Michaela Vorlíčková mifi@ibp.cz Institute of Biophysics Czech Academy of Sciences Brno Laboratory of Biophysics of nucleic acids Conformational Properties of DNA revealed by CD Spectroscopy [USEMAP] U:\MIFI\obrazky\sbírka\kolostruktur.png C:\WINDOWS\Plocha\image002.jpg [USEMAP] U:\MIFI\obrazky\sbírka\kolostruktur.png [USEMAP] Kypr, J., Kejnovska, I., Renciuk, D., Vorlickova, M.: Nucleic Acids Res. 37 (2009) 1713-1725. [USEMAP] 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 - the rotation of the plane of linearly polarized light traveling through chiral materials 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 Specific rotation – characteristic quantity U:\MIFI\obrazky\sbírka\Macek-poekresl..jpg glycin Specific rotation [α]Tλ = α/cl Optical rotatory dispersion - ORD is the the variance of specific rotation with wavelength [USEMAP] 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 φ [θ] tg θ = b/a = εL – ε R/ εL + ε R = difference/sum [USEMAP] 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) 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 [USEMAP] V:\mifi-obr\nové obr pro CD presentaci\obrF.bmp Cottonův efekt λ[nm] Optická rotační disperse (ORD) [USEMAP] V:\mifi-obr\obr12.bmp l ORD + CD ORD - CD [USEMAP] V:\mifi-obr\obr1.bmp α-helix β-sheet β-turn CD of proteins [USEMAP] Preconditions of the origin of CD • ABSORBCE CHIRALITY + BASE SUGAR CD http://biology-forums.com/gallery/33_23_06_11_4_12_30.jpeg [USEMAP] V:\mifi-obr\obr2.bmp [USEMAP] Conditions of the origin of CD • ABSORBCE CHIRALITA + BÁZE * CUKR CD [USEMAP] Circular dichroism and optical activity of biopolymers ) CD – principle, quantities - ellipticity, Δε, relation between ORD and CD ) Advantages and disadvantages of CD spectroscopy Advantages Enormous sensitivity - low concecntration of studied substances easy solubility even in extreme conditions Easy manipulation - titraton transition between different structures whole conformational space Disadvantages no explicit relation between CD spectrum and structure of complex molecules experience Discrimination between cooperative and non-cooperative changes [USEMAP] 40dichro Main component Alternately depressed and spread out Interior of the CD apparatus [USEMAP] 02fibryA 03fibryB 04fibryC A corpulent B C,D,E,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 [USEMAP] 02fibryA 03fibryB 04fibryC A corpulent B C,D,E,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 [USEMAP] 05BCA Tunis-Schneider, M.J.B. + Maestre, M.F. [USEMAP] Cooperative changes between discrete structures 06a Examples of cooperative and non-cooperative structural changes 06a Non-cooperative changes within the same structure Ivanov, V.I [USEMAP] V:\Iva\učebnicováB.jpg U:\MIFI\obrazky\sbírka\ucebnicovaA.jpg [USEMAP] V:\Iva\učebnicováB.jpg U:\MIFI\obrazky\sbírka\ucebnicovaA.jpg [USEMAP] 220 300 R L GCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGC l Pohl, F,. Jovin T.: J.Mol Biol 1972 [USEMAP] 220 300 R L GCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGC l Pohl, F,. Jovin T.: J.Mol Biol 1972 [USEMAP] 06Olson Vilma Olson [USEMAP] V:\mifi-obr\sejmout.jpg V:\mifi-obr\sejmout0005.jpg Sasisekharan [USEMAP] 09Z 08 Z Dickerson [USEMAP] 11B 10B B Dickerson [USEMAP] V:\mifi-obr\sejmout0001.jpg [USEMAP] CD spectral changes accompanying B-Z transition of poly(dG-dC) V:\mifi-obr\ZDFB51-biol1_files\ZDFB51-biol1.jpg B-forma Z-forma wavelength [nm] [USEMAP] V:\mifi-obr\obr7.bmp 12Ivanov V:\mifi-obr\BDJB49-biol1_files\BDJB49-biol1.jpg V:\mifi-obr\ZDFB51-biol1_files\ZDFB51-biol1.jpg V:\mifi-obr\ADH020-biol1.jpg [USEMAP] 13BZ,BX ATATATATATATATATATATATATATATATATATATA Vorlíčková, M., Sklenář, V., Kypr, J.: J. Mol. Biol. 166 (1983) 85-92 [USEMAP] V:\mifi-obr\obr5.tif X-DNA ATATATATATATATATATATATATATATATATATATA Vorlíčková, M., Sklenář, V., Kypr, J.: J. Mol. Biol. 166 (1983) 85-92 [USEMAP] 14zvyraz B B-A A A-X B-X X [USEMAP] 41 LD X - DNA V:\mifi-obr\Fig2.jpg ALTERNATING A-T FRAGMENT WITH HOOGSTEEN BASE PAIRING Subirana, J. Proc.Nat.Acad.Sci.USA , 99, pp. 2806, 2002. Biochemistry , 43, pp. 4092 - 4100, 2004. V:\mifi-obr\Fig1.jpg [USEMAP] 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 … … poly(dG).poly(dC) poly(dA).poly(dT) poly(dA-dG).poly(dC-dT) [USEMAP] C:\WINDOWS\Plocha\149D-inset.jpg DNA Triplex Radhakrishnan, I., Patel, D.J. (1994) Pyrimidine. Purine. Pyrimidine V:\mifi-obr\sejmout0004.jpg [USEMAP] 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 [USEMAP] T Gray, D.M., Hung,S-H., Johnson, K.H.: Methods Enzymol. 246 (1995) 19-34. (GA)12 (TC)12 C:\Documents and Settings\mifi\Plocha\Gray_30.jpg The triplex formation determined by mixing curves [USEMAP] U:\MIFI\presentace\Bez názvu 1.tif U:\MIFI\presentace\Bez názvu 2.tif U:\MIFI\presentace\Bez názvu 3.tif [USEMAP] Quadruplexes Wang, Y., Patel, D.J. (1994) Kang, C.H. et al.(1994) frequently occur in promoters of genes and were shown to control their expression. [USEMAP] 27i-tetr i - motif Two parallel-bonded duplexes are intercalated in the antiparallel fashion U:\MIFI\obrazky\sbírka\C.C+.jpg Leroy, J.L., Gueron, M.,1995 V:\mifi-obr\UDF043-inset.jpg [USEMAP] 28PY copy (1) TCCCCACCTTCCCCACCCTCCCCACCCTCCCCA Fragmenty promotoru c-myc [USEMAP] 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 [USEMAP] WAVELENGTH [nm] Fragment Pu-27 promotoru c-myc: TGGGGAGGGTGGGGAGGGTGGGGAAGG Pan, A.T. et al.: J.Am.Chem.Soc.126(2004)8710 V:\Iva\obr3.JPG V:\mifi-obr\Fig8.jpg V:\mifi-obr\G_Q_Elsevier.JPG [USEMAP] 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] 22 [USEMAP] 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 [USEMAP] 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 17 3+1 [USEMAP] 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 [USEMAP] obr8 Long telomere molecules have a beads on a string- like arrangement Xu, et al. Angev. Chemie (2009) How does the structure of the long telomere DNA look like? Nucleic Acids Res. 33 (2005) 5851-5860 What is the structure of the bead? AFM [USEMAP] 3-50 mM strand concentration in CD 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 0.2-5 mM strand concentration in NMR BASKET two tetrads AG3(TTAG3 G3(TTAG3)3 AG3(TTAG3)3 TTAG3(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. K+ PARALLEL What may be the reason that different quadruplex structures were observed by various methods? [USEMAP] telomerni telomerni telomerni telomerni telomerni telomerni In nucleosides: 200 mM 120 mM 55 mM 16 mM 0.8 mM ( 0.1 mM) What may be the reason that different quadruplex structures were observed by various methods? G3(TTAG3)16 •Nucleic Acids Research 37 (2009) 6625-6634. Renčiuk et al.: Nucleic Acids Research 37 (2009) 6625-6634 [USEMAP] CD spectroscopy and conformational properties of nucleic acids ) What is optical activity, chiral metters, optical rotation, circular dichroism ) Wat are the (two) conditions for the origin of CD effect ) What components are responsible for CD of nucleic acids and proteins ) What are the advantages of CD spectroscopy as compared with other methods of the structural studies of biopolymers ) What is the substance of the unique sensitivity of CD to structural changes in NA ) What is optical rotatory dispersion and Cotton effect ) What is the difference between cooperative and non-cooperative changes ) Global characteristics of the forms B, A and Z DNA (particulary the grooves, an inverse topology of base pairs in the case of the Z-form ) some examples of non-canonical forms of DNA Types of four-stranded arrangements of NA mifi@ibp.cz [USEMAP] CHIROPTICKÉ METODY Optická rotační disperze-ORD Závislost úhlu stočení roviny polarizace lineárně polarizovaného světla průchodem opticky aktivní látkou na vlnové délce procházejícího záření. (180-800 nm) Cirkulární dichroismus-CD Závislost rozdílu absorpce pro vlevo a vpravo kruhově polarizované světlo na vlnové délce absorbovaného záření v oblasti energií elektronových přechodů. (180-1000 nm) Infračervený cirkulární dichroismus-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) Fluorescenčně detegovaný cirkulární dichroismus-FDCD Závislost rozdílu intenzity fluorescence, excitované vlevo a vpravo kruhově polarizovaným světlem na vlnové délce excitačního záření. (~ 200 nm až vlnová délka emise) Cirkulárně polarizovaná luminiscence (emise)-CPL (CPE) Spektrální průběh rozdílu intenzit (spontánní) emise vlevo a vpravo cirkulárně polarizovaného světla. (Interval vlnových délek emise chromoforu) Cirkulární diferenciální Ramanův rozptyl-Raman CID Spektrální průběh rozdílů intenzit Ramanova rozptylu vlevo a vpravo kruhově polarizovaného dopadajícího záření. (Interval vlnových délek Ramanova jevu)