THz-BRIDGE Workshop 2002 THz-SPECTROSCOPY OF BIOLOGICAL MOLECULES T. R. Globus 1, D. L. Woolard 2,...

THz-BRIDGE Workshop 2002

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

h


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 .


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

Bond angles

>700 cm-1

200-900 cm-1

<300 cm-1Torsion angles


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.


hvibr

I2

I1hlight

hlight

hvibr.=hlight

Absorption Raman scattering

h1

Ground state Exited state

h2

hvibr.

hvibr. = h1-h2

h1, h2 >> hvibr.

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 .


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.


“THE WORLD OF THE DEAD OR OF FUTURE PUNISHMENT".

Low energy of sourcesLow energy of sources..

Low absorption of biological material requires samples with Low absorption of biological material requires samples with large area and thickness which is difficult to make because large area and thickness which is difficult to make because samples are too fragilesamples are too fragile..

High absorption by waterHigh absorption by water masks absorption by biological masks absorption by biological materials in solutions.materials in solutions.

Many reasons for very Many reasons for very poor reproducibilitypoor reproducibility of of experimental results. The most serious reason – multiple reflection experimental results. The most serious reason – multiple reflection in measurement systems, responsible for artificial features.in measurement systems, responsible for artificial features.

Low energy of sourcesLow energy of sources..

Low absorption of biological material requires samples with Low absorption of biological material requires samples with large area and thickness which is difficult to make because large area and thickness which is difficult to make because samples are too fragilesamples are too fragile..

High absorption by waterHigh absorption by water masks absorption by biological masks absorption by biological materials in solutions.materials in solutions.

Many reasons for very Many reasons for very poor reproducibilitypoor reproducibility of of experimental results. The most serious reason – multiple reflection experimental results. The most serious reason – multiple reflection in measurement systems, responsible for artificial features.in measurement systems, responsible for artificial features.

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:


Questions to answer:

Is there something in the very far IR spectra?Is there something in the very far IR spectra? ((initial prediction of vibrational 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 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, B. F. Putnam, Phys Lett., 70 a, 492 1979; K.V. Devi Prasad and E.W.Prohofsky, Biopolymers, 23,1795, 1984]. 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.

Questions to answer:

Is there something in the very far IR spectra?Is there something in the very far IR spectra? ((initial prediction of vibrational 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 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, B. F. Putnam, Phys Lett., 70 a, 492 1979; K.V. Devi Prasad and E.W.Prohofsky, Biopolymers, 23,1795, 1984]. 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.


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


THEORETICAL PREDICTION OF ABSORPTION SPECTRA

Maria Bykhovskaia, B. Gelmont

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?

IR active modes are calculated directly from the base pair sequence and topology of a molecule.


ENERGY MINIMIZATION AND NORMAL MODE ANALYSISin internal coordinates of a molecule

Conformational energy: E total = E Van der Waals + EElectrostatic + E HBonds + E Torsion + E Bond angles

Variables (q): torsion and bond angles

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.

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 Initial approximation for the B-helical conformation of the fragment (TA)12B-helical conformation of the fragment (TA)12

and for the and for the A-helix of double stranded RNA Poly[C]A-helix of double stranded RNA Poly[C]..Poly[G]Poly[G] are generated are generated

and optimized by the and optimized by the program package JUMNAprogram package JUMNA


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

Hij = ma (ra /qi ) 0 (ra /qi ) 0

Energy is minimized using the Energy is minimized using the program package LIGANDprogram package LIGAND

W is a diagonal matrix with elementsis a diagonal matrix with elements k22 (vibrational frequencies), (vibrational frequencies),

(eigenfrequencies W and eigenvectors A)

FF is a matrix of second derivatives of potential energy andis a matrix of second derivatives of potential energy and HH is a matrix of is a matrix of second derivatives of kinetic energy. second derivatives of kinetic energy.


A double stranded 12 base pair RNA homopolymer fragment Poly[C]-Poly[G]

Absorption spectra for two values of oscillator decay =0.5 cm -1 and

=1 cm –1, () 2 (pk)2 / ( (k2 - 2 )2 + 2 2 ); the dipole moment p

2422201816141210100

20

40

60

80(x+y)

z

Frequency, cm

Abs

orpt

ion

coef

fici

ent,

rel

. u

nits

-1

=0.5 cm -1

2422201816141210100

20

40

60

80

(x+y)

z

Frequency, cm

Abs

orpt

ion

coef

fici

ent,

rel

. un

its

-1

=1 cm -1

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.

iiii me/ap ,


Spectral range for Spectral range for detaileddetailed study in the study in the rangerange 10 cm10 cm-1-1 -25 cm -25 cm -1-1..

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 We extended measurements to as low as 2 cm 2 cm -1-1.. Theory predicts modes here. Theory predicts modes here.

A New unique Martin-Pupplett Polarizing Spectrometer built in England (Specac).


Martin-Pupplett Polarizing Spectrometer


Sample preparationSample 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.


Image (40X) of the Salmon DNA sample in polarizing microscope (free standing).

Film thickness about 10 m.

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.

Sample‘s texture


Material for studyMaterial for study

Herring, salmon and calf thymus DNAHerring, salmon and calf thymus DNA sodium salts with 6 % Na content, sodium salts with 6 % Na content, from Sigma Chemical Co. from Sigma Chemical Co.

Artificial 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].


Sensitivity Resolution

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 .

What is different in our approach?

10 15 20 25

Frequency, cm

0.87

0.89

0.91

0.93

0.95

0.97

0.99

1.01

Tra

nsm

issi

on

CT DNA, 5 mg100%-linePC-membrane, d=5 mkm

-1 Frequency, cm-1

10 12 14 16 18 20 22 24

Tra

nsm

issi

on

0.97

0.98

0.99

1.00

1.01

# 206.83, Gor-Tex, d=3 mm

Check for false resonances


Poly[A], d~160 m

Frequency, cm-1

8 10 12 14 16 18 20 22 24

Tra

nsm

issi

on

0.50

0.52

0.54

0.56

0.58

0.60

02/0402/05

m=2

m=1

Resonance features resolved on the envelope of the wide interference pattern.

Reproducibility and interference effects,

Herring DNA, d=145 m

Frequency, cm-1

10 15 20 25

Tra

nsm

issi

on

0.46

0.48

0.50

0.52

0.54

0.56

0.58

0.60

0.62

E perpend. Z E // Z

Same orientation. Two different orientations

The estimated values of refractive index

varied between 1.7 and 2.3


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.

Sample position with electric fieldSample position with electric field (a) perpendicular and (b) parallel (a) perpendicular and (b) parallel to the long-axis of the molecule zto the long-axis of the molecule z

aa

EE EEX+YX+Y

X+YX+Y

ZZ

ZZ

ZZ

bb

Change with sample rotation.Change with sample rotation.

Poly[A]-Poly[U], d=16 m

Frequency, cm-1

10 15 20 25

Tra

nsm

issi

on

0.70

0.75

0.80

0.85

0.90

0.95

E perpend. ZE parall. Z


2422201816141210880.25

0.35

0.45

0.55

0.65

d=0.125 mm d=0.205 mm

Frequency, cm

Tra

nsm

issi

on

-1

n=2.2 n=1.9

Results over broad ranges in frequency are very sensitive to the thickness of the film

The interference pattern is not obvious in transmission of thin films (thickness between 15 and 70 µm).

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.

Experimental results on Absorption coefficient

2422201816141210100.45

0.55

0.65

0.75

0.85

0.95 PolyCPolyG

PolyC-PolyG

PolyAPolyUPolyA-PolyU

Frequency, cm

Tra

nsm

issi

on

-1

extr - the wavelength of transmission extremad - the film thicknessm is the order of extremum.


Poly [A]-Poly[U], d=16 m

Frequency, cm-1

10 15 20 25

Abs

orpt

ion

coef

ficie

nt, c

m-1

0

50

100

150

E perp Z E//Z

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.


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 werederived Normal modes were calculated and the predicted absorption spectrum was derived for this fragment. Some calculated absorption peaks areabsent 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 The estimated value is about =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.

Poly [C]- Poly [G], d=6.5 m

Frequency, cm-1

10 12 14 16 18 20 22 24

Ab

sorp

tion

coef

ficie

nt,

cm-1

0

10

20

30

40

measured

calculated, =0.5 cm-1 , E perpend. Zcalculated, E // Z


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

CONCLUSIONS


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).


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

CONCLUSIONS

THz-BRIDGE Workshop 2002 THz-SPECTROSCOPY OF BIOLOGICAL MOLECULES T. R. Globus 1, D. L. Woolard 2,...

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