THz-SPECTROSCOPY OF BIOLOGICAL MOLECULES

1. THz-SPECTROSCOPY OF BIOLOGICAL MOLECULES T. R. Globus1, D. L. Woolard2, T. Khromova1, T. W. Crowe1, M. Bykhovskaia3, B. L. Gelmont1, J. Hesler1, and A. C. Samuels4 1 University of Virginia, Charlottesville, VA; 2 U.S. Army Research Laboratory, RTP, NC; 3 Dept. of Biological Sciences, Lehigh University, Bethlehem, PA; 4 U.S. Army SCBRD, Edgewood, MD hn THz-BRIDGE Workshop 2002

2. Objectives • To discover and understand the fundamental physical principles governing the interactions of biological molecules with electromagnetic radiation in the terahertz region of the spectrum, called “the dead land”. • To establish the initialfoundation for the future use of terahertz • spectroscopy in the identification and characterization of • biological macromolecules and related materials. THz-BRIDGE Workshop 2002

3. Introduction The far-infrared region of DNA and RNA absorption spectra (2 - 300 cm-1) reflects low-frequency molecular internal motions. The resonant frequencies of such motions – phonon modes- are strongly dependent on the weak hydrogen bonds of the double-helix base-pairs and non-bonded interactions between different functional groups.1 INTERNAL MOLECULAR VIBRATIONS AND FAR IR ABSORPTION SPECTRA Bonds >700 cm-1 Bond angles 200-900 cm-1 Torsion angles <300 cm-1 THz-BRIDGE Workshop 2002

4. MOTIVATION • Sub-mm wave range of DNA absorption spectra (2-300 cm-1) is predicted to be fairly rich with spectral features which can give information regarding three-dimensional structure and flexibility of DNA double helix. • DNA three-dimensional structure and flexibility determines • biological function • DNA low-frequency deformations are extremely sensitive to its nucleotide composition and structure, have an impact on the main processes related to transferring of genetic information and should reflect features specific to the DNA code. • Submillimeter-wave spectroscopy combined with theoretical prediction has a potential to become a powerful tool for investigating DNA and RNA topology and internal motions. THz-BRIDGE Workshop 2002

5. Scattering or absorption spectroscopy? Scattering and absorption spectroscopy utilize the interactionof an applied electromagnetic (EM) field with the phonon (vibration) field of the material to provide useful structural information . Absorption Raman scattering hn1 hnlight I1 hnvibr Exited state Ground state hnvibr. hn2 hnlight I2 hnvibr. = hn1-hn2 hn1, hn2 >> hnvibr. hnvibr.=hnlight THz-BRIDGE Workshop 2002

6. Scattering or absorption spectroscopy? • The absorption and scattering of light can be used for characterization of bioparticles. • The Raman effect is a second-order optical process that produces frequency shifts in the optical spectra of radiation. Raman is especially effective in the characterization of periodic microstructures process. • DNA polymers are of low symmetry, that is not highly periodic in space. Hence, a DNA chain represents multiple oscillator structure and none of the spectral line are truly forbidden. The oscillators contributing to any individual frequency have a low density per unit volume. • The detection of DNA phonons via resonant absorption is not a very strong process. However, resonant absorption is a first order optical process that is much more favorable in comparison with a second order- Raman scattering. THz-BRIDGE Workshop 2002

7. “THE WORLD OF THE DEAD OR OF FUTURE PUNISHMENT". The spectral range between the upper end of the microwave and the lower end of the extreme far IR Many challenges for experimentator arise from: • Low energy of sources. • Low absorption of biological material requires samples with large area and thickness which is difficult to make because samples are too fragile. • High absorption by water masks absorption by biological materials in solutions. • Many reasons for very poor reproducibility of experimental results. The most serious reason – multiple reflection in measurement systems, responsible for artificial features. THz-BRIDGE Workshop 2002

8. Questions to answer: • Is there something in the very far IR spectra?(initial prediction of vibrational modes in polymer DNA in the 1-100 cm-1 frequency range [ E.W.Prohofsky, K.C. Lu, L.L.Van Zandt and B. F. Putnam, Phys Lett., 70 a, 492 1979; K.V. Devi Prasad and E.W.Prohofsky, Biopolymers, 23,1795, 1984]. • If yes, have the observed resonances a fundamental character?(Observed spectra are mostly wide and featureless. Resolution 2cm-1 . Results are sensitive to everything: to environment, to a sample geometry, to time and others) • What are the reasons why other researchers failed?Experimental results are not reproducible and are contradictive. It was not clear what to expect. • Can we receive reproducible results? • Can we use the observed features for DNA characterization and • identification and discriminate between species? • So, we focus our attempts on receiving highly resolved and reproducible spectra in the very low frequency end which is the most difficult to achieve and where reliable data are very poor. THz-BRIDGE Workshop 2002

9. GOALS • Primary goals • to observethe resonant modes in absorption spectra within the submillimeter wave range. • to demonstrate the fundamental character of the submillimeter-wave spectral absorption, thereby, • establishing a physical confirmation that very long wavelength resonant features result from electromagnetic wave interactions with the DNA and RNA macromolecules – phonon modes • Secondary goal • possible assignments to the modes THz-BRIDGE Workshop 2002

10. THEORETICAL PREDICTION OF ABSORPTION SPECTRA Maria Bykhovskaia, B. Gelmont IR active modes are calculated directly from the base pair sequence and topology of a molecule. Energy minimum Normal modes Oscillator strengths Spectra • QUESTIONS: • What do we expect to find in the submillimeter wave range? • What is the predictive power of the method? • How sensitive are far IR absorption spectra to DNA structure? THz-BRIDGE Workshop 2002

11. ENERGY MINIMIZATION AND NORMAL MODE ANALYSIS in internal coordinates of a molecule Molecular potential energy approximated as a function of dynamic variables. In the low frequency range, long distance interactions contribute significantly to the force field of a macromolecule. Variables (q): torsion and bond angles Conformational energy: E total = E Van der Waals + EElectrostatic + E HBonds+ E Torsion + E Bond angles van der Waals and electrostatic interactions; the energy of hydrogen bonds deformations; torsion rotation potentials; stretching deformations of bond angles and of bond length Initial approximation for the B-helical conformation of the fragment (TA)12 and for the A-helix of double stranded RNA Poly[C].Poly[G] are generated and optimized by the program package JUMNA THz-BRIDGE Workshop 2002

12. Energy is minimized using the program package LIGAND Molecule vibrations or normal modes (frequencies k and eigenvectors Aik) are calculated: qi(t)=q0i + Aik k cos(kt+k) k from Matrix equation HAW = FA Fij = E / (qi qj )0 Wis a diagonal matrix with elementsk2(vibrational frequencies), (eigenfrequenciesWand eigenvectorsA) Fis a matrix of second derivatives of potential energy andHis a matrix of second derivatives of kinetic energy. Hij =  ma (ra /qi ) 0 (ra /qi ) 0 THz-BRIDGE Workshop 2002

13. A double stranded 12 base pair RNA homopolymer fragment Poly[C]-Poly[G] Absorption spectra for two values of oscillator decay g =0.5 cm -1 and g =1 cm –1, () gw2(pk)2 / ( (k2 -2 )2 + 2 2 ); the dipole momentp for electric fieldEperpendicular to the long axes of a molecule (ax+y) and parallel to the long axes (a z). The maximum absorption corresponds to the electric field perpendicular to the long molecular axis z. THz-BRIDGE Workshop 2002

14. Spectral range for detailed study in the range10 cm-1 -25 cm -1. • A commercial FTS (Bruker IFS-66) system. The FTS system is equipped with mercury-lamp ( < 100 cm-1) and liquid-helium-cooled Si-bolometer (T = 1.7 o K) for the signal detection. The sample chamber of the spectrometer was placed under vacuum to eliminate any influence of water-absorption lines. • The measurements with polarized light were performed using wire polarizer • with wire diameter 25 µm and spacing between wires 75 µm. • We extended measurements to as low as 2 cm -1. Theory predicts modes here. A New unique Martin-Pupplett Polarizing Spectrometer built in England (Specac). THz-BRIDGE Workshop 2002

15. Martin-Pupplett Polarizing Spectrometer THz-BRIDGE Workshop 2002

16. Sample preparation • Free-standing films and films on substrates are prepared from the water gel. • Film thickness 2 µm - 250 µm. • The ratio of water to dry material content in the gel from 5:1 to 30:1. • Thin polycarbonate membraneor polyethylene with 98% transmission is used as a supporting substrate in some cases. • To receive good resolution, samples of at least 1/2" diameter are fabricated. • Samples are aligned to receive preferable orientation of long molecule axes in one direction. Good alignment enhances the sensitivity. THz-BRIDGE Workshop 2002

17. Sample‘s texture Image (40X) of the Salmon DNA sample in polarizing microscope (free standing). Film thicknessabout 10 mm. Gel concentration 1:10. RNA, as a rod-like polymer, spontaneously forms ordered liquid crystalline phases in aqueous solution with the long molecular axis preferentially aligned in one direction. In drying process, DNA solution undergoes a series of transitions and film samples are characterized by their microscopic textures with periodic variations in refractive index and fringe patterns observed in polarizing microscope. The film texture depends on the concentration of molecules in solution and on drying conditions. THz-BRIDGE Workshop 2002

18. Material for study • Herring, salmon and calf thymus DNA sodium salts with 6 % Na content, from Sigma Chemical Co. • Artificial short-chained oligonucleotides of known base-pair sequences from Sigma Chemicals: • • Single stranded RNA - potassium salts with the different nucleotide • composition: poly (G), poly (C), poly (A), poly (U), • (Guanine (G), Cytosine (C), Adenine (A), Uracyl (U)). • • Double helical RNA - sodium salts: Poly [C] * Poly [G] and • Poly [A] * Poly [U]. THz-BRIDGE Workshop 2002

19. What is different in our approach? Better resolution and higher sensitivity We use a resolution not worse than 0.25cm–1, and we discovered features that are small on the frequency scale. The most serious obstacles are effects of multiple reflection or standing waves in measurement systems. These effects cause artificial false resonances . Sensitivity Resolution Check for false resonances THz-BRIDGE Workshop 2002

20. Reproducibility and interference effects, Same orientation. Two different orientations Resonance features resolved on the envelope of the wide interference pattern. The estimated values of refractive index varied between 1.7 and 2.3 THz-BRIDGE Workshop 2002

21. Change with sample rotation. E E X+Y Z X+Y Z a b Sample position with electric field (a) perpendicular and (b) parallel to the long-axis of the molecule z Z Optical characteristicsdependenton the orientation of aligned DNA fibers of the film samples in electromagnetic field of radiation. Sample rotation changes the fine spectral structure because of coupling change between the electromagnetic field of radiation and the dipole moment of the DNA oscillators. THz-BRIDGE Workshop 2002

22. Experimental results on Absorption coefficient The interference pattern is not obvious in transmission of thin films (thickness between 15 and 70 µm). Results over broad ranges in frequency are very sensitive to the thickness of the film lextr - the wavelength of transmission extrema d - the film thickness m is the order of extremum. Estimated valuesofrefractive indexn varied between 1.7 and 2.3. Absorption coefficient spectra of biological material are derived by applying an interference spectroscopy technique (IST) for proper modeling of the multiple reflection behavior. THz-BRIDGE Workshop 2002

23. Absorption coefficient at two orientations Strong anisotropy of optical characteristics of biological molecules in Terahertz range. Absorption is higher and resonance structure is much more pronounced with electric field of radiation E perpendicular to the long axes of molecules Z. THz-BRIDGE Workshop 2002

24. Direct comparison of predicted and experimental absorption spectra to validate both. • We have measured transmission spectra of polynucleotide with known base-pair sequences including 12 base pair RNA fragment PolyG-PolyC. • Experimental absorption spectra were derived • Normal modes were calculated and the predicted absorption spectrum was derived for this fragment. • Some calculated absorption peaks are • absent in experimental spectra (for example, peaks at 16.5 cm -1 and 19.9 cm -1). • Possible reasons:high sensitivity of peak intensity to the actual value of oscillator dissipation g. • The estimated value isaboutg =0.5 cm -1 • Direct comparisonof experimental spectrum(red)with theoretical prediction (blue)for longititude and transverse absorption modes. • The best resonance structure is observed in the most thin films. THz-BRIDGE Workshop 2002

25. CONCLUSIONS • The transmission spectra of partially aligned films of DNA and polynucleotide molecules were carefully measured in the terahertz frequency range. • The effect of multiple reflections in films were carefully considered and were easily distinguished from the actual phonon modes. • The effect of the orientation of the macromolecules relative to the electromagnetic field of the terahertz radiation was also analyzed. The results were consistent with the theoretical predictions of the dependence of the mode strength on the polarization of the terahertz field with respect to the molecule alignment. • Absorption spectra from experimental data were then compared with the modeling results for polinucleotides of known base-pair sequences - the 12 base pair RNA fragment Poly[G]-Poly[C]. A significant correlation between the calculated and experimentally observed spectra of RNA homopolymers was achieved. • Taken together these results confirm the fundamental physical nature of the observed resonance structure that is caused by the internal vibrational modes in the DNA macromolecules. • Furthermore, these experimental results indicate that theoretical modeling, combined with measured data, may be used to directly assign vibrational modes to specific structural features of the macromolecules THz-BRIDGE Workshop 2002

26. Our publications T. Globus, D. L. Woolard, A. C. Samuels, B. L. Gelmont, J. Hesler, T. W. Crowe and M. Bykhovskaia, “Submillimeter-Wave FTIR Spectroscopy of DNA Macromolecules and Related Materials”, J. Appl. Phys,6106-6113 (May 2002). D. L. Woolard, T. R. Globus, B. L. Gelmont, M. Bykhovskaia, A. C. Samuels, D. Cookmeyer, J L. Hesler, T. W. Crowe, J. O. Jensen, J. L. Jensen and W.R. Loerop, "Submillimeter-Wave Phonon Modes in DNA Macromolecules", Phys. Rev E 65, 051903 (May 2002) T. Globus, M. Bykhovskaia, B. Gelmont, D.L.Woolard , “Far-infrared phonon modes of selected RNA molecules”, in Instrumentation for Air Pollution and Global Atmospheric Monitoring, James O. Jensen, Robert L. Spellicy, Editors, Proceedings of SPIE, Vol. 4574, pp.119-128 (2002). M. Bykhovskaia, B. Gelmont, T. Globus, D.L.Woolard, A. C. Samuels, T. Ha-Duong, and K. Zakrzewska, "Prediction of DNA Far IR Absorption Spectra Basing on Normal Mode Analysis", Theoretical Chemistry Accounts, 106, 22-27 (2001). T. Globus, L. Dolmatova-Werbos, D. Woolard, A.Samuels, B. Gelmont, and M. Bykhovskaia, "Application of Neural Network Analysis to Submillimeter-Wave Vibrational Spectroscopy of DNA Macromolecules", International Symposium on Spectral Sensing Research Proceedings (ISSSR), p.439 (2001), Canada, Quebec, June 2001. D. Woolard, T. Globus, E. Brown, L.Werbos, B. Gelmont, A. Samuels, "Sensitivity limits & discrimination capability of thz transmission spectroscopy as a technique for biological agent detection", Proc. of 5th Joint Conference on Standoff Detection for Chemical and Biological Defence (5JCSD), Williamsburg, VA, Sept. 2001. T. Globus, G. Ganguly, P. Roca i Cabarrocas,"Optical characterization of hydrogenated silicon films using interference technique", J. Appl. Phys.88, 1907 (2000). T. R.Globus, D.L.Woolard, M. Bykhovskaia, B. Gelmont, J.L.Hesler, T.W.Crowe, A.C.Samuels, Proc of International Semiconductor Device Researsh Symposium (ISDRS), p.485, Charlottesville VA (1999). THz-BRIDGE Workshop 2002

27. CONCLUSIONS • We measured transmission spectra of partially aligned films of DNA and polynucleotides in the submillimeter-wave range. • Effects of multiple reflection in films and orientation of macromolecules relative to the electromagnetic field of radiation studied and analyzed. • Experimental results are used to derive optical characteristics of biological materials. • Absorption spectra from experimental data compared with the modeling results for polinucleotides of known base-pair sequences - the 12 base pair RNA fragment Poly[G]-Poly[C]. • These studies indicate strong anisotropy of optical characteristics in Terahertz according to the theoretical prediction. • There is a correlation between calculated and experimentally observed spectra of RNA homopolymers. • The results confirm the fundamental physical nature of the observed resonance structure which is caused by the internal vibration modes in macromolecules. • Furthermore, experimental results may be combined with theoretical modeling to directly assign vibrational modes to specific structural features and topology of the macromolecules THz-BRIDGE Workshop 2002