Browse
Submit Data
Databases
API
Help

Dataset Information

31 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Pinpointing RNA-Protein Cross-Links with Site-Specific Stable Isotope-Labeled Oligonucleotides.

ABSTRACT: High affinity RNA-protein interactions are critical to cellular function, but directly identifying the determinants of binding within these complexes is often difficult. Here, we introduce a stable isotope mass labeling technique to assign specific interacting nucleotides in an oligonucleotide-protein complex by photo-cross-linking. The method relies on generating site-specific oxygen-18-labeled phosphodiester linkages in oligonucleotides, such that covalent peptide-oligonucleotide cross-link sites arising from ultraviolet irradiation can be assigned to specific sequence positions in both RNA and protein simultaneously by mass spectrometry. Using Lin28A and a let-7 pre-element RNA, we demonstrate that mass labeling permits unambiguous identification of the cross-linked sequence positions in the RNA-protein complex.

SUBMITTER: Lelyveld VS

PROVIDER: S-EPMC4697197 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Pinpointing RNA-Protein Cross-Links with Site-Specific Stable Isotope-Labeled Oligonucleotides.

Lelyveld Victor S VS Björkbom Anders A Ransey Elizabeth M EM Sliz Piotr P Szostak Jack W JW

Journal of the American Chemical Society 20151203 49

High affinity RNA-protein interactions are critical to cellular function, but directly identifying the determinants of binding within these complexes is often difficult. Here, we introduce a stable isotope mass labeling technique to assign specific interacting nucleotides in an oligonucleotide-protein complex by photo-cross-linking. The method relies on generating site-specific oxygen-18-labeled phosphodiester linkages in oligonucleotides, such that covalent peptide-oligonucleotide cross-link si ...[more]

PMID: 26583201

Similar Datasets

Production and Application of Stable Isotope-Labeled Internal Standards for RNA Modification Analysis.

Project description:Post-transcriptional RNA modifications have been found to be present in a wide variety of organisms and in different types of RNA. Nucleoside modifications are interesting due to their already known roles in translation fidelity, enzyme recognition, disease progression, and RNA stability. In addition, the abundance of modified nucleosides fluctuates based on growth phase, external stress, or possibly other factors not yet explored. With modifications ever changing, a method to determine absolute quantities for multiple nucleoside modifications is required. Here, we report metabolic isotope labeling to produce isotopically labeled internal standards in bacteria and yeast. These can be used for the quantification of 26 different modified nucleosides. We explain in detail how these internal standards are produced and show their mass spectrometric characterization. We apply our internal standards and quantify the modification content of transfer RNA (tRNA) from bacteria and various eukaryotes. We can show that the origin of the internal standard has no impact on the quantification result. Furthermore, we use our internal standard for the quantification of modified nucleosides in mouse tissue messenger RNA (mRNA), where we find different modification profiles in liver and brain tissue.

| S-EPMC6356711 | biostudies-literature

Site-specific inter-strand cross-links of DNA duplexes.

Project description:We report the development of technology that allows inter-strand coupling across various positions within one turn of DNA. Four 2'-modified nucleotides were synthesized as protected phosphoramidites and incorporated into DNA oligonucleotides. The modified nucleotides contain either 5-atom or 16-atom linker components, with either amine or carboxylic acid functional groups at their termini, forming 10 or 32 atom (11 or 33 bond) linkages. Chemical coupling of the amine and carboxylate groups in designed strands resulted in the formation of an amide bond. Coupling efficiency as a function of trajectory distance between the individual linker components was examined. For those nucleotides capable of forming inter-strand cross-links (ICLs), coupling yields were found to depend on temperature, distance, and linker length, enabling several approaches that can control regioselective linkage. In the most favorable cases, the coupling yields are quantitative. Spectroscopic measurements of strands that were chemically cross-linked indicate that the global structure of the DNA duplex does not appear to be distorted from the B form after coupling. Thermal denaturing profiles of those strands were shifted to somewhat higher temperatures than those of their respective control duplexes. Thus, the robust amide ICLs formed by this approach are site-specific, do not destabilize the rest of the duplex, and only minimally perturb the secondary structure.

| S-EPMC3719409 | biostudies-literature

Site-specific orientation of an α-helical peptide ovispirin-1 from isotope-labeled SFG spectroscopy.

Project description:Sum-frequency generation (SFG) vibrational spectroscopy is often used to probe the backbone structures and orientations of polypeptides at surfaces. Using the ovispirin-1 polypeptide at the solid/liquid interface of polystyrene, we demonstrate for the first time that SFG can probe the polarization response of a single-isotope-labeled residue. To interpret the spectral intensities, we simulated the spectra using an excitonic Hamiltonian approach. We show that the polarization dependence of either the label or the unlabeled amide I band alone does not provide sufficient structural constraints to obtain both the tilt and the twist of the ovispirin helix at a solid/liquid interface, but that both can be determined from the polarization dependence of the complete spectrum. For ovispirin, the detailed analysis of the polarized SFG experimental data shows that the helix axis is tilted at roughly 138° from the surface normal, and the transition dipole of the isotope-labeled C═O group is tilted at 23° from the surface normal, with the hydrophobic region facing the polystyrene surface. We further demonstrate that the Hamiltonian approach is able to address the coupling effect and the structural disorder. For comparison, we also collected the FTIR spectrum of ovispirin under similar conditions, which reveals the enhanced sensitivity of SFG for structural studies of single monolayer peptide surfaces. Our study provides insight into how structural and environmental effects appear in SFG spectra of the amide I band and establishes that SFG of isotope-labeled peptides will be a powerful technique for elucidating secondary structures with residue-by-residue resolution.

| S-EPMC3896219 | biostudies-literature

Amyloid-beta isoform metabolism quantitation by stable isotope-labeled kinetics.

Project description:Abundant evidence suggests a central role for the amyloid-beta (A?) peptide in Alzheimer's disease (AD) pathogenesis. Production and clearance of different A? isoforms have been established as targets of proposed disease-modifying therapeutic treatments of AD. However, previous studies used multiple sequential purification steps to isolate the isoforms individually and quantitate them based on a common mid-domain peptide. We created a method to simultaneously purify A? isoforms and quantitate them by the specific C-terminal peptides in order to investigate A? isoform physiology in the central nervous system. By using standards generated from in vitro metabolic labeling, the relative quantitation of four peptides representing total amount of A? (A?-Total), A?38, A?40, and A?42 were achieved both in cell culture and in human cerebrospinal fluid (CSF). Standard curves for each isoform demonstrated good sensitivity with very low limits of detection and high accuracy. Because the assay does not require antibody development for each A? isoform peptide, significant improvements in the throughput and accuracy of isoform quantitation were achieved.

| S-EPMC3749773 | biostudies-literature

Stable Isotope-Labeled Lipidomics to Unravel the Heterogeneous Development Lipotoxicity.

Project description:Non-alcoholic fatty liver disease (NAFLD) as a global health problem has clinical manifestations ranging from simple non-alcoholic fatty liver (NAFL) to non-alcoholic steatohepatitis (NASH), cirrhosis, and cancer. The role of different types of fatty acids in driving the early progression of NAFL to NASH is not understood. Lipid overload causing lipotoxicity and inflammation has been considered as an essential pathogenic factor. To correlate the lipid profiles with cellular lipotoxicity, we utilized palmitic acid (C16:0)- and especially unprecedented palmitoleic acid (C16:1)-induced lipid overload HepG2 cell models coupled with lipidomic technology involving labeling with stable isotopes. C16:0 induced inflammation and cell death, whereas C16:1 induced significant lipid droplet accumulation. Moreover, inhibition of de novo sphingolipid synthesis by myriocin (Myr) aggravated C16:0 induced lipoapoptosis. Lipid profiles are different in C16:0 and C16:1-treated cells. Stable isotope-labeled lipidomics elucidates the roles of specific fatty acids that affect lipid metabolism and cause lipotoxicity or lipid droplet formation. It indicates that not only saturation or monounsaturation of fatty acids plays a role in hepatic lipotoxicity but also Myr inhibition exasperates lipoapoptosis through ceramide in-direct pathway. Using the techniques presented in this study, we can potentially investigate the mechanism of lipid metabolism and the heterogeneous development of NAFLD.

| S-EPMC6278256 | biostudies-literature

Examining the mechanism of action of a kinesin inhibitor using stable isotope labeled inhibitors for cross-linking (SILIC).

Project description:It is difficult to determine a chemical inhibitor's binding site in multiprotein mixtures, particularly when high-resolution structural studies are not straightforward. Building upon previous research involving photo-cross-linking and the use of mixtures of stable isotopes, we report a method, Stable Isotope Labeled Inhibitors for Cross-linking (SILIC), for mapping a small molecule inhibitor's binding site in its target protein. In SILIC, structure-activity relationship data is used to design inhibitor analogues that incorporate a photo-cross-linking group along with either natural or 'heavy' stable isotopes. An equimolar mixture of these inhibitor analogues is cross-linked to the target protein to yield a robust signature for identifying inhibitor-modified peptide fragments in complex mass spectrometry data. As a proof of concept, we applied this approach to an ATP-competitive inhibitor of kinesin-5, a widely conserved motor protein required for cell division and an anticancer drug target. This analysis, along with mutagenesis studies, suggests that the inhibitor binds at an allosteric site in the motor protein.

| S-EPMC3309614 | biostudies-literature

Stable isotope labeled 4-(dimethylamino)benzoic acid derivatives of glycerophosphoethanolamine lipids.

Project description:A set of four (D(0), D(4), D(6), and D(10)) deuterium enriched 4-(dimethylamino)benzoic acid (DMABA) N-hydroxysuccinimide (NHS) ester reagents was developed that react with the primary amine group of glycerophosphoethanolamine (PE) lipids to create derivatives where all subclasses of DMABA labeled PE are detected by a common precursor ion scan. The positive ion collision induced dissociation data from (D(0), D(4), D(6), and D(10))-DMABA labeled PE standards indicated that a precursor ion scan of m/z 191.1, 195.1, 197.1, and 201.1 could be used to selectively detect (D(0), D(4), D(6), and D(10))-DMABA modified PE, respectively, in a complex biological mixture. The PE lipids from a time course (0, 30, 60, and 300 min) of 2,2'-azobis-(2-amidinopropane) hydrochloride (AAPH) treatment of liposomes made of RAW 264.7 cell phospholipids were each labeled with the D(0)-, D(4)-, D(10)-, and D(6)-DMABA NHS ester reagents, respectively. The DMABA derivatives revealed loss of endogenous PE lipids and an increase in oxidized PE lipid throughout the time course of AAPH treatment. These DMABA NHS ester reagents provide a universal scan for diacyl, ether, and plasmalogen PE lipids that cannot be readily observed otherwise, enable differential labeling, and provide an internal standard for each PE lipid.

| S-EPMC2929906 | biostudies-literature

A new method for measuring cholesterol efflux capacity uses stable isotope-labeled, not radioactive-labeled, cholesterol.

Project description:The incidence of cardiovascular events correlates inversely with cholesterol efflux capacity (CEC) more than with HDL-cholesterol level. The measurement of CEC is used to qualify cardiovascular disease risk and is conventionally performed with radioisotope (RI)-labeled cholesterol. Here, we established a CEC measurement technique using stable isotope-labeled cholesterol as an alternative, and we compared the new method with RI and fluorescence (boron dipyrromethene difluoride-cholesterol) methods in cells and in patient serum. We incubated J774 cells labeled with [d 7]cholesterol ([d 7]C) with patient serum depleted of apoB, and [d 7]C extracted from the culture medium was quantified by liquid chromatography/quadrupole time-of-flight mass spectrometry. [d 7]C efflux increased with greater apoB-depleted serum concentration and longer incubation time. The assay coefficient of variation (CV) of five consecutive measurements of three sets of samples ranged from 7.3% to 9.5%, and the interassay CV determined by measuring three samples four times ranged from 4.1% to 8.5%, both indicating good precision. We then measured CEC levels of 41 outpatients with serum HDL-cholesterol levels between 36 and 94 mg/dl (mean: 61.7 ± 18.0 mg/dl); in the presence of cAMP, we observed a significant, positive correlation between CEC levels determined with the stable isotope and RI methods that was stronger than the correlation between measurements obtained by the fluorescence and RI methods (r = 0.73, P < 0.0001 vs. r = 0.55, P < 0.001). Therefore, our stable isotope method can be considered useful as a non-RI method and thus deserves evaluation in future clinical studies.

| S-EPMC6824490 | biostudies-literature

Site-specific isotope labeling of long RNA for structural and mechanistic studies.

Project description:A site-specific isotope labeling technique of long RNA molecules was established. This technique is comprised of two simple enzymatic reactions, namely a guanosine transfer reaction of group I self-splicing introns and a ligation with T4 DNA ligase. The trans-acting group I self-splicing intron with its external cofactor, 'isotopically labeled guanosine 5'-monophosphate' (5'-GMP), steadily gave a 5'-residue-labeled RNA fragment. This key reaction, in combination with a ligation of 5'-remainder non-labeled sequence, allowed us to prepare a site-specifically labeled RNA molecule in a high yield, and its production was confirmed with (15)N NMR spectroscopy. Such a site-specifically labeled RNA molecule can be used to detect a molecular interaction and to probe chemical features of catalytically/structurally important residues with NMR spectroscopy and possibly Raman spectroscopy and mass spectrometry.

| S-EPMC3245953 | biostudies-literature

In vivo calibration of microdialysis using infusion of stable-isotope labeled neurotransmitters.

Project description:In vivo calibration of microdialysis probes is required for interpreting measured concentrations. The most popular method of in vivo calibration is no-net-flux (NNF), which requires infusing several concentrations of neurotransmitters to determine in vivo recoveries (extraction fraction or Ed) and extracellular concentrations. A new method for in vivo calibration of microdialysis of neurotransmitters using glutamate (GLU) and dopamine (DA) as model analytes is reported. (13)C6-DA and (13)C5-GLU were perfused through microdialysis probes as internal calibrators. Using liquid chromatography with mass spectrometry, it was possible to distinguish the (13)C-forms from the endogenous forms of each neurotransmitter. Ed was directly calculated by measuring the loss of the (13)C-forms during infusion. The measured endogenous (12)C forms of the neurotransmitters could be corrected for Ed to give calibrated extracellular concentrations in vivo. Retrodialysis of stable-isotope-labeled (SIL) neurotransmitters gave Ed and extracellular concentrations of (13)C5-GLU and (13)C6-DA that matched no-net-flux measurements; however, the values were obtained in a fraction of time because no added measurements were required to obtain the calibration. Ed was reduced during uptake inhibition for GLU and DA when measured by SIL retrodialysis. Because Ed is directly measured at each microdialysis fraction, it was possible to monitor changes in Ed under transient conditions created by systemic injection of uptake inhibitors. The results show that DA and GLU concentrations are underestimated by as much as 50% if not corrected for Ed during uptake inhibition. SIL retrodialysis provides equivalent information to NNF at much reduced time and animal use.

| S-EPMC3656751 | biostudies-other

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data