Title: THzSPECTROSCOPY 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
2Objectives
- 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 initial foundation for the
future use of terahertz - spectroscopy in the identification and
characterization of - biological macromolecules and related
materials .
3Introduction 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
gt700 cm-1
Bond angles
200-900 cm-1
Torsion angles
lt300 cm-1
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.
5Scattering or absorption spectroscopy?
Scattering and absorption spectroscopy utilize
the interaction of an applied electromagnetic
(EM) field with the phonon (vibration) field of
the material to provide useful structural
information .
Absorption
Raman scattering
hn1
I1
hnlight
hnvibr
Ground state
Exited state
hnvibr.
hn2
hnlight
I2
hnvibr. hn1-hn2 hn1, hn2 gtgt hnvibr.
hnvibr.hnlight
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.
7THE 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.
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.
9GOALS
- Primary goals
- to observe the 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
10THEORETICAL 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?
11ENERGY 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 PolyC.PolyG
are generated and optimized by the program
package JUMNA
12Energy is minimized using the program package
LIGAND
Molecule vibrations or normal modes (frequencies
?k and eigenvectors Aik) are calculated qi(t)q
0i ? Aik ?k cos(?kt?k) k
from Matrix equation HAW FA Fij
?E / (?qi ?qj )0
W is a diagonal matrix with elements ?k2
(vibrational frequencies), (eigenfrequencies W
and eigenvectors A) F is a matrix of second
derivatives of potential energy and H is a matrix
of second derivatives of kinetic energy.
Hij ? ma (?ra /?qi ) 0 (?ra /?qi ) 0
13A double stranded 12 base pair RNA homopolymer
fragment PolyC-PolyG
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 moment p
for electric field E perpendicular to the long
axes of a molecule ( axy) and parallel to
the long axes (a z). The maximum absorption
corresponds to the electric field perpendicular
to the long molecular axis z.
14- Spectral range for detailed study in the range
10 cm-1 -25 cm -1. - A commercial FTS (Bruker IFS-66) system. The
FTS system is equipped with mercury-lamp ( lt 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). -
15 Martin-Pupplett Polarizing
Spectrometer
16Sample 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 51 to 301. - Thin polycarbonate membrane or 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.
17Samples texture
Image (40X) of the Salmon DNA sample in
polarizing microscope (free standing). Film
thickness about 10 mm. Gel concentration 110.
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.
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.
-
19What is different in our approach?
Better resolution and higher sensitivity We use a
resolution not worse than 0.25 cm1, 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
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
21Change with sample rotation.
E
E
XY
Z
XY
Z
a
b
Sample position with electric field (a)
perpendicular and (b) parallel to the long-axis
of the molecule z
Z
Optical characteristics dependent on 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.
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 values of refractive index n
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.
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.
24Direct 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 is about g 0.5 cm -1
- Direct comparison of experimental spectrum (red)
with theoretical prediction (blue) for
longititude and transverse absorption modes. - The best resonance structure
- is observed in the most thin films.
25CONCLUSIONS
- 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 PolyG-PolyC. 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 -
26Our 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).
27CONCLUSIONS
- 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 PolyG-PolyC. - 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