ABSTRACT
Assaying Dicer-mediated miRNA maturation is a valuable tool not only for validating miRNA maturation itself but also for testing Dicer activity in cell lysate and for screening small molecules inhibiting miRNA maturation in a high-throughput format. The classical assay for miRNA maturation relies on radioactive labeling of a pre-miRNA and subsequent gel electrophoresis and autoradiography. Here we present a fluorescently labeled and quenched pre-miRNA beacon that can be ligated easily out of two single labeled RNA strands. Upon Dicer cleavage of the beacon, fluorophore and quencher are separated, which results in an increase of fluorescence over time. Unlike (32)P-labeled probes, our fluorescently labeled pre-miRNA beacon is stable for at least 5 years under storage conditions. Dicer or miRNA maturation assays can be easily performed in a 384-well plate format, consuming less than 1 pmol of RNA beacon per reaction.
Subject(s)
Fluorescent Dyes/metabolism , MicroRNAs/metabolism , RNA Cleavage , Ribonuclease III/metabolism , HEK293 Cells , Humans , Spectrometry, FluorescenceABSTRACT
Several viral and non-viral vectors have been developed for exogenous protein expression in specific cells. Conventionally, this purpose is achieved through the use of recombinant DNA. But mainly due to the risks associated with permanent genetic alteration of cells, safety and ethical concerns have been raised for the use of DNA-based vectors in human clinical therapy. In the last years, synthetic messenger RNA has emerged as powerful tool to deliver genetic information. RNA vectors exhibit several advantages compared to DNA and are particularly interesting for applications that require transient gene expression. RNA stability and translation efficiency can be increased by cis-acting structural elements in the RNA such as the 5'-cap, the poly(A)-tail, untranslated regions and the sequence of the coding region. Here we review recent developments in the optimization of messenger RNA as vector for modulation of protein expression emphasizing on stability, transfection and immunogenicity. In addition, we summarize current pre-clinical and clinical studies using RNA-based vectors for immunotherapy, T cell, stem cell as well as gene therapy.
Subject(s)
Gene Transfer Techniques , RNA, Messenger/genetics , Animals , Gene Expression , Genetic Therapy , Genetic Vectors , Humans , Immunity, Innate , Protein Processing, Post-Translational , RNA Stability , RNA, Messenger/immunology , RNA, Messenger/metabolismABSTRACT
The total synthesis of the natural occurring polyamides proximicin A-C (3-5) has been accomplished. A short and efficient synthesis of a thus far unknown 4-amino-2-furan carboxylic acid was developed. Furthermore, this unique heterocyclic gamma-amino-acid was used for the synthesis of a new class of AT-selective DNA-binding agents derived from the natural products combining structural features of the proximicins with those from the known DNA-binding natural products netropsin (1) and distamycin (2).
Subject(s)
DNA/chemistry , Furans/chemistry , Netropsin/analogs & derivatives , Nylons/chemical synthesis , Amino Acids/chemical synthesis , Amino Acids/chemistry , DNA/metabolism , Distamycins/chemistry , Distamycins/pharmacology , Molecular Structure , Netropsin/chemical synthesis , Netropsin/chemistry , Netropsin/pharmacology , Nylons/chemistryABSTRACT
A class of small RNAs known as micro-RNAs (miRNAs) has been shown to play an important role in development and cellular regulation in eukaryotes. Recent evidence also associates aberrant expression of various miRNAs with multiple diseases in humans including many types of cancer. An up- or downregulation of certain miRNAs may play a significant role in the course of these diseases. We were interested in whether binding of small molecules to the inactive miRNA precursors would block their cleavage into the active miRNAs by the enzyme Dicer. The inhibition of miRNA maturation might provide a new rationale for future therapeutic strategies. We have developed a fluorescence beacon with which to study the maturation of miRNAs. Details are provided of a homogeneous assay for detecting potential inhibitors of miRNA maturation in a high-throughput format.
Subject(s)
Fluorescent Dyes , MicroRNAs/analysis , Molecular Probe Techniques , RNA Processing, Post-Transcriptional , Disease Susceptibility , Gene Expression Regulation , MicroRNAs/antagonists & inhibitors , MicroRNAs/geneticsABSTRACT
The palladium catalysed coupling of aryl chlorides and amines can be readily achieved with short reaction times when carried out at high temperatures under thermal or microwave conditions. These coupling protocols are successful using two co-ordinate palladium-N-heterocyclic carbene complexes, or imidazolium salt protocols.
