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.

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.

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)).

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.

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.

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.

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.

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].

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.

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.

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.

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.

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.

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.

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).