RESUMO
Single cell transcriptomics provides a powerful discovery tool for identifying new cell types and functions as well as a means to probe molecular features of the etiology and treatment of human diseases, including cancer. However, such analyses are limited by the difficulty of isolating mRNA from single cells within biological samples. We recently introduced a photochemical method for isolating mRNA from single living cells, Transcriptome In Vivo Analysis (TIVA). The TIVA probe is a "caged" polyU : polyA oligonucleotide hairpin designed to enter live tissue, where site-specific activation with 405 nm laser reveals the polyU-biotin strand to bind mRNA in a target cell, enabling subsequent mRNA isolation and sequencing. The TIVA method is well suited for analysis of living cells in resected tissue, but has not yet been applied to living cells in whole organisms. Adapting TIVA to this more challenging environment requires a probe with higher thermal stability, more robust caging, and greater nuclease resistance. In this paper we present modifications to the original TIVA probe with multiple aspects of enhanced stability. These newer probes utilize an extended 22mer polyU capture strand with two 9mer polyA blocking strands ("22/9/9") for higher thermal stability pre-photolysis and improved mRNA capture affinity post-photolysis. The "22/9/9 GC" probe features a terminal GC pair to reduce pre-photolysis interactions with mRNA by more than half. The "PS-22/9/9" probe features a phosphorothioated backbone, which extends serum stability from <1 h to at least 48 h, and also mediates uptake into cultured human fibroblasts.
Assuntos
Perfilação da Expressão Gênica , Sondas Moleculares/química , Oligonucleotídeos/química , Análise de Célula Única , Células Cultivadas , Fibroblastos/citologia , Humanos , Sondas Moleculares/síntese química , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificaçãoRESUMO
Genetically encoded magnetic resonance imaging (MRI) contrast agents enable non-invasive detection of specific biomarkers in vivo. Here, we employed the hyper-CEST 129Xe NMR technique to quantify maltose (32 nM to 1 mM) through its modulation of conformational change and xenon exchange in maltose binding protein (MBP). Remarkably, no hyper-CEST signal was observed for MBP in the absence of maltose, making MBP an ultrasensitive "smart" contrast agent. The resonance frequency of 129Xe bound to MBP was greatly downfield-shifted (Δδ = 95 ppm) from the 129Xe(aq) peak, which facilitated detection in E. coli as well as multiplexing with TEM-1 ß-lactamase. Finally, a Val to Ala mutation at the MBP-Xe binding site yielded 34% more contrast than WT, with 129Xe resonance frequency shifted 59 ppm upfield from WT. We conclude that engineered MBPs constitute a new class of genetically encoded, analyte-sensitive molecular imaging agents detectable by 129Xe NMR/MRI.
RESUMO
Photochemical approaches afford high spatiotemporal control over molecular structure and function, for broad applications in materials and biological science. Here, we present the first example of a visible light responsive ruthenium-based photolinker, Ru(bipyridine)2(3-ethynylpyridine)2 (RuBEP), which was reacted stoichiometrically with a 25mer DNA or morpholino (MO) oligonucleotide functionalized with 3' and 5' terminal azides, via Cu(I)-mediated [3+2] Huisgen cycloaddition reactions. RuBEP-caged circular morpholinos (Ru-MOs) targeting two early developmental zebrafish genes, chordin and notail, were synthesized and tested in vivo. One-cell-stage zebrafish embryos microinjected with Ru-MO and incubated in the dark for 24 h developed normally, consistent with caging, whereas irradiation at 450 nm dissociated one 3-ethynylpyridine ligand (Ï = 0.33) and uncaged the MO to achieve gene knockdown. As demonstrated, Ru photolinkers provide a versatile method for controlling structure and function of biopolymers.
RESUMO
In collaboration with the University of Pennsylvania, a (222)Rn emanation source was used for the determination of the binding affinity of radon to a cryptophane molecular host. This source was similar to a (222)Rn emanation standard that was developed and disseminated by the National Institute of Standards and Technology (NIST). The novel experimental design involved performing the reactions at femtomole levels, developing exacting gravimetric sampling methods and making precise (222)Rn assays by liquid scintillation counting. A cryptophane-radon association constant was determined, K(A)=(49,000±12,000) L mol(-1) at 293 K, which was the first measurement of radon binding to a molecular host.
Assuntos
Compostos Policíclicos/química , Radiometria/normas , Radônio/química , Radônio/normas , Meia-Vida , Internacionalidade , Doses de Radiação , Radiometria/instrumentação , Radônio/análise , Padrões de Referência , Valores de ReferênciaRESUMO
Cytochromes P450 play key roles in drug metabolism and disease by oxidizing a wide variety of natural and xenobiotic compounds. High-resolution crystal structures of P450cam bound to ruthenium sensitizer-linked substrates reveal an open conformation of the enzyme that allows substrates to access the active center via a 22-A deep channel. Interactions of alkyl and fluorinated biphenyl linkers with the channel demonstrate the importance of exploiting protein dynamics for specific inhibitor design. Large changes in peripheral enzyme structure (F and G helices) couple to conformational changes in active center residues (I helix) implicated in proton pumping and dioxygen activation. Common conformational states among P450cam and homologous enzymes indicate that static and dynamic variability in the F/G helix region allows the 54 human P450s to oxidize thousands of substrates.
Assuntos
Cânfora 5-Mono-Oxigenase/química , Rutênio/metabolismo , Sítios de Ligação , Cânfora 5-Mono-Oxigenase/metabolismo , Catálise , Conformação ProteicaRESUMO
Molecules with photosensitizers attached to substrates (Wilker et al., Angew. Chem. Int. Ed. 38 (1999) 90-92) or cofactors (Hamachi et al., J. Am. Chem. Soc. 121 (1999) 5500-5506) can rapidly deliver redox equivalents to buried active sites. The structure of cytochrome P450cam (P450) co-crystallized with a prototypal sensitizer-substrate, [Ru-C9-Ad]Cl2, has been determined (Dmochowski et al., Proc. Natl. Acad. Sci. USA 96 (1999) 12987-12990); and, in separate UV-vis absorption and time-resolved luminescence experiments, the binding of the lambda and delta enantiomers of Ru-C9-Ad to P450 has been measured. The results, KD(delta/lambda) approximately 2, indicate that the bipyridyl ligands of the lambda isomer interact more favorably with hydrophobic residues at the entrance to the substrate channel. We conclude that enantiospecific interactions may be exploited in the design of enzyme-metallosubstrate conjugates.
Assuntos
Cânfora 5-Mono-Oxigenase/química , Cânfora 5-Mono-Oxigenase/metabolismo , Rutênio/química , Sítios de Ligação , Dicroísmo Circular , Cristalografia por Raios X , Escherichia coli/metabolismo , Isomerismo , Cinética , Espectroscopia de Ressonância Magnética , Modelos Químicos , Modelos Moleculares , Oxirredução , Plasmídeos/metabolismo , Espectrofotometria , Fatores de Tempo , Raios UltravioletaRESUMO
The ability to detect, characterize, and manipulate specific biomolecules in complex media is critical for understanding metabolic processes. Particularly important targets are oxygenases (cytochromes P450) involved in drug metabolism and many disease states, including liver and kidney dysfunction, neurological disorders, and cancer. We have found that Ru photosensitizers linked to P450 substrates specifically recognize submicromolar cytochrome P450(cam) in the presence of other heme proteins. In the P450:Ru-substrate conjugates, energy transfer to the heme dramatically accelerates the Ru-luminescence decay. The crystal structure of a P450(cam):Ru-adamantyl complex reveals access to the active center via a channel whose depth (Ru-Fe distance is 21 A) is virtually the same as that extracted from an analysis of the energy-transfer kinetics. Suitably constructed libraries of sensitizer-linked substrates could be employed to probe the steric and electronic properties of buried active sites.