Michaela Vorlíčková   mifi@ibp.cz
Institute of Biophysics
Czech Academy of Sciences Brno
Laboratory of Biophysics of Nucleic Acids
CD Spectroscopy and its Role in the History of our Knowledge of DNA Conformation
 Czech Republic
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tralala

U:\MIFI\obrazky\sbírka\kolostruktur.png
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Circular dichroism and optical activity of biopolymers
) CD – principle, quantities - ellipticity, ΔA, Δε, relation between ORD and CD
Optical activity property of a chiral molecule
Chiral molecules (aminoacids, sugars) are those lacking mirror symmetry
Optical rotation of the plane of polarization (difference in refraction indexes –difference in
propagation velocity) may be either to the right (dextrorotatory -D)
or to the left (levorotatory –L) depending on the stereoisomer (enantiomer) present
U:\MIFI\obrazky\sbírka\Macek-poekresl..jpg
Specific rotation – characteristic quantity
[α]Tλ = α/cl
Optical rotatory dispersion - ORD
is the dependednce of specific rotation on the  wavelength
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Circular dichroism and optical activity of biopolymers
CD phenomenon – different absorption of the left-handed and right-haned circularly polarized
light.
U:\MIFI\obrazky\sbírka\Macek-poekresl..jpg
 Circular dichroism   Δε               Δε = εL – ε R    =   ΔA/cl,   θ=3300. Δε
quantity- ellipticity        F  [θ]      tg θ    = b/a  = εL – ε R/ εL + ε R = difference/sum
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V:\mifi-obr\nové obr pro CD presentaci\obrA.bmp V:\mifi-obr\nové obr pro CD presentaci\obrC.bmp
[α]
λ[nm]
Cotton effect
Optical rotatory dispersion                    (ORD) is the dependednce of specific rotation on
the  wavelength
The Cotton effect is the characteristic change in optical rotatory dispersion (and/or circular
dichroism) in the vicinity of an absorption band of a substance.
dextrorotatory
levorotatory
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V:\mifi-obr\nové obr pro CD presentaci\obrF.bmp
Cotton effect
λ[nm]
Optical rotatory dispersion
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V:\mifi-obr\obr12.bmp
l
ORD
  +
CD
ORD
   -
CD
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V:\mifi-obr\obr1.bmp
α-helix
β-sheet
β-turn
CD of proteins
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Preconditions for an appearance of CD of DNA
•
ABSORBANCE
CHIRALITY
+
BASE
* SUGAR
CD
http://biology-forums.com/gallery/33_23_06_11_4_12_30.jpeg
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V:\mifi-obr\obr2.bmp
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Conditions of the origin of CD

•
ABSORPTION
CHIRALITY
+
BASE
* SUGAR
CD
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Kypr, J., Kejnovska, I., Renciuk, D., Vorlickova, M.:
Circular dichroism and conformational polymorphism of DNA. Nucleic Acids Res. 37 (2009) 1713-1725.
  Tetraplexes
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02fibryA 03fibryB 04fibryC
  A

B
C,D,T…
Long DNA molecules can be oriented by mechanical stroking. X ray diffraction pattern obtained on
these semicrystaline matter enables to determine some periodicities of the  DNA arrangement
M. Wilkins, R. Franklin, W+C
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05BCA Tunis-Schneider, M.J.B., Maestre, M.F.:
Circular dichroism spectra of oriented and unoriented deoxyribonucleic acid films - a preliminary
study. J. Mol. Biol. 52 (1970) 521-541.
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 Cooperative changes between discrete structures
06a
06a
    Non-cooperative changes
within the same global structure
Ivanov, V. I., Minchenkova, L. E., Minyat, E. E, Frank-Kamenetskii, M. D.,
Schyolkina,  A. K.:. The B to A transition of DNA in solution. J. Mol. Biol. 87 (1974) 817-833.
B
C,D
B
A
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Kypr, J., Chladkova, J., Zimulova, M. Vorlickova, M.:
Aqueous trifluoroethanol solutions simulate the environment  of DNA in the crystaline state.
Nucleic Acids Res. 27 (1999) 3466-3473.
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V:\Iva\učebnicováB.jpg U:\MIFI\obrazky\sbírka\ucebnicovaA.jpg
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220
300
R
L
GCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGC
l
Pohl, F.M., Jovin T.M.: Salt-induced co-operative conformational change of a synthetic DNA:
Equilibrium and kinetic studies with poly(dG-dC) J.Mol. Biol. 67 (1972) 375-396
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V:\mifi-obr\sejmout.jpg V:\mifi-obr\sejmout0005.jpg
Rodley et al., 1976; Sasisekharan and Pattabiraman, 1976, 1978; Bates et al., 1977, 1980a; Albiser
and Premilat, 1980, 1982; Millane and Rodley, 1981;
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06Olson
Vilma Olson:
Spatial configuration of ordered polynucleotide chains: A novel double helix.
Proc. Natl. Acad. Sci. USA 74 (1977) 1775-1779.
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09Z 08
Z
Wang, A. H.; Quigley, G. J.; Kolpak, F. J.; Crawford, J. L.; van Boom, J. H.; van der Marel, G.;
Rich, A. Molecular structure of a left-handed double helical DNA fragment at atomic resolution.
Nature. 282 (1979) 680–686.
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11B 10B
B
Wing, R., Drew, H., Takano, T., Broka, Ch., Tanaka, S., Itakura, K., Dickerson, R.E.:
Crystal structure analysis of a complete turn of B-DNA  Nature 287 (1980) 755–758.
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V:\mifi-obr\sejmout0001.jpg
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V:\mifi-obr\obr7.bmp 12Ivanov
Valery I. Ivanov, Elvira E. Minyat
The transitions between left- and right-handed forms of poly(dG-dC)
Nucleic Acids Res. 9 (1981) 4783-4798
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ATATATATATATATATATATATATATATATATATATA
Vorlíčková, M., Sklenář, V., Kypr, J.: Salt-induced Conformational Transition of Poly[d(A-T)]
J.  Mol. Biol. 166 (1983) 85-92
13BZ,BX
X
31P NMR
Z
ApT
TpA
mC-G
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14zvyraz
B
B-A
A
A-X
B-X
X
ATATATATATATATATATATATATATATATATATATA
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Antiparallel duplex of alternating A-T fragment with Hoogsteen base pairing.
Abrescia, N.G.A., Thompson, A., Huynh-Dinh, T., Subirana, J.A.:  Alternating A-T fragment with
Hoogsteen base pairing. Proc.Nat.Acad.Sci.USA , 99 (2002) 2806 – 2811.
V:\mifi-obr\Fig1.jpg
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(TA)n
Z forms
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G
C
G
C
G
C
C
G
C
G
C
G
…
…
A
T
A
T
A
T
T
A
T
A
T
A
…
…
A
C
A
C
A
C
T
G
T
G
T
G
…
…
(Pu)n . (Py)n complexes
Alternating (Pu-Py)n
B
A
Z
B
A
X
Z
B
A
Z
X
G
G
G
G
G
G
C
C
C
C
C
C
…
…
A
A
A
A
A
A
T
T
T
T
T
T
…
…
A
G
A
G
A
G
T
C
T
C
T
C
…
…
(dG)n. (dC)n
(dA)n.(dT)n
   (dA-dG)n.(dC-dT)n
V:\mifi-obr\sejmout0004.jpg
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C:\WINDOWS\Plocha\149D-inset.jpg
                                               DNA Triplex




Radhakrishnan, I., Patel, D.J. (1994)
Pyrimidine. Purine. Pyrimidine
V:\mifi-obr\sejmout0004.jpg
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C:\WINDOWS\Plocha\135D-model.jpg
DNA TRIPLEX
                  T C C T C C T T T T T T A G G A G G A T T T T T T G G T G G T
Radhakrishnan, I., Patel, D.J. (1993)
Pyrimidine. Purine. Purine
V:\mifi-obr\sejmout0004.jpg
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U:\MIFI\presentace\Bez názvu 1.tif U:\MIFI\presentace\Bez názvu 2.tif U:\MIFI\presentace\Bez názvu
3.tif
.
.
A  Casasnovas, J.M., et al.  Azorin, F.:. Structural polymorphism of d(GA.TC)n DNA sequences.
Intramolecular and intermolecular associations of the individual strands. J. Mol. Biol. 233 (1993)
671-6811.
B   Rippe, K., Fritch, V., Westhof, E., Jovin, T.M.: Alternating d(G-A) sequences form a
parallel-stranded DNA homoduplex.  EMBO J. 11 (1992) 3777-3786.
C   Dolinnaya, N. G., Fresco, J. R: Single-stranded nucleic acid helical secondary structure
stabilized by ionic bonds: d(A+-G)10. Proc. Natl. Acad. Sci. USA 89 (1992) 9242-9246.
C Vorlícková, M., Kejnovská, I., Kovanda, J., Kypr, J.: Dimerization of the guanine-adenine repeat
strands of DNA. Nucleic Acids Research, 1999, Vol. 27, No. 2 581–586.
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Quadruplexes
Kang, C.H. et al.(1994)
frequently occur in promoters of genes and were shown to control their expression.
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V:\mifi-obr\UDF062-inset.jpg
CD spectra reflecting formation of a parallel and antiparallel
                            guanine quadruplex


V:\mifi-obr\UD0014-inset.jpg
RNA
DNA
DNA
K
Penazova, H., Vorlickova, M.
Guanine tetraplex formation by short DNA fragments containing runs of guanine and cytosine.
Biophys. J. 73 (1997) 2054-2063
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WAVELENGTH [nm]
Fragment of Pu-27 promoter c-myc:   (1998)
       TGGGGAGGGTGGGGAGGGTGGGGAAGG
V:\mifi-obr\G_Q_Elsevier.JPG
 K+
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V:\mifi-obr\Figure1.jpg V:\mifi-obr\Figure2.jpg V:\mifi-obr\Figure5.jpg
Parkinson, G.N., Lee, M.P.H, Neidle, S.
Nature 417 (2002) 876-880.
d(TAGGGTTAGGGT)              12
d[AGGG(TTAGGG)3]
Phan, A.T. et al.:
J.Am.Chem.Soc.126(2004)8710
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Human telomeric DNA forms quadruplex

Xu, Y.: Chem. Soc. Rev. (2011)
C:\WINDOWS\Plocha\143D-model.jpg
The telomere quadruplex became a target for developing anticancer drugs
Telomeric DNA is associated with aging
Telomerase – does not get older – ageless, immortal
Quadruplex does not allow telomerase to get on the sequence
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Vorlíčková, M., Chládková, J., Kejnovská, I., Fialová, M., Kypr, J.:
Guanine quadruplex topology of human telomere DNA is governed by the number of (TTAGGG) repeats.
Nucleic Acids Res.  33 (2005) 5851-5860.
wavelength [nm]
     G3(TTAG3)n in 150 mM K+
1mM Na phosphate
Number of
G3 blocks
2
 3     4      5     6     7     8    9     10   12  14   16
3+1
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AG3(TTAG3)3  TAG3(TTAG3)3                 AAAG3(TTAG3)3AA
U:\Iva_U\2007\Mifi_Albany\3+1_lsam.jpg U:\Iva_U\2007\Mifi_Albany\3+1_sam.png
TAG3(TTAG3)3TT
3 + 1

Luu, K.N., Phan, A.T., Kuryavyi, V., Lacroix, L.,      Patel, D.J. (2006)  J.Am.Chem.Soc., 128,
9963-9970.
Ambrus, A., Chen, D., Dai, J., Bialis, T.,  Jones, R.A., Yang, D. (2006) Nucleic Acids Res. 34,
2723–2735.
Phan, A. T., Luu, K.N., Patel, D.J. (2006) Nucleic Acids Res., 34, 5715-5719.
anti
syn
K+
3 + 1
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Vorlíčková, M., Chládková, J., Kejnovská, I., Fialová, M., Kypr, J.:
Guanine quadruplex topology of human telomere DNA is governed by the number of (TTAGGG) repeats.
Nucleic Acids Res.  33 (2005) 5851-5860.
wavelength [nm]
     G3(TTAG3)n in 150 mM K+
1mM Na phosphate
Number of
G3 blocks
 2
  3    4     5    6     7     8    9   10   12  14   16
3+1
How does the structure of the long telomere DNA look like?
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obr8
Long telomere molecules have a beads on a string- like arrangement
Xu, Yet al.
Angev. Chemie (2009)
Vorlíčková, M., Chládková, J., Kejnovská, I., Fialová, M., Kypr, J.:
Guanine quadruplex topology of human telomere DNA is governed by the number of (TTAGGG) repeats.
Nucleic Acids Res.  33 (2005) 5851-5860.
                      What is the structure of the bead?
enthalpy
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3 +1
AG3(TTAG3)3  TAG3(TTAG3)3
  AAAG3(TTAG3)3AA
U:\Iva_U\2007\Mifi_Albany\3+1_lsam.jpg U:\Iva_U\2007\Mifi_Albany\3+1_sam.png
TAG3(TTAG3)3TT
3 + 1
U:\Iva_U\2007\Mifi_Albany\chair.jpg
CHAIR
AG3(TTAG3)3
BASKET
Phan, at al.: Nucleic Acids Res. 34 (2006) 5715-5719.
He et al.:Nucleic Acids Res. 32 (2004)             5359-5367.
Matsugami, et al.:. Nucleic acids symp. Series         50 (2006) 45-46.
Xu et al.: Bioorg.& Medicinal Chem.
14 (2006)5584 – 5591.
Lim, et al.: J.Am.Chem.Soc. 131           (2009) 4301–4309.
anti
syn
BASKET
two tetrads
                   acidic form
AG3(TTAG3) 3

G3(TTAG3) 3T
Luu, et al.: J.Am.Chem.Soc., 128 (2006) 9963-9970.
Ambrus, et al.: Nucleic Acids Res. 34 (2006) 2723–2735.
Parkinson, Lee, Neidle:
Nature 417 (2002) 876-880.
Balagurumoorthy, Brahmachari: J. Biol. Chem. 269 (1994) 21858-21869.
Redon et al.: Nucleic Acids Res. 31 (2003) 1605-1613.
   PARALLEL
     G3(TTAG3)3
     AG3(TTAG3)3 TTAG3(TTAG3)3
Galer et all.: Angewandte Chem. 55 (2016) 1993-1997.
TAG3(TTAG3)3
2 + 2
Lim et al.: Nucleic Acids Res. 41 (2013) 10556-10562.
TTAG3(TTAG3)3TTA
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telomerni telomerni telomerni
telomerni telomerni telomerni
nucleoside conc:
 200 mM
 120 mM
 55 mM
 16 mM
 0.8 mM
( 0.1 mM)
      Different quadruplex structures observed for the same sequence at the same solvent conditions
The arrangement of the human telomere quadruplex is polymorphic
   AG3(TTAG3)16
•
Renciuk, D., Kejnovska, I., Skolakova, P., Bednarova, K., Vorlickova, M.:
Arrangements of human telomere DNA quadruplex
in physiologically relevant K+ solutions
Nucleic Acids Research 37 (2009) 6625-6634
K+
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27i-tetr

          i - motif
Two parallel duplexes bound by C.C+ pairs
are intercalated in the antiparallel fashion
U:\MIFI\obrazky\sbírka\C.C+.jpg
Gehring, K., Leroy, J.L., Gueron, M.:
A tetrameric DNA structure with protonated cytosine.cytosine base pairs. Nature 363 (1993) 561-565.
V:\mifi-obr\UDF043-inset.jpg
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28PY copy (1)
TCCCCACCTTCCCCACCCTCCCCACCCTCCCCA
Fragmenty promotoru c-myc
                                           Human c-myc Promoter Fragment
Simonsson, T., Přibylová, M., Vorlíčková, M.: Biochem. Biophys. Res.Commun.278 (2000) 158–166
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)  Školáková, P., Renčiuk, D.,  Palacký, J., Krafčík, D., Dvořáková, Z., Kejnovská. I., Bednářová,
K.,  Vorlíčková, M.: Systematic investigation of sequence requirements  for DNA i-motif formation.
Nucleic Acids Research 47 (2019) 2177–2189.
)  Dvořáková, Z., Renčiuk, D., Kejnovská, I., Školáková, P., Bednářová, K., Sagi, J. Vorlíčková,
M.: i-Motif of cytosine-rich human telomere DNA fragments containing natural base lesions. Nucleic
Acids Research Nucleic Acids Research 46 (2018) 1624-1634.
(C3Tx)3Cn
Three nucleotides in the loop are optimal for intramolecular iM formationare
Single nucleotides in the loop result in bimolecular iM
One C is spent for loop in the case of two non-C nucleotides in the loop
Strictly alternating C.C+ pairing
Is required for iM stability
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)  Školáková, P., Renčiuk, D.,  Palacký, J., Krafčík, D., Dvořáková, Z., Kejnovská. I., Bednářová,
K.,  Vorlíčková, M.: Systematic investigation of sequence requirements  for DNA i-motif formation.
Nucleic Acids Research 47 (2019) 2177–2189.
)  Dvořáková, Z., Renčiuk, D., Kejnovská, I., Školáková, P., Bednářová, K., Sagi, J. Vorlíčková,
M.: i-Motif of cytosine-rich human telomere DNA fragments containing natural base lesions. Nucleic
Acids Research Nucleic Acids Research 46 (2018) 1624-1634.
(CnT3)3Cn
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)  Školáková, P., Renčiuk, D.,  Palacký, J., Krafčík, D., Dvořáková, Z., Kejnovská. I., Bednářová,
K.,  Vorlíčková, M.: Systematic investigation of sequence requirements  for DNA i-motif formation.
Nucleic Acids Research 47 (2019) 2177–2189.
)  Dvořáková, Z., Renčiuk, D., Kejnovská, I., Školáková, P., Bednářová, K., Sagi, J. Vorlíčková,
M.: i-Motif of cytosine-rich human telomere DNA fragments containing natural base lesions. Nucleic
Acids Research Nucleic Acids Research 46 (2018) 1624-1634.
(CnT3)3Cn
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Iva Kejnovská
Jan Palacký
Petra Školáková
Klára Bednářová
Zuzana Dvořáková
Daniel Renčiuk
Jaroslav Kypr
Jean-Louis Mergny
Jiří Šponer
Lukáš Trantírek
Jiří  Fajkus
Miroslav Fojta
mifi@ibp.cz
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CHIROPTICKÉ METODY
Optical Rotatory Dispersion - ORD
Závislost úhlu stočení roviny polarizace lineárně polarizovaného světla průchodem opticky aktivní
látkou na vlnové délce procházejícího záření.     (180-800 nm)
Circular Dichroism-CD
Závislost rozdílu absorpce pro vlevo a vpravo kruhově polarizované světlo na vlnové délce
absorbovaného záření v oblasti energií elektronových přechodů.  (180-1000 nm)
Infrared Circular Dichroism-IRCD (VCD)
Závislost rozdílu absorpce pro vlevo a vpravo kruhově polarizované světlo na vlnové délce
absorbovaného záření v oblasti energií vibračních přechodů.   (1-5 um)
Fluorescence Detected circular Dichroism -FDCD
Závislost rozdílu intenzity fluorescence, excitované vlevo a vpravo kruhově polarizovaným světlem
na vlnové délce excitačního záření.    (~ 200 nm až vlnová délka emise)
Circulary Polarized Luminiscence (emission) - CPL (CPE)
Spektrální průběh rozdílu intenzit (spontánní) emise vlevo a vpravo cirkulárně polarizovaného
světla.  (Interval vlnových délek emise chromoforu)
Circular Diferential Raman Dispersion - Raman CID
Spektrální průběh rozdílů intenzit Ramanova rozptylu vlevo a vpravo kruhově polarizovaného
dopadajícího záření.   (Interval vlnových délek Ramanova jevu)
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