Structural and dynamic changes of the serum response element and the core domain of serum response factor induced by their association.

Transcriptional activity of serum response factor (SRF) is dependent on its binding to the CC(A/T)(6)GG box (CArG box) of serum response element (SRE). By Raman spectroscopy, we carried out a comparative analysis, in solution, of the complexes obtained from the association of core-SRF with 20-mer SREs bearing wild-type and mutated c-fos CArG boxes. In case of association with the wild type c-fos CArG box, the complex does not bring out the expected Raman signature of a stable bending of the targeted SRE but keeps a bend-linear conformer oligonucleotide interconversion. The linear conformer population is larger than that of free oligonucleotide. In the core-SRF moiety of the wild-type complex a large spectral change associated with the CO-groups from Asp and/or Glu residues shows that their ionization states and the strength of their interactions decrease as compared to those of mutated non-specific complexes. Structural constraints evidenced on the free core-SRF are released in the wild-type complex and environmental heterogeneities appear in the vicinity of Tyr residues, due to higher water molecule access. The H-bonding configuration of one Tyr OH-group, in average, changes with a net transfer from H-bond acceptor character to a combined donor and acceptor character. A charge repartition distributed on both core-SRF and targeted SRE stabilizes the specific complex, allowing the two partners to experience a variety of conformations.

Raman tweezers microspectroscopy of circa 100 nm extracellular vesicles.

The technique of Raman tweezers microspectroscopy (RTM) for the global biomolecular content characterization of a single extracellular vesicle (EV) or a small number of EVs or other nanoscale bioparticles in an aqueous dispersion in the difficult-to-access size range of near 100 nm is described in detail. The particularities and potential of RTM are demonstrated using the examples of DOPC liposomes, exosomes from human urine and rat hepatocytes, and a mixed sample of the transfection reagent FuGENE in diluted DNA solution. The approach of biomolecular component analysis for the estimation of the main biomolecular contributions (proteins, lipids, nucleic acids, carotenoids, etc.) is proposed and discussed. Direct Raman evidence for strong intra-sample biomolecular heterogeneity of individual optically trapped EVs, due to variable contributions from nucleic acids and carotenoids in some preparations, is reported. On the basis of the results obtained, we are making an attempt to convince the scientific community that RTM is a promising method of single-EV research; to our knowledge, it is the only technique available at the moment that provides unique information about the global biomolecular composition of a single vesicle or a small number of vesicles, thus being capable of unravelling the high diversity of EV subpopulations, which is one of the most significant urgent challenges to overcome. Possible RTM applications include, among others, searching for DNA biomarkers, cancer diagnosis, and discrimination between different subpopulations of EVs, lipid bodies, protein aggregates and viruses.

Time-resolved microspectrofluorometry and fluorescence imaging techniques: study of porphyrin-mediated cellular uptake of oligonucleotides.

Time-resolved confocal microspectrofluorometry and fluorescence microscopy imaging were applied to monitor the cellular uptake of fluorescent-labeled oligonucleotides (ONs) delivered by a porphyrin molecule. The fate of porphyrin-ON complexes inside living cells has also been monitored. Due to intrinsic fluorescence of the porphyrin and sensitivity of its characteristics to microenvironment, multicomponent analysis of time-resolved fluorescence provides unique information about stability of the porphyrin-ON complexes, ON interactions with their target sequences, and ON and porphyrin distributions after delivery inside the cells. Time-resolved confocal microspectrofluorometry indeed delivers additional information compared with fluorescence confocal microscopy imaging widely employed to study ON uptake.

C-->G base mutations in the CArG box of c-fos serum response element alter its bending flexibility. Consequences for core-SRF recognition.

By binding to the CArG box sequence, the serum response factor (SRF) activates several muscle-specific genes, as well as genes that respond to mitogens. The core domain of the SRF (core-SRF) binds as a dimer to the CArG box C-5C-4A-3T-2A-1T+1T+2A+3G+4G+5 of the c-fos serum response element (SREfos). However, previous studies using 20-mer DNAs have shown that the binding stoichiometry of core-SRF is significantly altered by mutations C-5-->G (SREGfos) and C-5C-4-->GG (SREGGfos) of the CArG box [A Huet, A Parlakian, M-C Arnaud, J-M Glandières, P Valat, S Fermandjian, D Paulin, B Alpert & C Zentz (2005) FEBS J272, 3105-3119]. To understand these effects, we carried out a comparative analysis of the three 20-mer DNAs SREfos, SREGfos and SREGGfos in aqueous solution. Their CD spectra were of the B-DNA type with small differences generated by variations in the mutual arrangement of the base pairs. Analysis by singular value decomposition of a set of Raman spectra recorded as a function of temperature, revealed a premelting transition associated with a conformational shift in the DNA double helices from a bent to a linear form. Time-resolved fluorescence anisotropy shows that the fluorescein reporter linked to the oligonucleotide 5'-ends experiences twisting motions of the double helices related to the interconversion between bent and linear conformers. The three SREs present various bent populations submitted, however, to particular internal dynamics, decisive for the mutual adjustment of binding partners and therefore specific complex formation.

DNA Electric Charge Oscillations Govern Protein-DNA Recognition.

The transcriptional activity of the serum response factor (SRF) protein is triggered by its binding to a 10-base-pair DNA consensus sequence designated the CArG box, which is the core sequence of the serum response element (SRE). Sequence-specific recognition of the CArG box by a core domain of 100 amino acid residues of SRF (core-SRF) was asserted to depend almost exclusively on the intrinsic SRE conformation and on the degree of protein-induced SRE bending. Nevertheless, this paradigm was invalidated by a temperature-dependent Raman spectroscopy study of 20-mer oligonucleotides involved in bonding interactions with core-SRF that reproduced both wild type and mutated c-fos SREs. Indeed, the SRE moieties that are complexed with core-SRF exhibit permanent interconversion dynamics between bent and linear conformers. Thus, sequence-specific recognition of the CArG box by core-SRF cannot be explained only in terms of the three-dimensional structure of the SRE. A particular dynamic pairing process discriminates between the wild type and mutated complexes. Specific oscillations of the phosphate charge network of the SRE govern the recognition between both partners rather than an intrinsic set of conformations of the SRE.

Organization of the MADS box from human SRF revealed by tyrosine perturbation.

MADS box family transcription factors are involved in signal transduction and development control through DNA specific sequence recognition. The DNA binding domain of these proteins contains a conservative 55-60 amino acid sequence which defines the membership of this large family. Here we present a thorough study of the MADS segment of serum response factor (MADS(SRF)). Fluorescence, UV-absorption, and Raman spectroscopy studies were performed in order to disclose its behavior and basic functional properties in an aqueous environment. The secondary structure of MADS(SRF) estimated by analysis of Raman spectra and supported by CD has revealed only the C-terminal part as homologous with those of free core-SRF, while the N-terminal part has lost the stable α-helical structure found in both the free core-SRF and its specific complex with DNA. The three tyrosine residues of the MADS(SRF) were used as spectroscopic inner probes. The effect of environmental conditions, especially pH variations and addition of variously charged quenchers, on their spectra was examined. Two-component fluorescence quenching was revealed using factor analysis and corresponding Stern-Volmer constants determined. Factor analysis of absorbance and fluorescence pH titration led to determination of three dissociation constants pKa1 = 6.4 ± 0.2, pKa2 = 7.3 ± 0.2, and pKa3 = 9.6 ± 0.6. Critical comparison of all experiments identified the deprotonation of His193 hydrogen bonded to Tyr195 as a candidate for pKa1 (and that of Tyr158 as a candidate for pKa2). Within MADS(SRF), His193 is a key intermediary between the N-terminal primary DNA binding element and the hydrophobic C-terminal protein dimerization element.

Interaction of phospholipid dispersions with water-soluble porphyrins as monitored by their Raman temperature profiles.

Raman scattering spectra of 1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DPPG) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) dispersions, mixed with water-soluble porphyrins, i.e. cationic copper(II)-5,10,15,12-tetrakis(4-N-methylpyridyl) and anionic silver(II)-5,10,15,20-tetrakis(4-carboxyphenyl)porphyrins, were measured in the 2800-3100 cm(-1) C-H stretching vibration region as a function of the temperature within the 5-55 degrees C range. Temperature profiles of Raman data were constructed from a quantitative data treatment based on factor analysis. This method is shown to be more efficient than the commonly used approach employing peak intensity ratios. Parameters of the gel phase to liquid crystal phase transition determined from Raman temperature profiles were used to monitor the porphyrin influence on DPPG and DPPC structures. Both negatively and positively charged porphyrins significantly perturb DPPC and DPPG dispersions, causing significant downshift of the transition temperature and broadening of the transition region. Water-soluble porphyrins are assumed to set at the outside part of phospholipid dispersions and interact via coulombic forces with charged lipid heads. For the cationic CuTMPyP, the strongest effect has been observed for negatively charged DPPG. In contrast, anionic AgTPPC4 has been found to interact more efficiently with DPPC possessing both positive and negative charges.

Protonation effect of tyrosine in a segment of the SRF transcription factor: a combined optical spectroscopy, molecular dynamics, and density functional theory calculation study.

The high sensitivity to pH of a short segment (an octamer) of serum response factor (SRF), an important member of the MADS box family of transcription factors, was investigated by Raman scattering, infrared and circular dichroism spectroscopies. Molecular dynamics (MD) and density functional theory (DFT) calculations enabled interpretation of spectral changes in close detail. Although there was a negligible difference between spectra in acidic and neutral environments, the spectrum in basic pH was substantially different. The major changes were attributed to the deprotonation of tyrosine. The secondary structure of the SRF octamer fragment was estimated experimentally as well as predicted theoretically by MD. All techniques proved that it exists in a dynamical equilibrium among several conformations mostly close to ß turn, unordered conformations, and extended structure, in contrast to the stable secondary structure it possesses as a part of SRF. Generally, this approach represents a useful tool for the study of various short oligopeptides.

Fast characterisation of cell-derived extracellular vesicles by nanoparticles tracking analysis, cryo-electron microscopy, and Raman tweezers microspectroscopy.

The joint use of 3 complementary techniques, namely, nanoparticle tracking analysis (NTA), cryo-electron microscopy (Cryo-EM) and Raman tweezers microspectroscopy (RTM), is proposed for a rapid characterisation of extracellular vesicles (EVs) of various origins. NTA is valuable for studying the size distribution and concentration, Cryo-EM is outstanding for the morphological characterisation, including observation of vesicle heterogeneity, while RTM provides the global chemical composition without using any exogenous label. The capabilities of this approach are evaluated on the example of cell-derived vesicles of Dictyostelium discoideum, a convenient general model for eukaryotic EVs. At least 2 separate species differing in chemical composition (relative amounts of DNA, lipids and proteins, presence of carotenoids) were found for each of the 2 physiological states of this non-pathogenic microorganism, that is, cell growth and starvation-induced aggregation. These findings demonstrate the specific potency of RTM. In addition, the first Raman spectra of human urinary exosomes are reported, presumably constituting the primary step towards Raman characterisation of EVs for the purpose of human diseases diagnoses.

Structural features of a central mismatch in oligonucleotide hybrid duplexes visualized via Raman spectroscopy: model system for evaluation of potential "antisense" drugs.

Structural features of mismatched base pairs were studied on four nonamer hybrid duplexes formed between the 5'-d(GTGATATGC)-3' complement and its 5'-r(GCAUNUCAC)-3' (N = A, C, G, U) counterparts. This oligonucleotide set is considered a model molecular system for future systematic studies of various modifications of internucleotide linkages with respect to their impact on the structure of mismatched base pairs. Raman spectra, measured at 15 degrees C, revealed the prevailing A-like structure of the RNA strand and mixed A-like and B-like characteristics for the DNA strand. All three mismatches disturb only weakly the overall conformation of the hybrid duplex in contrast to analogous mismatched DNA duplexes. In particular, the dT x rG mismatch influences the global hybrid duplex geometry almost negligibly. The dT x rC and dT x rU mismatches induce somewhat more pronounced distortions of the backbone structure and of the thymine position, the latter being expressed by a change of the surrounding methylene group without effect on the carbonyl's vibrations. Structural effects of the mismatches correlate well with the duplex thermodynamic stabilities obtained by ultraviolet (UV) absorption, i.e., the dT x rG mismatch decreases the hybrid duplex stability very weakly while the effect of both pyrimidine-pyrimidine mismatches is considerable.

Probing of porphyrin surface chemistry in systems with laser-ablated Ag nanoparticle hydrosol: role of thiosulfate anions.

The influence of sodium thiosulfate (THS) concentration in Ag colloid/THS/H(2)TMPyP and Ag colloid/H2TMPyP/THS systems (H2TMPyP = 5,10,15,20-tetrakis(1-methyl-4-pyridyl)porphyrin) was investigated by a combination of surface-enhanced resonance Raman scattering (SERRS) spectroscopy, surface plasmon extinction (SPE) measurements, and transmission electron microscopy (TEM). THS was found to have a strong impact on Ag nanoparticle surface structure and aggregation state and on interaction with H2TMPyP probe molecules, as evidenced by variations of the SERRS spectrum. In the Ag colloid/THS/H2TMPyP system, when laser-ablated Ag colloid was THS pretreated prior to the porphyrin addition, a critical threshold THS concentration (4 x 10(-5) M) was discovered. At concentrations below the threshold, THS mainly reduces the number of Ag+ adsorption sites. This leads to increased Ag nanoparticle aggregation prior to the porphyrin addition and significant weakening of the overall SERRS signal. Dominant contributions in the SERRS spectrum correspond to free base H2TMPyP and Ag+ containing the AgTMPyP form. At concentrations above the threshold, THS mediates also the formation and stabilization of new adsorption sites, probably Ag(0) sites. This induces a turn in the aggregation state of the pretreated Ag-c/THS system, an increase of the overall SERRS signal, and the appearance of a new spectral form of Ag metalated porphyrin.

Raman study of potential "antisense" drugs: nonamer oligonucleotide duplexes with a central mismatch as a model system for the binding selectivity evaluation.

A set of four 9-mer oligonucleotide duplexes formed between the 5'-GCATNTCAC-3', N=A,C,T,G, and the 5'-GTGATATGC-3' complement has been proposed as a model system for the investigation of novel oligonucleotide analogues (candidates for antisense use) binding selectivity. Raman measurements were carried out on a set of natural DNA 9-mer in order to verify suitability of the model and to obtain reference spectral data. Difference Raman spectra between the mismatch and match duplexes obtained at 15 degrees C exhibited numerous spectral features sensitively indicating the structural changes. All the three mismatches only very weakly disturb the overall B-form conformation of the duplex. Significant structural changes that occurred at the mismatch site are reflected mainly by the neighboring thymidine Raman bands at 1377, 1650, and 1675 cm(-1). The intensity change of the two latter bands is almost the same for the T:G and the T:T mismatch while in the case of the T:C mismatch it is just opposite, demonstrating a very different arrangement of the mismatched pair.

Structural features of two distinct molecular complexes of copper(II) cationic porphyrin and deoxyribonucleotides.

The associations of the water-soluble cationic copper(II)-5,10,15,20-meso-tetrakis(4-N-methylpyridyl) porphyrin (CuP) with d(pT)9 oligothymidylate and its building blocks deoxythymidine (dT) and deoxythymidine 5'-monophosphate (dTMP) were investigated by spectrophotometric titration [absorption, nanosecond transient resonance Raman (ns-RR) and picosecond time-resolved resonance Raman (ps-TR3) spectroscopies] to elucidate the structural requirements for the CuP exciplex formation in molecular complexes with unchained mononucleotides. In the d(pT)9 a factor analysis and global fit of the CuP absorption spectra revealed the formation of a single spectral species attributable to a 1 : 1 CuP. d(pT)9 complex throughout a wide range of d(pT)9/CuP ratios (0-10). Using ps-TR3 spectroscopy, the CuP. d(pT)9 complex was shown to be fully responsible for exciplex formation. In contrast, CuP mixed with dTMP ([dTMP]/[CuP] < 3000) yielded two spectroscopically distinct types of molecular complexes with 1 : 1 (CuP. dTMP) and 1 : 2 (CuP. (dTMP)2) (or even higher for [dTMP]/[CuP] > 3000) stoichiometry, the latter being spectroscopically identical to the CuP. d(pT)9 and providing a microenvironment favorable for exciplex formation to the same extent as the oligothymidylate. On the other hand, the 1 : 1 CuP. dTMP complex (prevailing for [dTMP]/[CuP] < 100) yielded no exciplex features. Similar behavior was observed for the CuP complexed with dT. To explain the difference in the ability of the CuP. dTMP and CuP. (dTMP)2 species to form the exciplex, two types of molecular complexes were suggested and discussed, differing in the orientation of the thymine planes with respect to the porphyrin macrocycle.

Intracellular uptake of modified oligonucleotide studied by two fluorescence techniques.

Interaction, i.e., cellular uptake and intracellular distribution, of synthetic modified antisense oligonucleotide with the B16 melanoma cell line was studied using cationic polyene antibiotic, amphotericin B 3-dimethylaminopropyl amide, as a carrier vector. The antisense oligonucleotide--dT(15) oligomer analogue containing isopolar, nonisosteric, phosphonate-based internucleotide linkages 3'-O-P-CH(2)-O-5'--was labeled with fluorescent tetramethylrhodamine marker. The oligonucleotide itinerancy across the cell membrane and its distribution inside the cell was visualized using fluorescence microimaging. During the first several hours a strong preference staining of the cell nucleus was found. Fluorescence lifetime measurements from the intracellular environment (confocal laser microspectrofluorimeter, frequency domain phase/modulation technique in 1 to 200 MHz frequency region) yielded two spectral components of 4.9 and 1.4 ns lifetime, respectively. While the former component correlates with the previously characterized effect of the fluorophore binding to biomolecular targets in membranes and/or cytoplasm, the latter component is newly observed and its possible origin is discussed.

Spectral decomposition of intracellular complex fluorescence using multiple-wavelength phase modulation lifetime determination: technical approach and preliminary applications.

Lifetime-based spectral decomposition using a frequency-domain phase/modulation technique is developed on a microspectrofluorimeter prototype. In a fluorescent mixture with strongly overlapping components, such measurements enable us to not only obtain excited state lifetimes of each fluorescent component but also determine the specific spectral contribution of each species without the use of any model spectra. Examples of such applications are first given for complex mixtures of highly overlapping fluorescent components in solution. Preliminary results concerning cellular applications are also reported. This allows us to follow the cellular uptake and intracellular stability of fluorescent labeled modified oligonucleotides in the context of antisense strategy studies. Indeed, the intracellular signal from the fluorescent label bound to oligonucleotides can be distinguished from those of the free label by its specific excited state lifetime.