RNA tertiary folding monitored by fluorescence of covalently attached pyrene.
AuthorsSilverman SK, Cech TR
JournalBiochemistry
PubMed ID10571996
'The pathways by which large RNAs adopt tertiary structure are just beginning to be explored, and new methods that reveal RNA folding are highly desirable. Here we report an assay for RNA tertiary folding in which the fluorescence of a covalently incorporated chromophore is monitored. Folding of the 160-nucleotide Tetrahymena ... More
Effects of Mg2+ and the 2' OH of guanosine on steps required for substrate binding and reactivity with the Tetrahymena ribozyme reveal several local folding transitions.
AuthorsLi Y, Turner DH
JournalBiochemistry
PubMed ID9287156
'Transient kinetic studies with fluorescence detection were used to determine the effects of Mg2+ concentration and the 2' OH group of guanosine monophosphate, prG, substrate on various steps in the transesterification reaction of prG with 5' pyrene-labeled oligonucleotides as catalyzed by the L-21 ScaI ribozyme. The effect of increasing Mg2+ ... More
Human plasma fibronectin structure probed by steady-state fluorescence polarization: evidence for a rigid oblate structure.
'In order to more clearly define the structure of human plasma fibronectin (PFn) under physiologic buffer conditions, we determined the mean harmonic rotational relaxation times (rho H) of PFn and the thrombin-derived 190/170-kDa PFn fragment using steady-state fluorescence polarization. These measurements utilized the long lifetime emission (tau = 1.2 X ... More
Spontaneous and plasma factor-mediated transfer of pyrenyl cerebrosides between model and native lipoproteins.
AuthorsVia DP, Massey JB, Vignale S, Kundu SK, Marcus DM, Pownall HJ, Gotto AM
JournalBiochim Biophys Acta
PubMed ID3931685
'A series of pyrenyl glucocerebrosides was synthesized by reacylation of psychosine with pyrene-labeled fatty acids having 3-11 methylene units. When incorporated into model high-density lipoproteins consisting of dimyristoylphosphatidylcholine-apolipoprotein A-II complexes and incubated with unlabeled complexes, these lipids exhibited spontaneous transfer. Half times of transfer varied from 1.5 min to 365 ... More
Productive and nonproductive intermediates in the folding of denatured rhodanese.
AuthorsPanda M, Gorovits BM, Horowitz PM
JournalJ Biol Chem
PubMed ID10617586
'The competition between protein aggregation and folding has been investigated using rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) as a model. During folding from a urea-denatured state, rhodanese rapidly forms associated species or intermediates, some of which are large and/or sticky. The early removal of such particles by filtration results in a ... More
Single nucleotide polymorphism detection by combinatorial fluorescence energy transfer tags and biotinylated dideoxynucleotides.
AuthorsTong AK, Ju J
JournalNucleic Acids Res
PubMed ID11861924
'Combinatorial fluorescence energy transfer (CFET) tags, constructed by exploiting energy transfer and combinatorial synthesis, allow multiple biological targets to be analyzed simultaneously. We here describe a multiplex single nucleotide polymorphism (SNP) assay based on single base extension (SBE) using CFET tags and biotinylated dideoxynucleotides (biotin-ddNTPs). A library of CFET-labeled oligonucleotide ... More
Ionic-strength- and pH-dependent conformational states of human plasma fibronectin.
'In order to provide a more detailed understanding of human plasma fibronectin (PFn) solution structure, we examined the effects of pH and ionic strength (mu) variation on the sedimentation velocities (s20,w), fluorescence polarization-derived mean harmonic rotational relaxation times (rho H), far-ultraviolet (UV) circular dichroism (CD), and intrinsic tryptophan fluorescence of ... More
Specificity of aminoglycoside binding to RNA constructs derived from the 16S rRNA decoding region and the HIV-RRE activator region.
AuthorsWang Y, Hamasaki K, Rando RR
JournalBiochemistry
PubMed ID9020774
'Aminoglycoside antibiotics can bind to many different types of RNA molecules. It was of interest to determine the nature of the selectivity of binding of aminoglycosides to important, biologically relevant RNA targets. Fluorescence anisotropy methods were developed to quantitatively measure aminoglycoside affinities to constructs of the HIV-1 RRE transcriptional activation ... More
Structural changes induced by binding of the high-mobility group I protein to a mouse satellite DNA sequence.
AuthorsSlama-Schwok A, Zakrzewska K, Léger G, Leroux Y, Takahashi M, Käs E, Debey P
JournalBiophys J
PubMed ID10777751
'Using spectroscopic methods, we have studied the structural changes induced in both protein and DNA upon binding of the High-Mobility Group I (HMG-I) protein to a 21-bp sequence derived from mouse satellite DNA. We show that these structural changes depend on the stoichiometry of the protein/DNA complexes formed, as determined ... More
Structure and drug interactions of parallel-stranded DNA studied by infrared spectroscopy and fluorescence.
AuthorsFritzsche H, Akhebat A, Taillandier E, Rippe K, Jovin TM
JournalNucleic Acids Res
PubMed ID7504812
'The infrared spectra of three different 25-mer parallel-stranded DNAs (ps-DNA) have been studied. We have used ps-DNAs containing either exclusively dA x dT base pairs or substitution with four dG x dC base pairs and have them compared with their antiparallel-stranded (aps) reference duplexes in a conventional B-DNA conformation. Significant ... More
Time-resolved detection of energy transfer: theory and application to immunoassays.
AuthorsMorrison LE
JournalAnal Biochem
PubMed ID3218725
'Energy-transfer measurements based upon acceptor fluorophore emission are plagued with background fluorescence resulting from absorption of the excitation light by the acceptor fluorophore. The present work examines the use of a long-lifetime donor fluorophore and a short-lifetime acceptor fluorophore, combined with pulsed-laser excitation and electronic gating of detector signals, to ... More
Novel cytotoxic DNA sequence and minor groove targeted photosensitizers: conjugates of pyrene and netropsin analogues.
AuthorsHartley JA, Webber J, Wyatt MD, Bordenick N, Lee M
JournalBioorg Med Chem
PubMed ID7582941
'The design, syntheses, photochemical and biological properties of conjugates of pyrene with pyrrole- (1) and imidazole-containing (2) analogues of netropsin are reported. The results of an ethidium displacement assay and circular dichroism (CD) titration studies show both compounds bind with a higher affinity to poly(dA-dT) than to poly(dG-dC). In addition ... More
Alternating d(G-A) sequences form a parallel-stranded DNA homoduplex.
AuthorsRippe K, Fritsch V, Westhof E, Jovin TM
JournalEMBO J
PubMed ID1396571
'The oligonucleotides d[(G-A)7G] and d[(G-A)12G] self-associate under physiological conditions (10 mM MgCl2, neutral pH) into a stable double-helical structure (psRR-DNA) in which the two polypurine strands are in a parallel orientation in contrast to the antiparallel disposition of conventional B-DNA. We have characterized psRR-DNA by gel electrophoresis, UV absorption, vacuum ... More
Osmotic response in Lactobacillus casei ATCC 393: biochemical and biophysical characteristics of membrane.
AuthorsMachado MC, López CS, Heras H, Rivas EA
JournalArch Biochem Biophys
PubMed ID14725858
'The biochemical and biophysical properties of the membrane and some general characteristics of the response of Lactobacillus casei ATCC 393 (reclassified Lactobacillus zeae) to hyperosmotic conditions were studied. Under hypertonic conditions, the hydrophobicity and the bile salt sensitivity of the cultures were increased. The glycolipid AcylH3DG is only present in ... More
Noncovalent sidewall functionalization of single-walled carbon nanotubes for protein immobilization.
AuthorsChen RJ, Zhang Y, Wang D, Dai H
JournalJ Am Chem Soc
PubMed ID11457124
Fluorescence assays to study structure, dynamics, and function of RNA and RNA-ligand complexes.
AuthorsWalter NG, Burke JM
JournalMethods Enzymol
PubMed ID10829293
Coumarin-phalloidin: a new actin probe permitting triple immunofluorescence microscopy of the cytoskeleton.
AuthorsSmall JV, Zobeley S, Rinnerthaler G, Faulstich H
JournalJ Cell Sci
PubMed ID2458368
7-Diethylamino-3-(4-isothiocyanotophenyl)-4-methylcoumarin (CPITC) was coupled to amino-methyldithiolanophalloidin to produce a new phalloidin derivative, coumarin-phalloidin, fluorescent in the blue region of the spectrum. Coumarin-phalloidin binds to actin with around 100-fold less affinity than unconjugated phalloidin, but with enough avidity to make it a useful stain for actin filaments. Appropriate filter combinations permit ... More
Simultaneous determination of histamine and histidine by liquid chromatography following intramolecular excimer-forming fluorescence derivatization with pyrene-labeling reagent.
AuthorsYoshida H, Ichinose F, Yoshitake T, Nakano Y, Todoroki K, Nohta H, Yamaguchi M
JournalAnal Sci
PubMed ID15068305
A highly sensitive and selective fluorometric method for the determination of histamine and histidine has been developed. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent followed by reversed-phase liquid chromatography. The analytes, containing two amino moieties in a molecule, were converted to the corresponding ... More
Optimal configuration of pyrene excimer induced by the excimer-forming two-probe nucleic acid hybridization method.
AuthorsMasuko M, Toyoda S, Suwa M, Mitaku S, Shimazu A, Ohtani H
JournalNucleic Acids Symp Ser
PubMed ID9586010
Excimer-forming two-probe nucleic acid hybridization (ETPH) method with pyrene as a fluorophore enables homogeneous hybridization assays. We examined the effect of linker length between a pyrene residue and a terminal sugar moiety on Tm of hybrids in the presence of 20% dimethylformamide (DMF). The results including those of CD measurements ... More
Ultrasensitive carbon nanotube-based biosensors using antibody-binding fragments.
AuthorsKim JP, Lee BY, Hong S, Sim SJ,
JournalAnal Biochem
PubMed ID18640089
We report a method to build ultrasensitive carbon nanotube-based biosensors using immune binding reaction. Here carbon nanotube-field effect transistors (CNT-FETs) were functionalized with antibody-binding fragments as a receptor, and the binding event of target immunoglobulin G (IgG) onto the fragments was detected by monitoring the gating effect caused by the ... More
Targeted generation of DNA strand breaks using pyrene-conjugated triplex-forming oligonucleotides.
AuthorsBenfield AP, Macleod MC, Liu Y, Wu Q, Wensel TG, Vasquez KM,
JournalBiochemistry
PubMed ID18473480
Gene targeting by triplex-forming oligonucleotides (TFOs) has proven useful for gene modulation in vivo. Photoreactive molecules have been conjugated to TFOs to direct sequence-specific damage in double-stranded DNA. However, the photoproducts are often repaired efficiently in cells. This limitation has led to the search for sequence-specific photoreactive reagents that can ... More
Selective fluorometric detection of polyamines using micellar electrokinetic chromatography with laser-induced fluorescence detection.
AuthorsPaproski RE, Roy KI, Lucy CA
JournalJ Chromatogr A
PubMed ID11873974
The polyamines putrescine, cadaverine, spermine and spermidine were separated and quantified by micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence detection. The derivatization reagent, 1-pyrenebutanoic acid succinimidyl ester (PSE), allowed for the selective detection of the polyamines at 490 nm. Multiple labeling of the polyamines with PSE allows the formation of ... More
Triple helix formation and homologous strand exchange in pyrene-labeled oligonucleotides.
AuthorsMohammadi S, Slama-Schwok A, Léger G, el Manouni D, Shchyolkina A, Leroux Y, Taillandier E
JournalBiochemistry
PubMed ID9398205
The orientations of the symmetrical third strands (G3A4G3) and (G3T4G3) within the triplexes (C3T4C3) - (G3A4G3) x (G3A4G3) and (C3T4C3) - (G3A4G3) x (G3T4G3) were investigated by fluorescence spectroscopy and thermal denaturation using pyrene-labeled oligodeoxynucleotides. In the two triplex structures, both parallel and antiparallel orientations of the third strand with ... More
Binary system for selective photoaffinity labeling of base excision repair DNA polymerases.
AuthorsLavrik OI, Kolpashchikov DM, Prasad R, Sobol RW, Wilson SH
JournalNucleic Acids Res
PubMed ID12136121
A system of photoaffinity reagents for selective labeling of DNA polymerases in extracts has been examined. To create the photoreactive DNA probe in situ, DNA substrates containing a synthetic abasic site are incubated in mouse embryonic fibroblast (MEF) cellular extract in the presence of base-substituted arylazido derivatives of dNTPs. This ... More
Highly selective fluorometric determination of polyamines based on intramolecular excimer-forming derivatization with a pyrene-labeling reagent.
AuthorsNohta H, Satozono H, Koiso K, Yoshida H, Ishida J, Yamaguchi M
JournalAnal Chem
PubMed ID10994984
We introduce a novel approach in highly selective and sensitive fluorescence derivatization of polyamines. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester (PSE), followed by reversed-phase high-performance liquid chromatography (HPLC). Polyamines, having two to four amino moieties in a molecule, ... More
Solution-phase detection of polynucleotides using interacting fluorescent labels and competitive hybridization.
AuthorsMorrison LE, Halder TC, Stols LM
JournalAnal Biochem
PubMed ID2624314
DNA was assayed in a homogeneous format using DNA probes containing hybridization-sensitive labels. The DNA probes were prepared from complementary DNA strands in which one strand was covalently labeled on the 5'-terminus with fluorescein and the complementary strand was covalently labeled on the 3'-terminus with a quencher of fluorescein emission, ... More
Conformational changes of tarantula (Eurypelma californicum) haemocyanin detected with a fluorescent probe, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole.
AuthorsLeidescher T, Decker H
JournalEur J Biochem
PubMed ID2406137
Different fluorescent labels were tested in order to monitor conformational transitions of the four-hexamer haemocyanin from the tarantula Eurypelma californicum during the oxygenation process. When the four-hexamer was labelled with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, the maximum wavelength lambda max of the fluorescence emission spectrum was significantly shifted up to 5 nm, depending on ... More
Biotin-pyrene conjugates with poly(ethylene glycol) spacers are convenient fluorescent probes for avidin and streptavidin.
AuthorsMarek M, Kaiser K, Gruber HJ
JournalBioconjug Chem
PubMed ID9258456
Conventional biotin-fluorophore conjugates with approximately 14 atom spacers are strongly quenched when bound to avidin or streptavidin, whereas fluorescence becomes insensitive to receptor binding if typical fluorophores are linked to biotin via poly(ethylene glycol) (PEG) chains (Gruber et al., see the second of three papers in this issue). In the ... More
Molecular mechanisms of peptide loading by the tumor rejection antigen/heat shock chaperone gp96 (GRP94).
AuthorsSastry S, Linderoth N
JournalJ Biol Chem
PubMed ID10207025
Complexes of gp96/GRP94 and peptides have been shown to elicit immunogenicity. We used fluorescence to understand peptide association with gp96. A pyrene-peptide conjugate was complexed with gp96 under a variety of conditions. At room temperature in low salt (20 mM NaCl), the peptide binds gp96 with a strong affinity (approximately ... More
Conditions for nucleotide-dependent GroES-GroEL interactions. GroEL14(groES7)2 is favored by an asymmetric distribution of nucleotides.
A still unresolved question regarding the mechanism of chaperonin-assisted protein folding involves the stoichiometry of the GroEL-GroES complex. This is important, because the activities of the Escherichia coli chaperonin GroEL are modulated by the cochaperonin GroES. In this report, the binding of GroES to highly purified GroEL in the presence ... More
Reactive oxygen species-producing site in hydrogen peroxide-induced apoptosis of human peripheral T cells: involvement of lysosomal membrane destabilization.
AuthorsOgawa Y, Kobayashi T, Nishioka A, Kariya S, Ohnishi T, Hamasato S, Seguchi H, Yoshida S
JournalInt J Mol Med
PubMed ID14767567
In our previous study, we examined the effect of exogenous hydrogen peroxide, which causes a potent oxidative stress and has been demonstrated to be a potent apoptosis-inducer in many kinds of cells. We found that the addition of 1 or 10 mM hydrogen peroxide induced reactive oxygen species (ROS) formation, ... More
Multiple folding pathways for the P4-P6 RNA domain.
AuthorsSilverman SK, Deras ML, Woodson SA, Scaringe SA, Cech TR
JournalBiochemistry
PubMed ID11015228
We recently described site-specific pyrene labeling of RNA to monitor Mg(2+)-dependent equilibrium formation of tertiary structure. Here we extend these studies to follow the folding kinetics of the 160-nucleotide P4-P6 domain of the Tetrahymena group I intron RNA, using stopped-flow fluorescence with approximately 1 ms time resolution. Pyrene-labeled P4-P6 was ... More