affinity can be attributed to the isoquinoline moieties and amines on the surface of m-CDs, which can bind to RNA through π-π stacking and electrostatic bonding, respectively. The cellular internalization of m-CDs is time-, temperature-, ATP-, caveolar, and microtubule-dependent. Additionally, investigations on the in vivo behavior of m-CD suggest that they can be efficiently and rapidly excreted from the zebrafish larvae body after 48 h. Our results provide a powerful tool for clarifying complex relationships between RNA dynamics and basic biological processes, disease development, or drug interactions.

A ratiometric fluorescent composite nanomaterial for RNA detection based on graphene quantum dots and molecular probes.

RNA plays a central role in controlling cellular functions. Research of the content and distribution of RNA in living cells is of great significance to both biochemistry and biomedicine. However, ratiometric fluorescent probes for the detection of RNA in the cytoplasm and nucleoli are still rarely reported. We herein present the first example of a novel ratiometric fluorescent composite nanomaterial by using graphene quantum dots (GQDs) and a fluorescent probe molecule for the sensitive and selective detection of RNA. HVC-6 was selected as the detection group. The fluorescence was excited at 365 nm and the fluorescence emission at 470 and 610 nm increased gradually with the addition of RNA. The fluorescence intensity ratio of I610/I470 displayed a linear response to RNA. Furthermore, the developed nanomaterial HVC-6@GQDs showed potential for utilization as a fluorescent RNA probe in living cells.

Synthesis of Wavelength-Shifting Fluorescent DNA and RNA with Two Photostable Cyanine-Styryl Dyes as the Base Surrogate Pair.

Two nucleic acid building blocks were synthesized, consisting of two photostable green‐ and red‐emitting cyanine–styryl dyes and (S)‐3‐amino‐1,2‐propanediol as a substitute for the ribofuranoside, and incorporated as base‐pair surrogates by using automated phosphoramidte chemistry in the solid phase. The optical properties and, in particular, the energy‐transfer properties were screened in a range of DNA duplexes, in which the “counter bases” of the two dyes were varied and the distance between the two dyes was enlarged to up to three intervening adenosine–thymidine pairs. The DNA duplex with the best optical properties and the best red/green emission ratio as the readout bore adenosine and thymidine opposite to the dyes, and the two dyes directly adjacent to each other as the base surrogate pair. This structural arrangement can be transferred to RNA to obtain similarly fluorescent RNA probes. Representatively, the positively evaluated DNA duplex was applied to verify the fluorescence readout in living HeLa cells by using fluorescence confocal microscopy.

Spatiotemporal analysis with a genetically encoded fluorescent RNA probe reveals TERRA function around telomeres.

Telomeric repeat-containing RNA (TERRA) controls the structure and length of telomeres through interactions with numerous telomere-binding proteins. However, little is known about the mechanism by which TERRA regulates the accessibility of the proteins to telomeres, mainly because of the lack of spatiotemporal information of TERRA and its-interacting proteins. We developed a fluorescent probe to visualize endogenous TERRA to investigate its dynamics in living cells. Single-particle fluorescence imaging revealed that TERRA accumulated in a telomere-neighboring region and trapped diffusive heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), thereby inhibiting hnRNPA1 localization to the telomere. These results suggest that TERRA regulates binding of hnRNPA1 to the telomere in a region surrounding the telomere, leading to a deeper understanding of the mechanism of TERRA function.

A two-photon fluorescent RNA probe screened from a series of oxime-functionalized 2,2':6',2''-terpyridine ZnX2 (X = Cl, Br, I) complexes.

Imaging of RNA in living cells is a potential tool to understand intracellular RNA function. Therefore, an effective two-photon fluorescent probe with a reasonable two-photon action cross-section to label RNA is now urgently required. In this work, a series of novel two-photon absorbing terpyridine ZnX2 (X = Cl, Br, I) complexes have been designed and an effective RNA imaging probe has been obtained. The results revealed that OTP-ZnCl2 possesses large Stokes shift and two-photon action cross-section. Furthermore, live cell imaging experiments indicated that OTP-ZnCl2 could stain nucleoli in living cells by binding with nucleoli RNA. The mechanism of selective nucleoli staining of OTP-ZnCl2 was studied systematically via both experiments and molecular modeling calculations. Due to its low cytotoxicity, good membrane permeability and counterstain compatibility with the commercial fluorescent nucleic acid dye Hoechst 33342, as well as its ability to label RNA in living cells, OTP-ZnCl2 is a promising candidate for the detection of nucleic acid in living cells.

Photophysical processes of the spectroscopic RNA probe 2-(1-ethynylpyrene)-adenosine (PyA)

We examine the photoinduced excited state dynamics of pyrene modified adenosine, a versatile probe for folding and hybridization of ribonucleic acids. Measurements in different solvents revealed complex ultrafast dynamics, but high robustness since the overall fluorescence quantum yield (Φ f ) is hardly affected. The result is a strong fluorescent RNA-probe whose spectral properties change in a defined way upon environmental changes.

In situ assessment of active Thiobacillus species in corroding concrete sewers using fluorescent RNA probes

Culture-dependent studies have implicated sulfur-oxidizing bacteria as the causative agents of concrete corrosion in sanitary sewers. Thiobacillus species are often considered the major representative of the acid-producing bacteria in these environments, and members of the genus Acidiphilium have been implicated to support their growth. Active populations of selected Thiobacillus, Leptospirillum, and Acidiphilium species were compared to total bacterial populations growing on the surfaces of corroding concrete using three oligonucleotide probes that have been confirmed to recognize unique sequences of 16S rRNA in the following acidophilic bacteria: Thiobacillus ferrooxidans and Thiobacillus thiooxidans (probe: Thio820), Leptospirilium ferrooxidans (Probe: Lept581) and members of the genus Acidiphilium (probe: Acdp821). With these genetic probes, fluorescent in situ hybridizations (FISH) were used to identify and enumerate selected bacteria in homogenized biofilm samples taken from the corroding crowns of concrete sewer collection systems operating in Houston, Texas, USA. Direct epifluorescent microscopy demonstrated the ability of FISH to identify significant numbers of active acidophilic bacteria among concrete particles, products of concrete corrosion (e.g. CaSO 4), and other mineral debris. As judged by FISH analyses with the species-specific probe Thio820, and a domain-level probe that recognizes all Bacteria (Eub338), T. ferrooxidans and T. thiooxidans comprised between 12% and 42% of the total active Bacteria present in corroding concrete samples. Although both Acidiphilium and Leptospirillum have also been postulated to have ecological significance in acidic sulfur-oxidizing environments, neither genera was detected using genus-specific probes (Lept581 and Acdp821).

Fluorochrome-labeled RNA as a sensitive, strand-specific probe for direct fluorescence in situ hybridization.

Detection of target RNA by in situ hybridization (ISH) in the classic and confocal fluorescence microscope was performed using strand-specific single-stranded RNA probes labeled directly with the fluorochromes fluorescein isothiocyanate or Texas red. The probes, produced by in vitro transcription from PCR-generated templates with T7 RNA polymerase and fluorochromized UTP, gave ISH signals directly visible by fluorescence microscopy without the use of any immunological detection step. In avoiding antibodies, it was possible to strongly increase the sensitivity of the ISH since antibodies may contain RNase which can reduce hybridization signals considerably, even beyond the detection limit. Fluorescent RNA probes thus allowed for the detection of low numbers of target molecules per cell, such as minus strand intermediates in picornavirus RNA replication. Using appropriate denaturing conditions, the targets could be visualized in a double-stranded configuration as well as in the presence of a 100-fold excess of complementary RNA. Furthermore, double ISH for the simultaneous detection of two different RNA species, such as plus and minus strand RNA of poliovirus, or of different regions of the viral genomic RNA was possible with appropriate fluorescent strand-specific probes labeled with different fluorochromes. Combination of ISH and immunofluorescence was found feasible if RNA was present in relatively large amounts. In addition to the investigation of virus replication, possible applications of fluorochromized RNA probes might include antisense RNA detection as well as plant virus resistance and gene silencing.

Flow cytometric detection of yeast by in situ hybridization with a fluorescent ribosomal RNA probe

An in situ hybridization procedure with a fluorescent RNA probe applied to yeast detection by flow cytometry is described. Saccharomyces ceerevisiae cell were permeabilized to allow the use of a biotinylated rRNA probe of about ≈ 100 nucleotides, by formaldehyde treatment, followed by a limited enzymatic hydrolysis of the cell wall. Hybridization was performed with cells in suspension and hybrids were detected with streptavidin-FITC (fluorescein isothiocyanate) by microscopy and flow cytometry. The signal obtained with yeast antisense rRNA probe was 30 times higher than the nonspecific signal obtained with a bacterial sense rRNA probe. The yeast-specific signal was not eliminated after posthybridization RNase treatment althoug it completely disappeared when cells were treated with RNase before hybirdization, confirming the rRNA was the target. This study is the first demonstration of the use of in situ hybridization for yeast detection by flow cytomertry. With appropriate probes, the procedure could be used also for detection of specific yeast populations.