共查询到20条相似文献,搜索用时 31 毫秒
1.
Dangerfield JA Windbichler N Salmons B Günzburg WH Schröder R 《Electrophoresis》2006,27(10):1874-1877
StreptoTag is a novel affinity chromatography-based method for the isolation of high- and low-affinity RNA binding proteins. Originally it was shown possible to isolate recombinant protein from yeast or bacterial extracts using small, specific, well-characterised RNA binding targets. Here we show that using an enhanced aptamer it is not only possible to efficiently immobilise large, highly structured RNA binding targets onto the streptomycin columns but also that the StreptoTag method can be used for the isolation and purification of endogenously expressed regulatory proteins, with relatively low abundance, from eukaryotic extracts. As an example for this we uncover the identity of a karyophilic cellular protein which specifically binds to an area within the large, highly folded structure that characterises the mRNA from the unique 3' region (U3) of the mouse mammary tumour virus (MMTV) long terminal repeat (LTR). Hence, this method is now suitable for the quick and efficient isolation and identification of novel RNA binding proteins such as regulatory factors. 相似文献
2.
Stephen D. Fried Wolfgang H. Schmied Chayasith Uttamapinant Jason W. Chin 《Angewandte Chemie (Weinheim an der Bergstrasse, Germany)》2015,127(43):12982-12985
The creation of orthogonal large and small ribosomal subunits, which interact with each other but not with endogenous ribosomal subunits, would extend our capacity to create new functions in the ribosome by making the large subunit evolvable. To this end, we rationally designed a ribosomal RNA that covalently links the ribosome subunits via an RNA staple. The stapled ribosome is directed to an orthogonal mRNA, allowing the introduction of mutations into the large subunit that reduce orthogonal translation, but have minimal effects on cell growth. Our approach provides a promising route towards orthogonal subunit association, which may enable the evolution of key functional centers in the large subunit, including the peptidyl‐transferase center, for unnatural polymer synthesis in cells. 相似文献
3.
Dr. Klara Aradi Dr. Audrey Di Giorgio Dr. Maria Duca 《Chemistry (Weinheim an der Bergstrasse, Germany)》2020,26(54):12273-12309
Natural aminoglycosides are therapeutically useful antibiotics and very efficient RNA ligands. They are oligosaccharides that contain several ammonium groups able to interfere with the translation process in prokaryotes upon binding to bacterial ribosomal RNA (rRNA), and thus, impairing protein synthesis. Even if aminoglycosides are commonly used in therapy, these RNA binders lack selectivity and are able to bind to a wide number of RNA sequences/structures. This is one of the reasons for their toxicity and limited applications in therapy. At the same time, the ability of aminoglycosides to bind to various RNAs renders them a great source of inspiration for the synthesis of new binders with improved affinity and specificity toward several therapeutically relevant RNA targets. Thus, a number of studies have been performed on these complex and highly functionalized compounds, leading to the development of various synthetic methodologies toward the synthesis of conjugated aminoglycosides. The aim of this review is to highlight recent progress in the field of aminoglycoside conjugation, paying particular attention to modifications performed toward the improvement of affinity and especially to the selectivity of the resulting compounds. This will help readers to understand how to introduce a desired chemical modification for future developments of RNA ligands as antibiotics, antiviral, and anticancer compounds. 相似文献
4.
Dr. Alyssa C. Hill Dr. Daniël van Leeuwen Dr. Verena Schlösser Dr. Alok Behera Dr. Bogdan Mateescu Prof. Dr. Jonathan Hall 《Chemistry (Weinheim an der Bergstrasse, Germany)》2022,28(8):e202103995
RNA interference (RNAi) mediated by small interfering RNA (siRNA) duplexes is a powerful therapeutic modality, but the translation of siRNAs from the bench into clinical application has been hampered by inefficient delivery in vivo. An innovative delivery strategy involves fusing siRNAs to a three-way junction (3WJ) motif derived from the phi29 bacteriophage prohead RNA (pRNA). Chimeric siRNA-3WJ molecules are presumed to enter the RNAi pathway through Dicer cleavage. Here, we fused siRNAs to the phi29 3WJ and two phylogenetically related 3WJs. We confirmed that the siRNA-3WJs are substrates for Dicer in vitro. However, our results reveal that siRNA-3WJs transfected into Dicer-deficient cell lines trigger potent gene silencing. Interestingly, siRNA-3WJs transfected into an Argonaute 2-deficient cell line also retain some gene silencing activity. siRNA-3WJs are most efficient when the antisense strand of the siRNA duplex is positioned 5′ of the 3WJ (5′-siRNA-3WJ) relative to 3′ of the 3WJ (3′-siRNA-3WJ). This work sheds light on the functional properties of siRNA-3WJs and offers a design rule for maximizing their potency in the human RNAi pathway. 相似文献
5.
Schlippe YV Hartman MC Josephson K Szostak JW 《Journal of the American Chemical Society》2012,134(25):10469-10477
There is a great demand for the discovery of new therapeutic molecules that combine the high specificity and affinity of biologic drugs with the bioavailability and lower cost of small molecules. Small, natural-product-like peptides hold great promise in bridging this gap; however, access to libraries of these compounds has been a limitation. Since ribosomal peptides may be subjected to in vitro selection techniques, the generation of extremely large libraries (>10(13)) of highly modified macrocyclic peptides may provide a powerful alternative for the generation and selection of new useful bioactive molecules. Moreover, the incorporation of many non-proteinogenic amino acids into ribosomal peptides in conjunction with macrocyclization should enhance the drug-like features of these libraries. Here we show that mRNA-display, a technique that allows the in vitro selection of peptides, can be applied to the evolution of macrocyclic peptides that contain a majority of unnatural amino acids. We describe the isolation and characterization of two such unnatural cyclic peptides that bind the protease thrombin with low nanomolar affinity, and we show that the unnatural residues in these peptides are essential for the observed high-affinity binding. We demonstrate that the selected peptides are tight-binding inhibitors of thrombin, with K(i)(app) values in the low nanomolar range. The ability to evolve highly modified macrocyclic peptides in the laboratory is the first crucial step toward the facile generation of useful molecular reagents and therapeutic lead molecules that combine the advantageous features of biologics with those of small-molecule drugs. 相似文献
6.
HOFSTADLER Steven LOWERY Kristin DRADER Jared DING Yili JEFFERSON Elizabeth SWAYZE Eric GRIFFEY Rich HE Yun 《高等学校化学研究》2001,17(3)
RNAs have recently emerged as an exciting new target for small molecule therapeutics. Conventional HTS discovery strategies measuring disruption of RNAprotein interactions have proven unsuccessful. We describe a ligand-based drug discovery strategy that addresses the inherent difficulties RNA targets. The strategy is based on: 1) using a MS spectrometry (MS)-based assay to measure the affinity of compounds for a target; 2) performing competitive binding experiments and molecular modeling with the motifs to determine the binding site(s) of the ligands; 3) design and synthesis of derivatives of interesting binders to establish the linking sites; 4) identifying the appropriate linker group using MS; 5) fusing motifs into a more complex structure to afford higher affinity compounds. Example of applying this strategy to identify new classes of lead molecules with affinity and specificity for ribosomal RNA targets will be presented. 相似文献
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The suitability of various materials for messenger RNA isolation and possible fractionation was examined by comparing the chromatographic behaviour of synthetic poly(riboadenylic acid) [poly(A)] and ribosomal RNA on each suport. The ratios of poly(A) to ribosomal RNA capacities of the various materials under different chromatographic conditions were used to estimate the potential selectivity of the columns for messenger RNA isolation. Using this criterion, benzoylated cellulose and oligo(deoxythymidylate)-cellulose should be the most selective materials of those studied. The elution conditions (temperature or salt gradient) used to obtain bound poly(A) were investigated to determine the optimum conditions for possible messenger RNA fractionation. It is concluded that oligo(deoxythymidylate)-cellulose appears to be the most suitable support of those investigated for messenger RNA isolation and possible fractionation. 相似文献
9.
López-Senín P Gómez-Pinto I Grandas A Marchán V 《Chemistry (Weinheim an der Bergstrasse, Germany)》2011,17(6):1946-1953
We describe the use of dynamic combinatorial chemistry (DCC) to identify ligands for the stem-loop structure located at the exon 10-5'-intron junction of Tau pre-mRNA, which is involved in the onset of several tauopathies including frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17). A series of ligands that combine the small aminoglycoside neamine and heteroaromatic moieties (azaquinolone and two acridines) have been identified by using DCC. These compounds effectively bind the stem-loop RNA target (the concentration required for 50% RNA response (EC(50)): 2-58 μM), as determined by fluorescence titration experiments. Importantly, most of them are able to stabilize both the wild-type and the +3 and +14 mutated sequences associated with the development of FTDP-17 without producing a significant change in the overall structure of the RNA (as analyzed by circular dichroism (CD) spectroscopy), which is a key factor for recognition by the splicing regulatory machinery. A good correlation has been found between the affinity of the ligands for the target and their ability to stabilize the RNA secondary structure. 相似文献
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Recentlytherehasbeenconsiderableinterestindevel0pingcatalyststhathydrolyticallycleavethePhosPhodiesterbackbone0fRNAsite-specificallyforthepotentialaPplicati0nstomolecularbi0l0gyandgenetheraPy"2SmallmoleculeswithRNAcleavageactivityareusedasresearchtoolsfortheinVestigationofnucleicacidstrUctUres.3'4SeveralexamPlesofoxidativecleavagecatalystshavebeenrePortCd.'MuchefforthasbeendevotedtoRNAhydIolysiscatalystSduet0theadvatgesover0xidativecleavageones.'MetaLbasedhydrolysisofRNAhasbeenreport… 相似文献
12.
A highly convergent approach for the chemical synthesis of eight structurally related trisaccharides that contain 3 to 5 amino groups has been described. Fourier-transformation ion cyclotron resonance mass spectrometry (FT-ICR MS) has been employed to determine the dissociation constants (Kd) for the binding of the trisaccharides to a prototypical fragment of 16S ribosomal RNA. A compound that contained a 4,6-dideoxy-4-amino-beta-D-glucopyranoside moiety at C-3 displayed binding in the low micromolar range. It was found that small structural changes of the saccharides resulted in large differences in affinity. The described structure-activity relationship is expected to be valuable for the development of novel antibiotics that target rRNA. 相似文献
13.
Dr. Shiori Umemoto Seongwang Im Dr. Jinhua Zhang Dr. Masaki Hagihara Dr. Asako Murata Yasue Harada Dr. Takeo Fukuzumi Takahiro Wazaki Shin‐ichi Sasaoka Prof. Dr. Kazuhiko Nakatani 《Chemistry (Weinheim an der Bergstrasse, Germany)》2012,18(32):9999-10008
A series of xanthone and thioxanthone derivatives with aminoalkoxy substituents were synthesized as fluorescent indicators for a displacement assay in the study of small‐molecule–RNA interactions. The RNA‐binding properties of these molecules were investigated in terms of the improved binding selectivity to the loop region in the RNA secondary structure relative to 2,7‐bis(2‐aminoethoxy)xanthone (X2S) by fluorimetric titration and displacement assay. An 11‐mer double‐stranded RNA and a hairpin RNA mimicking the stem loop IIB of Rev response element (RRE) RNA of HIV‐1 mRNA were used. The X2S derivatives with longer aminoalkyl substituents showed a higher affinity to the double‐stranded RNA than the parent molecule. Introduction of a methyl group on the aminoethoxy moiety of X2S effectively modulated the selectivity to the RNA secondary structure. Methyl group substitution at the C1′ position suppressed the binding to the loop regions. Substitution with two methyl groups on the amino nitrogen atom resulted in reducing the affinity to the double‐stranded region by a factor of 40 %. The effect of methyl substitution on the amino nitrogen atom was also observed for a thioxanthone derivative. Titration experiments, however, suggested that thioxanthone derivatives showed a more prominent tendency of multiple binding to RNA than xanthone derivatives. The selectivity index calculated from the affinity to the double‐stranded and loop regions suggested that the N,N‐dimethyl derivative of X2S would be suitable for the screening of small molecules binding to RRE. 相似文献
14.
Ulf Birkedal Mikkel Christensen‐Dalsgaard Nicolai Krogh Radhakrishnan Sabarinathan Jan Gorodkin Prof. Henrik Nielsen 《Angewandte Chemie (International ed. in English)》2015,54(2):451-455
Ribose methylations are the most abundant chemical modifications of ribosomal RNA and are critical for ribosome assembly and fidelity of translation. Many aspects of ribose methylations have been difficult to study due to lack of efficient mapping methods. Here, we present a sequencing‐based method (RiboMeth‐seq) and its application to yeast ribosomes, presently the best‐studied eukaryotic model system. We demonstrate detection of the known as well as new modifications, reveal partial modifications and unexpected communication between modification events, and determine the order of modification at several sites during ribosome biogenesis. Surprisingly, the method also provides information on a subset of other modifications. Hence, RiboMeth‐seq enables a detailed evaluation of the importance of RNA modifications in the cells most sophisticated molecular machine. RiboMeth‐seq can be adapted to other RNA classes, for example, mRNA, to reveal new biology involving RNA modifications. 相似文献
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Dr. Matthieu Fonvielle Dénia Mellal Delphine Patin Maxime Lecerf Dr. Didier Blanot Dr. Ahmed Bouhss Dr. Marco Santarem Dr. Dominique Mengin‐Lecreulx Dr. Matthieu Sollogoub Dr. Michel Arthur Dr. Mélanie Ethève‐Quelquejeu 《Chemistry (Weinheim an der Bergstrasse, Germany)》2013,19(4):1357-1363
Peptidyl–RNA conjugates have various applications in studying the ribosome and enzymes participating in tRNA‐dependent pathways such as Fem transferases in peptidoglycan synthesis. Herein a convergent synthesis of peptidyl–RNAs based on Huisgen–Sharpless cycloaddition for the final ligation step is developed. Azides and alkynes are introduced into tRNA and UDP‐MurNAc‐pentapeptide, respectively. Synthesis of 2′‐azido RNA helix starts from 2′‐azido‐2′‐deoxyadenosine that is coupled to deoxycytidine by phosphoramidite chemistry. The resulting dinucleotide is deprotected and ligated to a 22‐nt RNA helix mimicking the acceptor arm of Ala‐tRNAAla by T4 RNA ligase. For alkyne UDP‐MurNAc‐pentapeptide, meso‐cystine is enzymatically incorporated into the peptidoglycan precursor and reduced, and L ‐Cys is converted to dehydroalanine with O‐(mesitylenesulfonyl)hydroxylamine. Reaction of but‐3‐yne‐1‐thiol with dehydroalanine affords the alkyne‐containing UDP‐MurNAc‐pentapeptide. The CuI‐catalyzed azide alkyne cycloaddition reaction in the presence of tris[(1‐hydroxypropyl‐1H‐1,2,3‐triazol‐4‐yl)methyl]amine provided the peptidyl‐RNA conjugate, which was tested as an inhibitor of non‐ribosomal FemXWv aminoacyl transferase. The bi‐substrate analogue was found to inhibit FemXWv with an IC50 of (89±9) pM , as both moieties of the peptidyl–RNA conjugate contribute to high‐affinity binding. 相似文献
17.
Deyuan Cong Dr. Yao Li Paul T. Ludford III Prof. Dr. Yitzhak Tor 《Chemistry (Weinheim an der Bergstrasse, Germany)》2022,28(35):e202200994
Ribosome-inactivating proteins, a family of highly cytotoxic proteins, interfere with protein synthesis by depurinating a specific adenosine residue within the conserved α-sarcin/ricin loop of eukaryotic ribosomal RNA. Besides being biological warfare agents, certain RIPs have been promoted as potential therapeutic tools. Monitoring their deglycosylation activity and their inhibition in real time have remained, however, elusive. Herein, we describe the enzymatic preparation and utility of consensus RIP hairpin substrates in which specific G residues, next to the depurination site, are surgically replaced with tzG and thG, fluorescent G analogs. By strategically modifying key positions with responsive fluorescent surrogate nucleotides, RIP-mediated depurination can be monitored in real time by steady-state fluorescence spectroscopy. Subtle differences observed in preferential depurination sites provide insight into the RNA folding as well as RIPs’ substrate recognition features. 相似文献
18.
DNA and RNA aptamers: from tools for basic research towards therapeutic applications 总被引:1,自引:0,他引:1
Ulrich H Trujillo CA Nery AA Alves JM Majumder P Resende RR Martins AH 《Combinatorial chemistry & high throughput screening》2006,9(8):619-632
The systematic evolution of ligands by exponential enrichment (SELEX) is a combinatorial oligonucleotide library-based in vitro selection approach in which DNA or RNA molecules are selected by their ability to bind their targets with high affinity and specificity, comparable to those of antibodies. Nucleic acids with high affinity for their targets have been selected against a wide variety of compounds, from small molecules, such as ATP, to membrane proteins and even whole organisms. Recently, the use of the SELEX technique was extended to isolate oligonucleotide ligands, also known as aptamers, for a wide range of proteins of importance for therapy and diagnostics, such as growth factors and cell surface antigens. The number of aptamers generated as inhibitors of various target proteins has increased following automatization of the SELEX process. Their diagnostic and therapeutic efficacy can be enhanced by introducing chemical modifications into the oligonucleotides to provide resistance against enzymatic degradation in body fluids. Several aptamers are currently being tested in preclinical and clinical trials, and aptamers are in the process of becoming a new class of therapeutic agents. Recently, the anti-VEGF aptamer pegaptanib received FDA approval for treatment of human ocular vascular disease. 相似文献
19.
Gene therapy is a promising tool for treatment of the human diseases that cannot be cured by rational therapies, and its primary
success depends on suitable vectors to deliver therapeutic genes. Adenoviruses (Ads) are among the most commonly used vectors
for gene therapy, second only to retroviruses. During the last decade, remarkable progress has been made in the development
of Ad vectors and in the understanding of the toxicity related to the Ad vector system. Ad vector has certain advantages such
as high transduction efficiency for different quiescent and dividing cell types and high levels of short-term expression to
provide therapeutic benefits. However, researchers are facing the challenges associated with tissue-specific targeting of
vectors and the vector-mediated immunogenicity. This review mainly focuses on the studies that have employed methods to improve
Ad vectors and reduce viral toxicity for different applications. These methods include minimization or elimination of viral
genes, retargeting of vector to the tissue of interest, and generation of immunocompromised recombinant vectors that lead
to safer use of Ad vector systems that improve persistence of transgene expression. Moreover, the therapeutic applications
of Ad vectors for liver-targeted gene therapy, suicide gene therapy, delivery of small interfering RNA, and production of
recombinant vaccine under regulated conditions used in clinical trials are discussed. 相似文献
20.
Aminoglycoside microarrays to explore interactions of antibiotics with RNAs and proteins 总被引:3,自引:0,他引:3
RNA is an important target for drug discovery efforts. Several clinically used aminoglycoside antibiotics bind to bacterial rRNA and inhibit protein synthesis. Aminoglycosides, however, are losing efficacy due to their inherent toxicity and the increase in antibiotic resistance. Targeting of other RNAs is also becoming more attractive thanks to the discovery of new potential RNA drug targets through genome sequencing and biochemical efforts. Identification of new compounds that target RNA is therefore urgent, and we report here on the development of rapid screening methods to probe binding of low molecular weight ligands to proteins and RNAs. A series of aminoglycosides has been immobilized onto glass microscope slides, and binding to proteins and RNAs has been detected by fluorescence. Construction and analysis of the arrays is completed by standard DNA genechip technology. Binding of immobilized aminoglycosides to proteins that are models for study of aminoglycoside toxicity (DNA polymerase and phospholipase C), small RNA oligonucleotide mimics of aminoglycoside binding sites in the ribosome (rRNA A-site mimics), and a large (approximately 400 nucleotide) group I ribozyme RNA is detected. The ability to screen large RNAs alleviates many complications associated with binding experiments that use isolated truncated regions from larger RNAs. These studies lay the foundation for rapid identification of small organic ligands from combinatorial libraries that exhibit strong and selective RNA binding while displaying decreased affinity to toxicity-causing proteins. 相似文献