Optical spectroscopic methods Daniel Renčiuk IBP AS CR S1001 / 2016 - CEITEC Contents 1.Brief background 2.Absorption spectroscopy (AS) 1.Electronic (UV/Vis) 2.Vibrational (IR) 3.Raman scattering 4.Emission spectroscopy 1.Fluorescence 2.FRET 3.Fluorescence polarisation / anisotropy 5.Chirooptical methods 1.Linear dichroism 2.Circular dichroism 2 Interaction of mass with EM radiation 3 QUANTUM MECHANICS SPECTROSCOPY Theory Experiment Wavefunction describes states of the molecule. Position of absorption and emission peaks correspond to differences in E between states. Phenomena X EM spectral regions Phenomenon Spectral region Wavelength Nuclear Gamma 0.1 nm Inner electrons X-rays 0.1 - 1.0 nm Ionisation UV 0 - 200 nm Valency electrons near UV / VIS 200 - 800 nm Molecular vibrations near IR / IR 0.8 - 25 μm Rotation and electron spin orientation in mag. fields Microwaves 400 μm – 30 cm Nuclear spin orientation in mag. fields Radiowaves > 100 cm 4 OPTICAL SPECTROSCOPY 5 the energy of molecular vibration is quantized rather than continuous Electron energy levels Molecular vibrations image026.png image030.png Transitions 6 0 1 2 3 4 5 6 2000 cm-1 Vibrational spectra measured in infrared region 0’ 1’ 2’ 3’ 4’ 5’ 6’ 100 cm-1 Additional structure to the vibrational and electronic spectra - rotations 0 rAB 40,000 cm-1 Electronic spectra measured in UV and VIS Morse potential – E vs rAB E = h·v = h·c/λ SPECTROSCOPIES MEASURES TRANSITION BETWEEN ENERGY STATES OF THE MOLECULE Background – Franck-Condon principle FC diagram.png UCDavis / Physical Chemistry course • Transition to an excited electronic state can be to any of the vibrational level • • Vibrational transitions are very slow, compared to electronic transitions • • Certain vertical transitions corresponding to no nuclear displacement during an electronic transition have the highest probability (Franck-Condon principle) • • Absorption band has the vibronic structure - one E0-E1 transition is a superposition of several transitions v0-vn’ characterized by different energy and probability (intensity of the peak) Vibronic structure of absorption spectra 8 wiki figure 3.jpg wiki figure 4.jpg Transition times 9 1st singlet 2nd singlet 1st triplet ground state ~ 10-8 sec In E1v0’ before fluorescence ~ 102 - 10-4 sec before fosforescence Van Holde et al., Principles of Physical Biochemistry, 2nd ed., 2006 Jablonski diagram Background – Kasha’s rule •Kasha’s rule: photon emission occurs only from the lowest excited level •As a consequence, the emission wavelength is independent of the excitation wavelength •Few exceptions from Kasha’s rule •Kasha’s rule + Franck-Condon principle stands behind the symmetry of absorption and fluorescence spectra (E0v0 to E1vn = E1v0’ to E0vn’) • 10 Background - Stokes and antistokes shift 11 11 Eex > Eem => vex > vem => λex < λem λ [nm] Stokes’s shift antistokes SYMETRY KASHA’S RULE Optical spectroscopy 12 Type of spectroscopy 13 Steady-state • continuous excitation • weak intensities of excitation light • highly populated ground state Time-resolved experiment • short excitation pulse (fs,ps) • higher intensities of excitation light (laser) • significantly populated excited states These types require different instrumentation and are used for different purposes. “pump” – light for excitation “probe” – light for measurement Either the same source or different. UV absorption spectroscopy 14 • All molecules absorb in UV – all atoms have electrons + UV has enough E to excite outer-shell electrons to higher energy orbitals • bottom λ limit – buffer absorption x O2 (<160 nm) absorption – vacuum UV – synchrotron up to 100 nm • Absorption bands are broad – vibronic structure + solution effects • Chromophore – part of the molecule that strongly absorbs in the desired region (UV/Vis) UV absorption spectroscopy 15 •Determination of concentration of nucleic acids – Beer-Lambert law • Determination of conformation of DNA – Thermal (TDS) and Isothermal (IDS) Differential Spectra • Measurement of renaturation and denaturation processes – determining of thermodynamic parameters using van’t Hoff equation • Following interactions of nucleic acids with ligands •Protonation of bases DNA absorption spectrum 16 Peak around 260 nm due to a conjugated π-bonding system (bases). Spectra of particular nucleotides depend on transition dipole moments of the bases. Final spectrum of unstructured DNA depends on primary sequence. Effect of structure 17 Sprecher et al., Biopolymers, 1977 d[G3(TTAG3)3] at 23°C in 1cm cell • final NA spectrum is based on contributions of individual monomers in primary sequence + contributions of their interactions • spectrum different for structured and non-structured NA (hypochromism around 260 nm after folding) Hypochromic effect Hyperchromic effect UV absorption – NA concentration 18 Beer-Lambert law A = c·ε·l = log10 I0/I I = I0·10-c·ε·l I0 – incident light I – output light Light intensity decreases exponentially when passing through sample thus absorbance (as log) increases linearly – 2x sample concentration or pathlength = 2x absorbance but 10x less light Optimal absorbance 0.6-0.8 Molar absorption coefficient - ε 19 A = c·ε·l ε – molar absorption coefficient [M-1.cm-1] • specific for each NA primary sequence • can be either: • calculated – 2*sum of ε of dimers minus sum of ε of monomers except the two terminal ones (Gray et al., 1995, Methods Enzymol) • • • • • • analytically determined – amount of phosphorus vs absorbance • usually calculated by DNA provider •http://eu.idtdna.com/calc/analyzer UV Absorbance – TDS (or IDS) 20 Mergny et al. Nucl. Acids Res. 2005 Normalized differential absorbance signatures: (A) DNA self-complementary duplexes, 100% AT; (B) DNA self-complementary duplexes 100% GC; (C) Z-DNA; (D) Parallel-stranded DNA; (E) GA DNA duplexes; (F) Hoogsteen DNA duplexes; (G) i-DNA; (H) Pyrimidine triplexes; (I) DNA G-quadruplexes in Na+. UV Absorbance – NA melting 21 Increasing temperature Guanine quadruplex G3(TTAG3)3 in 150 mM Na Thermodynamic parameters - Van’t Hoff 22 Mergny and Lacroix, Oligonucleotides, 2003 UV Absorbance – ligand interaction 23 NMM ligand titrated by guanine quadruplex http://orders.frontiersci.com/Orders/images/largemolecules/NMM580/mol.gif?x=200&y=200 Time-resolved absorption 24 IR absorption 25 • Measures the energies of vibration of atomic nuclei in the molecule • Each molecule has 3n-6 internal degrees of freedom (n=number of atoms in molecule) • specific absorption bands for various chemical groups • • • • • • modern IR spectrofotometers are Fourier transform instruments – Michelson interferometer + FT transformation of intensity to frequency – all frequencies taken simultaneously • water absorption in interesting IR regions – D2O (peak in other regions, films stretching In-plane bending IR absorption – group vibrations 26 4000 3000 2000 1000 0 2500 3333 5000 10000 ∞ v (cm-1) non H-bonded v (cm-1) H-bonded λ (nm) 4000 3000 2000 1000 0 H2O absorption H2O absorption OH NH CH C=O C=N PO3- SH IR absorption – Miles experiment 27 IR.TIF Miles, 1961, PNAS IR spectra in the 1750 to 1550 cm-1 region for two nontautomerizing methyl derivatives (c) and (d), and cytidine, now known to be in the first tautomeric form shown (a). Raman spectroscopy 28 Excited electronic stationary state Ground electronic stationary state 0’ 1’ 2’ 3’ 0 1 2 3 Nonstationary state vibrational absorption Rayleigh scattering Raman scattering resonance Raman scattering Band position: v01 = (Ein-Esc)/hc • when used light with E < E0-E1 – scattering • in most cases Ein = Esc – Rayleigh scattering • sometimes Ein <> Esc – Raman scattering • Ein > Esc – Stokes • Ein < Esc – antistokes • Raman photon incidence around 10-8 • Raman band position: v01 = (Ein-Esc)/hc • complementary to vibrational absorption – the same transition (0-1) • Raman – visible photon • vibrational – IR photon • some vib. transitions detected differently • nonstationary states are not quantized => any UV/Vis source may be used • practically lasers – intense monochromatic light • scattered light split by monochromator Ein Esc Raman spectroscopy 29 Deng et al., 1999, Biopolymers An external file that holds a picture, illustration, etc. Object name is gks1135f1p.jpg Palacky et al., 2013, NAR An external file that holds a picture, illustration, etc. Object name is gks1135f2p.jpg Raman spectra of G3(TTAG3)3 in 200 mM K+ (30 mM of PBS, pH 6.8, t = 5°C) at the nucleoside concentrations of 8 mM (bottom trace) and 200 mM (top trace). Intermediate traces show the differences between the spectra at indicated concentration and that of the lowest one CD Raman spectroscopy 30 Deng et al., 1999, Biopolymers poly(dA-dT) · poly(dA-dT) (0% G+C), C. perfringens DNA (27% G+C), calf thymus DNA (42% G+C), E. coli DNA (50% G+C), M. luteus DNA (72% G+C), and poly(dG-dC) · poly(dG-dC) (100% G+C). Fluorescence in nucleic acids 31 •Spontaneous emission of the photon followed by transition to electronic ground state (any vibrational state – Franck-Condon) • •Emission always from the vibrational ground state of the electronic excited state (Kasha’s rule) • •fluorescence itself very fast (10-15 s), but some time takes nonradiative conversion to v0’ • •Fluorophores – molecules/parts of the molecule that exhibit fluorescence • •Fluorescence lifetime – τ – average time from excitation of the molecule to emission of light [ns] • •Quantum yield – ratio between emitted and absorbed photons – “efficiency” of the fluorescence - max = 1, but usually lower (non-radiative transitions) 0’ 1’ 2’ 3’ 0 1 2 3 •Very low intrinsic fluorescence, thus: 1.Fluorescent base – 2-aminopurine 2. 2. 2. 2. 2.Fluorescent labels – FITC, TAMRA, … 3. 3. 3. 3. 3.Fluorescent ligand – EtBr, porphyrins, … Fluorescence in nucleic acids 32 http://www.atdbio.com/img/articles/2-aminopurine-large.png ThermoFisher Scientific Fluorescence spectra viewer Fluorescence – guanine quadruplex with 2-AP 33 An external file that holds a picture, illustration, etc. Object name is nihms164344f2.jpg Na+ and K+-dependent fluorescence emission spectra of 2-AP derivatives of Tel 22 as a function of cation concentration. Gray et al., 2010, Biochemistry An external file that holds a picture, illustration, etc. Object name is nihms164344f1.jpg An external file that holds a picture, illustration, etc. Object name is nihms164344f3.jpg Time-resolved fluorescence 34 Foerster (Fluorescence) resonance energy transfer (FRET) 35 0’ 1’ 2’ 3’ 0 1 2 3 0’ 1’ 2’ 3’ 0 1 2 3 Donor Acceptor FRET • FRET might occur when the emission band of the donor overlaps with the excitation band of the acceptor and the molecules are close enough. • FRET range 1-10 nm • Various FRET pairs, characterized by R0 (distance where FRET is 50% for this pair) • FRET efficiency E = 1 / (1 + r / R0)6 Foerster (Fluorescence) resonance energy transfer (FRET) 36 FAM – GQ – TAMRA 150 mM K Excitation: 480 nm TAMRA emission FAM emission Fluorescence polarization/anisotropy (FP/FA) 37 Difference in the intensity of the sample-emitted light with polarization parallel and perpendicular to the polarization of the excitation light. • requirements: molecules are fluorescent • FA provides information on molecular size (monomer x dimer) and shape, local viscosities of a fluorophore’s environment and allows measurement of kinetics parameters of reactions. • often as a time-resolved method for rotational velocities measurement– short pulse of light (10-9 sec) followed by fluorescence measurement over time • in this case the molecule must be spherical to avoid various rotational velocities in different directions and the fluorophore must be firmly attached to prevent rotation of the fluorophore only • • • Fluorescence polarization/anisotropy 38 x y z Iparallel Iperpendicular DETECTOR SOURCE SAMPLE Linearly polarized excitation light Fluorescence anisotropy r = Iparallel – Iperpendicular / Iparallel + 2Iperpendicular Parallel and perpendicular means orientation towards excitation light • significantly excited are only the molecules whose transition dipoles are parallel to the polarization of excitation light Fluorescence polarization anisotropy 39 Schematic illustration of the effect of rotational diffusion rate (tumbling and ... The effects of EDTA on the binding of Klentaq DNA polymerase to primed‐template ... The effects of EDTA on the binding of Klentaq DNA polymerase to primed‐template DNA (13/20‐mer DNA) LiCata et al., 2007, Methods Cell Biol Linear dichroism (LD) 40 Difference in absorption of the light linearly polarized parallelly and perpendicularly to the orientation of the molecules • requirements: molecules are oriented and molecules absorb in the region of interest • orienting the molecules: gel, electric field, flow (rotation) • LD is sensitive to the orientation of absorbing parts (nucleobases) towards the orientation of the molecule – e.g. base inclination in NA Bulheller et al., 2007, Phys Chem Chem Phys Rodger et al., 2006, Phys Chem Chem Phys LD http://pubs.rsc.org/services/images/RSCpubs.ePlatform.Service.FreeContent.ImageService.svc/ImageSer vice/Articleimage/2007/CP/b615870f/b615870f-f13.gif LD DNA + ligand 41 Dafforn et al., 2004, Curr Opin Struct Biol LD of DNA and DNA–ligand systems. (a) LD of calf thymus DNA (1000 μM base, dashed line) and the DNA plus an ethidium bromide intercalator (50 μM, solid line). (b) LD of calf thymus DNA (1000 μM base, dashed line) and the DNA plus a minor groove binder (diaminophenyl indole, 50 μM, solid line) LD of DNA and DNA–ligand systems. (a) LD of calf thymus DNA (1000μM base, dashed ... Circular dichroism (CD) 42 Difference in absorption of left-handed circularly polarized light and right-handed circularly polarized light by a molecule • requirements: molecules are chiral (sugar in NA), thus optically active and molecules absorb in the region of interest • CD is sensitive to the mutual orientation of absorbing parts (nucleobases) towards each other – base conformations (syn x anti) – secondary structure of DNA • • optical activity = ability of the molecule to differentially interact with left-handed and right-handed circularly polarized light • Optical rotatory dispersion (ORD) – angle of rotation of the linearly polarized light after passing through the optically active molecule – ORD in whole range of wavelenghts, with anomalous ORD, where molecule absorbs – more difficult interpretation than CD • Cotton effect – CD / ORD band – positive x negative • • • Circular dichroism (CD) 43 U:\MIFI\obrazky\sbírka\Macek-poekresl..jpg • Difference in absorbance: ΔA = AL – AR • • When known concentration, difference in molar absorption Δε = εL – εR = ΔA / lc (Beer-Lambert law) • • Ellipicity – the angle that describes the extent of change of the linearly polarized light into a elliptically polarized light (0 for linearly polarized, 45° for circularly polarized) tan φ = (EL – ER) / (EL + ER) = 3298 * Δε • CD can be calculated but the results do not fit well with the experiment • 44 cd2_0.gif Applied Photophysics Ltd. Zboku.tiff Circular dichroism – DNA / RNA 45 http://biology-forums.com/gallery/33_23_06_11_4_12_30.jpeg ABSORPTION CHIRALITY Shora.tiff MUTUAL ORIENTATION OF BASES 46 Transition cooperativity 47 06a 06a NON-COOPERATIVE TRANSITION COOPERATIVE TRANSITION ISODICHROIC POINT CD – NA melting 48 Molecularity 49 Johnson.TIF Vibrational / infrared CD (VCD/IRCD) 50 Keiderling et al., 1989, Biomol Spec The vibration CD and absorption spectra of homoduplex of d(GC)10 as the right-handed B-form and the left-handed Z-form. Difference in absorption of left-handed circularly polarized light and right-handed circularly polarized light in a region of vibrational transitions (λ = 1-5 um). • compared to eCD, IRCD shows well differentiated bands belonging to specific functional groups Thank you 51