01_umDNA Conformational properties of DNA viewed by CD spectroscopy Michaela Vorlíčková Institute of Biophysics Academy of Sciences of the Czech Republic, v.v.i. Brno Laboratory of CD spectroscopy of nucleic acids 01_umDNA Laboratoř CD spektroskopie nukleových kyselin 3.109 A C G T < 2 % ACACACACACACACACACACACACACAC U:\MIFI\obrazky\sbírka\kolostruktur.png C:\WINDOWS\Plocha\image002.jpg U:\MIFI\obrazky\sbírka\kolostruktur.png Cirkulární dichroismus a optická aktivita biopolymerů ) optická aktivita – chirální látky (aminokyseliny, cukry) úhel stočení roviny polarizovaného světla, ORD U:\MIFI\obrazky\sbírka\Macek-poekresl..jpg Cirkulární dichroismus a optická aktivita biopolymerů ) optická aktivita – chirální látky (aminokyseliny, cukry) úhel stočení roviny polarizovaného světla, ORD ) CD – princip, veličiny, elipticita, ΔA, Δε, vztah mezi ORD a CD U:\MIFI\obrazky\sbírka\Macek-poekresl..jpg Cirkulární dichroismus Δε Δε = εL – ε R = ΔA/lc Elipticita φ [ψ] tg φ = b/a = εL – ε R/ εL + ε R Cicular dichroism • ABSORPTION CHIRALITY + BASE * CUGAR CD obr2 Podmínky vzniku CD • ABSORBCE CHIRALITA + BÁZE * CUKR CD 02fibryA 03fibryB 04fibryC A B C,D,T Wilkins+ Franklin 05BCA Tunis-Schneider, M.J.B. + Maestre, M.F. Cooperative changes between discreste structures 06a Structural changes 06a Non-cooperative changes within the same structure B-A a RNA B-A DNA transition A B B-A a RNA A -forma of RNA B-forma A-forma A V:\Iva\učebnicováB.jpg U:\MIFI\obrazky\sbírka\ucebnicovaA.jpg Obtuse angle Acute angle 220 300 R L 06Olson Vilma Olson V:\mifi-obr\sejmout.jpg V:\mifi-obr\sejmout0005.jpg 09Z 08 Z A. Rich MIT, Dickerson CalTech 11B 10B B V:\mifi-obr\sejmout0001.jpg 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] 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 13BZ,BX V:\mifi-obr\obr5.tif 14zvyraz 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 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) C:\WINDOWS\Plocha\149D-inset.jpg DNA Triplex C C T A T T C G A A T AG G C T T G T C C Radhakrishnan, I., Patel, D.J. (1994) Pyrimidine. Purine. Pyrimidine V:\mifi-obr\sejmout0004.jpg 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 U:\MIFI\presentace\Bez názvu 1.tif U:\MIFI\presentace\Bez názvu 2.tif U:\MIFI\presentace\Bez názvu 3.tif C:\WINDOWS\Plocha\struktury-html\UDIB70-biol1.jpg A ZIPPER-LIKE DNA DUPLEX d(GCGAAAGCT) Shepard, W. et al., Structure 6, pp. 849 - 861, 1998. 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. 27i-tetr i - tetraplex 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 V:\mifi-obr\UDF043-inset.jpg D(C C C A A T) Intercalated tetraplex of human telomeric DNA Berger, I., Kang, C., Fredian, A., Ratliff, R., Moyzis, R., Rich, A. Nat.Struct.Biol., 2, pp. 416 - 25, 1995 28C-tetr 28PY copy (1) TCCCCACCTTCCCCACCCTCCCCACCCTCCCCA Fragment in c-myc promoter 25tetraplexy Q DNA G - quadruplex 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 WAVELENGTH [nm] Fragment Pu-27 of c-myc promoter: TGGGGAGGGTGGGGAGGGTGGGGAAGG Pan, A.T. et al.: J.Am.Chem.Soc.126(2004)8710 V:\mifi-obr\G_Q_Elsevier.JPG V:\Iva\obr3.JPG V:\mifi-obr\Fig8.jpg 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 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 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 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? 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? 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