RESUMO
Due to the mounting evidence that RNA structure plays a critical role in regulating almost any physiological as well as pathological process, being able to accurately define the folding of RNA molecules within living cells has become a crucial need. We introduce here 2-aminopyridine-3-carboxylic acid imidazolide (2A3), as a general probe for the interrogation of RNA structures in vivo. 2A3 shows moderate improvements with respect to the state-of-the-art selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) reagent NAI on naked RNA under in vitro conditions, but it significantly outperforms NAI when probing RNA structure in vivo, particularly in bacteria, underlining its increased ability to permeate biological membranes. When used as a restraint to drive RNA structure prediction, data derived by SHAPE-MaP with 2A3 yields more accurate predictions than NAI-derived data. Due to its extreme efficiency and accuracy, we can anticipate that 2A3 will rapidly take over conventional SHAPE reagents for probing RNA structures both in vitro and in vivo.
Assuntos
RNA/química , Células HEK293 , Humanos , Indicadores e Reagentes , Conformação de Ácido Nucleico , DNA Polimerase Dirigida por RNARESUMO
Isatoic anhydride (IA) has been shown to be a useful platform for quantifiable bioconjugation. The elaboration of a water-soluble isatoic anhydride-based platform with biotin offers readily quantifiable biotinylation reagents through nondestructive methods of quantification. The incorporation of functionality is directly quantified using the reagent's unique absorbance or fluorescence signature, located outside the biological window. Several biotinylation reagents are prepared with various linker lengths, and the quantification of biotinylated proteins is demonstrated and compared to results from the traditional HABA assay.
Assuntos
Oxazinas/química , Água/química , Biotinilação , SolubilidadeRESUMO
1-Methyl-7-nitroisatoic anhydride (1M7) and 2-methylnicotinic acid imidazolide (NAI) are two of the most commonly applied RNA-SHAPE electrophiles; 1M7 due to its high reactivity and NAI for its solubility and cell permeability. While the addition of a nitro group yields desirable activation of the reagent, it also leads to poorer water solubility. This limited solubility has motivated the development of water-soluble reagents. We present alternative, isatoic anhydride-based reagents possessing variable reactivities that are simultaneously water-soluble. Solubility is gained by using a quaternary ammonium, while modulation of the reactivity is obtained by functionalization of the aryl ring. The syntheses of the reagents are discussed, and the electrophiles are demonstrated to be suitable for use for an in vitro RNA SHAPE experiment when directly compared to 1M7.
Assuntos
Oxazinas/química , RNA/química , Água/química , Acilação , Sequência de Bases , Radical Hidroxila/química , RNA/genética , SolubilidadeRESUMO
N-(3-Iodopropyl)isatoic anhydride (IPIA) has been demonstrated to serve as an efficient substrate for the development of an extended bioconjugation platform. Derivatives of IPIA are water-soluble and adaptable and share a common chromophore, rendering them easily quantifiable. We demonstrate the preparation of the readily diversified bioconjugation platform technology and application of the reagents in RNA-SHAPE analysis.
Assuntos
Oxazinas/química , Proteínas/química , RNA/química , Estrutura Molecular , Solubilidade , Água/químicaRESUMO
BACKGROUND: Pancreatic ductal adenocarcinoma (PDA) remains the most aggressive cancers with a 5-year survival below 10%. Systemic delivery of chemotherapy drugs has severe side effects in patients with PDA and does not significantly improve overall survival rate. It is highly desirable to advance the therapeutic efficacy of chemotherapeutic drugs by targeting their delivery and increasing accumulation at the tumor site. MUC1 is a membrane-tethered glycoprotein that is aberrantly overexpressed in > 80% of PDA thus making it an attractive antigenic target. METHODS: Poly lactic-co-glycolic acid nanoparticles (PLGA NPs) conjugated to a tumor specific MUC1 antibody, TAB004, was used as a nanocarrier for targeted delivery into human PDA cell lines in vitro and in PDA tumors in vivo. The PLGA NPs were loaded with fluorescent imaging agents, fluorescein diacetate (FDA) and Nile Red (NR) or isocyanine green (ICG) for in vitro and in vivo imaging respectively or with a chemotherapeutic drug, paclitaxel (PTX) for in vitro cytotoxicity assays. Confocal microscopy was used to visualize internalization of the nanocarrier in vitro in PDA cells with high and low MUC1 expression. The in vivo imaging system (IVIS) was used to visualize in vivo tumor targeting of the nanocarrier. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay was used to determine in vitro cell survival of cells treated with PTX-loaded nanocarrier. One-sided t-test comparing treatment groups at each concentration and two-way ANOVAs comparing internalization of antibody and PLGA nanoparticles. RESULTS: In vitro, TAB004-conjugated ICG-nanocarriers were significantly better at internalizing in PDA cells than its non-conjugated counterpart. Similarly, TAB004-conjugated PTX-nanocarriers were significantly more cytotoxic in vitro against PDA cells than its non-conjugated counterpart. In vivo, TAB004-conjugated ICG-nanocarriers showed increased accumulation in the PDA tumor compared to the non-conjugated nanocarrier while sparing normal organs. CONCLUSIONS: The study provides promising data for future development of a novel MUC1-targeted nanocarrier for direct delivery of imaging agents or drugs into the tumor microenvironment.