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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
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 initial foundation for the future use of terahertz
spectroscopy in the identification and characterization of
biological macromolecules and related materials .
THz-BRIDGE Workshop 2002
Introduction
INTERNAL MOLECULAR VIBRATIONS AND FAR IR ABSORPTION SPECTRA
Bonds
>700 cm-1
Bond angles
200-900 cm-1
Torsion angles
<300 cm-1
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
THz-BRIDGE Workshop 2002
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
Scattering 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
hnlight
hn1
I1
hnvibr
hnlight
Raman scattering
I2
hnvibr.=hnlight
Exited state
Ground state
hnvibr.
hn2
hnvibr. = hn1-hn2
hn1, hn2 >> hnvibr.
THz-BRIDGE Workshop 2002
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 orderRaman scattering.
THz-BRIDGE Workshop 2002
“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
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
GOALS
 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
THz-BRIDGE Workshop 2002
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
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
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
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
THz-BRIDGE Workshop 2002
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,  ()  g2  (pk)2 / ( (k2 - 2 )2 + g2 2 ); the dipole moment p
p
e a
i
i
/ mi ,
80
g =0.5 cm
80
(x+y)
z
-1
Absorption coefficient, rel. units
Absorption coefficient, rel. units
i
60
40
20
0
10
12
14
16
18 20
-1
22
24
 (x+y)
z
-1
g =1 cm
60
40
20
0
10
12
14
16
18
20
22
24
-1
Frequency, cm
Frequency, cm
for electric field E perpendicular to the long axes of a molecule ( x+y) and
parallel to the long axes ( z).
The maximum absorption corresponds to the electric field perpendicular to the
long molecular axis z.
THz-BRIDGE Workshop 2002
 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 ( < 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
Martin-Pupplett Polarizing Spectrometer
THz-BRIDGE Workshop 2002
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 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.
THz-BRIDGE Workshop 2002
Sample‘s texture
Image (40X) of the Salmon DNA sample
in polarizing microscope (free standing).
Film thickness about 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
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
What is different in our approach?
Better resolution and higher sensitivity
We use a resolution not worse than 0.25 cm–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
1.01
1.01
# 206.83, Gor-Tex, d=3 mm
0.99
1.00
Transmission
Transmission
0.97
CT DNA, 5 mg
100%-line
PC-membrane, d=5 mkm
0.95
0.93
0.91
0.99
0.98
0.89
0.87
10
0.97
15
20
25
10
12
14
16
18
20
-1
Frequency, cm
-1
Frequency, cm
THz-BRIDGE Workshop 2002
22
24
Reproducibility and interference effects,
Same orientation.
Two different orientations
0.60
0.62
Poly[A], d~160 mm
Herring DNA, d=145 mm
0.60
02/04
02/05
E perpend. Z
E // Z
0.58
m=2
0.56
Transmission
Transmission
0.58
0.54
0.56
0.54
0.52
0.50
0.52
0.48
m=1
0.46
0.50
10
8
10
12
14
16
18
20
22
24
15
20
25
-1
Frequency, cm
Frequency, cm-1
Resonance features resolved on the envelope of the wide interference pattern.
nd =
m extr
4
The estimated values of refractive index
varied between 1.7 and 2.3
THz-BRIDGE Workshop 2002
Change with sample rotation.
X+Y
E
Z
E
0.95
Poly[A]-Poly[U], d=16 mm
Transmission
X+Y
Z
0.90
0.85
a
b
0.80
0.75
Sample position with electric field
(a) perpendicular and (b) parallel
to the long-axis of the molecule z
E perpend. Z
E parall. Z
0.70
10
15
20
25
-1
Frequency, cm
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.
THz-BRIDGE Workshop 2002
Experimental results on Absorption coefficient
0.95
0.65
n=2.2
Transmission
Transmission
0.85
0.55
n=1.9
0.45
0.35
d=0.125 mm
d=0.205 mm
10
12
14
16
18
Frequency, cm-1
20
22
0.65
P olyA
P olyU
P olyA-P olyU
0.45
24
10
12
14
16
18
20
22
24
Frequency, cm-1
Results over broad ranges in frequency
are very sensitive to the thickness of
the film
m extr
nd =
4
0.75
0.55
0.25
8
P olyC
P olyG
P olyC-P olyG
The interference pattern is not obvious
in transmission of thin films (thickness
between 15 and 70 µm).
extr - 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.
THz-BRIDGE Workshop 2002
Absorption coefficient at two orientations
150
Poly [A]-Poly[U], d=16 mm
Absorption coefficient, cm-1
E perp Z
E//Z
100
50
0
10
15
20
Frequency, cm
25
-1
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
Direct comparison of predicted and experimental absorption
spectra to validate both.
40
Poly [C]- Poly [G], d=6.5 mm
Absorption coefficient, cm-1
measured
calculated, g=0.5 cm-1 , E perpend. Z
calculated, E // Z
30
20
10
0
10
12
14
16
18
20
22
24
Frequency, 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.
 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
THz-BRIDGE Workshop 2002
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
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
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