Identification of proteins interacting with lncRNA Braveheart in mouse using protein microarray
ABSTRACT: The mouse lncRNA Braveheart (Bvht) as a non-coding transcript has been found to act in trans to regulate cardiovascular lineage commitment. However, the mechanism of Bvht action is still not clear. lncRNAs have been shown to regulate gene expression though cooperating with protein partners. Recently, we experimentally determine the secondary structure of Bvht containing a novel structural motif AGIL. AGIL motif deletion (BvhtdAGIL) in mouse embryonic stem cells prevents the transition from mesoderm cells to cardiac progenitors. To identify proteins that interact with the Bvht AGIL motif, we used a human protein microarray platform (Human ProtoArray, Life Technology). Full-length Bvht and BvhtdAGIL transcripts were generated by in vitro transcription and labeled with Cy5. 50pmol Cy5-labeled RNAs were individually incubated with the protein microarray.
Project description:Despite significant advances on fluorescent labeling of target proteins to study their structural dynamics and function, there has been need for labeling with high quantum yield ensuring high sensitivity and selectivity without sacrificing the biological function of the protein. Here as a technical advancement over non-canonical amino acid incorporation, we provided a conceptual design of the N-terminal fluorescent tagging of proteins. Cy5-labeled methionine (Cy5-Met) was chemically synthesized, and then the purified Cy5-Met was coupled with synthetic human initiator tRNA by methionine tRNA synthetase. Cy5-Met-initiator tRNA (Cy5-Met-tRNAi) was purified and transfected into HeLa cells with HIV-Tat plasmid, resulting in an efficient production of Cy5-labeled HIV-Tat protein. Based on the universal requirement in translational initiation, the approach provides co-translational incorporation of N-terminal probe to a repertoire of proteins in the eukaryote system. This methodology has potential utility in the single molecule analysis of human proteins in vitro and in vivo for addressing to their complex biological structural and functional dynamics.
Project description:Single-molecule fluorescence microscopy is a powerful tool for observing biomolecular interactions with high spatial and temporal resolution. Detecting fluorescent signals from individual labeled proteins above high levels of background fluorescence remains challenging, however. For this reason, the concentrations of labeled proteins in in vitro assays are often kept low compared to their in vivo concentrations. Here, we present a new fluorescence imaging technique by which single fluorescent molecules can be observed in real time at high, physiologically relevant concentrations. The technique requires a protein and its macromolecular substrate to be labeled each with a different fluorophore. Making use of short-distance energy-transfer mechanisms, only the fluorescence from those proteins that bind to their substrate is activated. This approach is demonstrated by labeling a DNA substrate with an intercalating stain, exciting the stain, and using energy transfer from the stain to activate the fluorescence of only those labeled DNA-binding proteins bound to the DNA. Such an experimental design allowed us to observe the sequence-independent interaction of Cy5-labeled interferon-inducible protein 16 with DNA and the sliding via one-dimensional diffusion of Cy5-labeled adenovirus protease on DNA in the presence of a background of hundreds of nanomolar Cy5 fluorophore.
Project description:For successful design of a nanoparticulate drug delivery system, the fate of the carrier and cargo need to be followed. In this work, we fluorescently labeled poly(n-butylcyanoacrylate) (PBCA) nanocapsules as a shell and separately an oligonucleotide (20 mer) as a payload. The nanocapsules were formed by interfacial anionic polymerization on aqueous droplets generated by an inverse miniemulsion process. After uptake, the PBCA capsules were shown to be round-shaped, endosomal structures and the payload was successfully released. Cy5-labeled oligonucleotides accumulated at the mitochondrial membrane due to a combination of the high mitochondrial membrane potential and the specific molecular structure of Cy5. The specificity of this accumulation at the mitochondria was shown as the uncoupler dinitrophenol rapidly diminished the accumulation of the Cy5-labeled oligonucleotide. Importantly, a fluorescence resonance energy transfer investigation showed that the dye-labeled cargo (Cy3/Cy5-labeled oligonucleotides) reached its target site without degradation during escape from an endosomal compartment to the cytoplasm. The time course of accumulation of fluorescent signals at the mitochondria was determined by evaluating the colocalization of Cy5-labeled oligonucleotides and mitochondrial markers for up to 48 hours. As oligonucleotides are an ideal model system for small interfering RNA PBCA nanocapsules demonstrate to be a versatile delivery platform for small interfering RNA to treat a variety of diseases.
Project description:Typical DNA microarrays utilize diffusion of dye-labeled cDNA probes followed by sequence-specific hybridization to immobilized targets. Here we experimentally estimated the distance typical probes travel during static 16-h hybridizations. Probes labeled with Cy3 and Cy5 were individually introduced to opposite sides of a microarray with minimal convective mixing. Oppositely labeled probes diffused across the initial front separating the two solutions, generating a zone with both dyes present. Diffusion-distance estimates for Cy3- and Cy5-labeled cDNAs were 3.8 mm and 2.6 mm, respectively, despite having almost identical molecular masses. In separate 16-h hybridization experiments with oppositely labeled probes premixed, arrays that were continuously mixed had 15-20% higher signal intensities than arrays hybridized statically. However, no change was observed in the Cy3/Cy5 signal intensity ratio between continuously mixed and static hybridizations. This suggests that the observed dye bias in diffusion-distance estimates results from differences in the detection limits of Cy3 and Cy5-labeled cDNA, a potential concern for array data on low-abundance transcripts. Our conservative diffusion-distance estimates indicate that replicate targets >7.6 mm apart will not compete for scarce probes. Also, raising the microarray gap height would delay the onset of diffusion-limited hybridization by increasing the amount of available probe.
Project description:We employ single-molecule fluorescence resonance energy transfer (smFRET) to study structural dynamics over the first two elongation cycles of protein synthesis, using ribosomes containing either Cy3-labeled ribosomal protein L11 and A- or P-site Cy5-labeled tRNA or Cy3- and Cy5-labeled tRNAs. Pretranslocation (PRE) complexes demonstrate fluctuations between classical and hybrid forms, with concerted motions of tRNAs away from L11 and from each other when classical complex converts to hybrid complex. EF-G?GTP binding to both hybrid and classical PRE complexes halts these fluctuations prior to catalyzing translocation to form the posttranslocation (POST) complex. EF-G dependent translocation from the classical PRE complex proceeds via transient formation of a short-lived hybrid intermediate. A-site binding of either EF-G to the PRE complex or of aminoacyl-tRNA?EF-Tu ternary complex to the POST complex markedly suppresses ribosome conformational lability.
Project description:Transcriptional profile of Streptococcus pyogenes stk mutant strain JRS2516 vs its wild type parent strain MGAS2221 The RNA prepared from triplicate cultures for the wild type and the stk mutant strains was each labeled with Cy3 and hybridized to duplicate arrays. Reference RNA consisted of pooled RNA for the wild type and stk mutant, labeled with Cy5.
Project description:A multicolor fluorescence imaging device was recently developed for image-guided surgery. However, conventional systems are typically bulky and function with two cameras. To overcome these issues, we developed an economical home-built fluorescence imaging device based on a single RGB-IR sensor that can acquire both color and fluorescence images simultaneously. The technical feasibility of RGB-IR imaging was verified ex vivo in chicken breast tissue using fluorescein isothiocyanate (FITC), cyanine 5 (Cy5), and indocyanine green (ICG) as fluorescent agents. The minimum sensitivities for FITC, Cy5, and ICG were 0.200 µM, 0.130 µM, and 0.065 µM, respectively. In addition, we validated the fluorescence imaging of this device in vitro during a minimally invasive procedure using smURFP-labeled probiotics, which emit a spectrum similar to that of Cy5. Our preliminary study of the ex vivo tissue suggests that Cy5 and ICG are good candidates for deep tissue imaging. In addition, the tumor-specific amplification process was visualized using cancer cells incubated with probiotics that had been labeled with a fluorescent protein. Our approach indicates the potential for in vivo screening of tumors in rodent tumor models.
Project description:Hybrid tracers containing both fluorescent and radioactive imaging labels have demonstrated clinical potential during sentinel lymph node procedures. To combine these two labels on a single targeting vector that allows tumor-targeted imaging, end-labeling strategies are often applied. For ?v?3-integrin-targeting hybrid tracers, providing an excellent model for evaluating tracer development strategies, end-labeling-based synthesis provides a rather cumbersome synthesis strategy. Hence, the aim of this study was to investigate the use of heterobifunctional cyanine dyes in a click-chemistry-based synthesis strategy for RGD-based hybrid tracers. The triazole-based hybrid tracers DTPA.DBCO.N 3 (SO 3 )-Cy5-c[RGDyK] and DTPA.BCN.N 3 (SO 3 )-Cy5-c[RGDyK] were obtained in fewer steps than DTPA-Lys(Cy5(SO 3 )methyl)-Cys-c[RGDyK] and had partition coefficients of log?P (o/w) = -2.55 ± 0.10, -1.45 ± 0.03, and -2.67 ± 0.12, respectively. Both tracers were chemically stable, and the brightnesses of DTPA.DBCO.N 3 (SO 3 )-Cy5-c[RGDyK] and DTPA.BCN.N 3 (SO 3 )-Cy5-c[RGDyK] were, respectively, 23 × 103 and 40 × 103 M-1 cm-1; lower than that of the reference tracer DTPA-Lys(Cy5(SO 3 )methyl)-Cys-c[RGDyK] (50 × 103 M-1 cm-1). Assessment of serum protein binding revealed no statistically significant difference (44 ± 2 and 40 ± 2% bound for DTPA.DBCO.N 3 (SO 3 )-Cy5-c[RGDyK] and DTPA.BCN.N 3 (SO 3 )-Cy5-c[RGDyK], respectively; 36 ± 5% bound for DTPA-Lys(Cy5(SO 3 )methyl)-Cys-c[RGDyK]; p > 0.05). DTPA.DBCO.N 3 (SO 3 )-Cy5-c[RGDyK] (K D = 17.5 ± 6.0) had a statistically significantly higher affinity than the reference compound DTPA-Lys(Cy5(SO 3 )methyl)-Cys-c[RGDyK] (K D = 30.3 ± 5.7; p < 0.0001), but DTPA.BCN.N 3 (SO 3 )-Cy5-c[RGDyK] had a statistically significantly lower affinity (K D = 76.5 ± 18.3 nM; p < 0.0001). Both [ 111 In]DTPA.DBCO.N 3 (SO 3 )-Cy5-c[RGDyK] and [ 111 In]DTPA.BCN.N 3 (SO 3 )-Cy5-c[RGDyK] enabled in vivo visualization of the 4T1 tumor via fluorescence and single-photon emission computed tomography (SPECT) imaging. Biodistribution data (% ID/g) revealed a significant increase in nonspecific uptake in the kidney, liver, and muscle for both [ 111 In]DTPA.DBCO.N 3 (SO 3 )-Cy5-c[RGDyK] and [ 111 In]DTPA.BCN.N 3 (SO 3 )-Cy5-c[RGDyK]. As a result of the higher background activity, the tumor-to-background ratio of the click-labeled RGD analogues was twofold lower compared to the end-labeled reference compound. The use of click chemistry labeling did not yield a pronounced negative effect on serum protein binding, in vitro stability, and receptor affinity; and tumors could still be visualized using SPECT and fluorescence imaging. However, quantitative in vivo biodistribution data suggest that the triazole and strained cyclooctyne moieties associated with this type of click chemistry negatively influence the pharmacokinetics of RGD peptides. Nevertheless, the design might still hold promise for other targets/targeting moieties.
Project description:Two dimensional-fluorescence difference gel electrophoresis (2D DIGE) is an emerging technique for comparative proteomics, which improves the reproducibility and reliability of differential protein expression analysis between samples. The purpose of this study was to investigate bovine pregnancy-specific proteins in the proteome between bovine pregnant and non-pregnant serum using DIGE technique. Serums of 2 pregnant Holstein dairy cattle at day 21 after artificial insemination and those of 2 non-pregnant were used in this study. The pre-electrophoretic labeling of pregnant and non-pregnant serum proteins were mixed with Cy3 and Cy5 fluorescent dyes, respectively, and an internal standard was labeled with Cy2. Labeled proteins with Cy2, Cy3, and Cy5 were separated together in a single gel, and then were detected by fluorescence image analyzer. The 2D DIGE method using fluorescence CyDye DIGE flour had higher sensitivity than conventional 2D gel electrophoresis, and showed reproducible results. Approximately 1,500 protein spots were detected by 2D DIGE. Several proteins showed a more than 1.5-fold up and down regulation between non-pregnant and pregnant serum proteins. The differentially expressed proteins were identified by MALDI-TOF mass spectrometer. A total 16 protein spots were detected to regulate differentially in the pregnant serum, among which 7 spots were up-regulated proteins such as conglutinin precursor, modified bovine fibrinogen and IgG1, and 6 spots were down-regulated proteins such as hemoglobin, complement component 3, bovine fibrinogen and IgG2a three spots were not identified. The identified proteins demonstrate that early pregnant bovine serum may have several pregnancy-specific proteins, and these could be a valuable information for the development of pregnancy-diagnostic markers in early pregnancy bovine serum.
Project description:The completeness of tumor removal during surgery is dependent on the surgeon's ability to differentiate tumor from normal tissue using subjective criteria that are not easily quantifiable. A way to objectively assess tumor margins during surgery in patients would be of great value. We have developed a method to visualize tumors during surgery using activatable cell-penetrating peptides (ACPPs), in which the fluorescently labeled, polycationic cell-penetrating peptide (CPP) is coupled via a cleavable linker to a neutralizing peptide. Upon exposure to proteases characteristic of tumor tissue, the linker is cleaved, dissociating the inhibitory peptide and allowing the CPP to bind to and enter tumor cells. In mice, xenografts stably transfected with green fluorescent protein show colocalization with the Cy5-labeled ACPPs. In the same mouse models, Cy5-labeled free ACPPs and ACPPs conjugated to dendrimers (ACPPDs) delineate the margin between tumor and adjacent tissue, resulting in improved precision of tumor resection. Surgery guided by ACPPD resulted in fewer residual cancer cells left in the animal after surgery as measured by Alu PCR. A single injection of ACPPD dually labeled with Cy5 and gadolinium chelates enabled preoperative whole-body tumor detection by MRI, intraoperative guidance by real-time fluorescence, intraoperative histological analysis of margin status by fluorescence, and postoperative MRI tumor quantification. Animals whose tumors were resected with ACPPD guidance had better long-term tumor-free survival and overall survival than animals whose tumors were resected with traditional bright-field illumination only.