Non-Natural Nucleobases for the Triplex DNA Formation as A New Strategy for Regulation of Gene Expression Duplex DNA TFO (triplex-forming ODN) Triplex DNA Parallel triplex DNA Anti-parallel triplex DNA A pyrimidine-rich TFO binds parallel to the homopurine strand of duplex DNA. A purine-rich TFO binds anti-parallel to the homopurine strand of duplex DNA. Formation in the major groove of the duplex. Inhibition of transcription (antigene) Recruitment of biological systems Site-specific modification of DNA Initiation of DNA repair events Probability in human genome (15 bases length, >50% G with one pyrimidine) At least one exist in the promoter/transcribed region of ~98% annotated human genes ~87% of them are the unique to one gene Limitation of Duplex Sequence for Triplex Formation TFO (triplex-forming ODN) Duplex DNA Stable Triplexes Form with HomopurineHomopyrimidine Strand TFO A A T G G C A A T A A T G G C G G C G G C Triplex DNA Pyrimidine Inserts Destabilize Triplexes Interrupting Site TFO A A T G G C A A T T A A G G C G G C G G C Non-Natural Base Analogs Needed for TA and CG Interrupting Sites Interrupting Sites Antiparallel Triplex T FO TFO ReverseHoogsteen H-bonds WatsonCrick pair Py Pu Py Pu T A A T T FO TFO ReverseHoogsteen H-bonds Pu Py Py WatsonCrick pair Pu Pu C H G Non-Natural Base Analogs for TA and CG Interrupting Sites In Parallel Triplex DNA Ohkubo, K. Yamada, Y. Ito, K. Yoshimura, K. Miyauchi, T. Kanamori, Y. Masaki, K. Seio, H. Yuasa, and M. Sekine, Nucleic Acids Res., 2015, 1–12. Y. Hari, M. Akabane, and S. Obika, Chem. Commun., 2013, 49, 7421. D. A. Rusling, Vicki E. C. Powers, R. T. Ranasinghe, Y. Wang, S. D. Osborne, T. Brown, and K. R. Fox, Nucleic Acids Res., 2005, 33, 3025–3032. Li, J.-S., Chen, F.-X., Shikiya, R., Marky, L. A. & Gold, B. J. Am. Chem. Soc. 127, 12657–12665 (2005). Difference in Geometry between Parallel and Antiparallel Triplexes Antiparallel Parallel Natural T-A:T TFO Natural A-A:T TFO b ReverseHoogsteen a Natural G-G:C b Design of Recognition Molecules for a TA and a CG Sites Stacking part •Hydrogen Bondings. DNAO O DNAO Recognition part •Stacking Interactions. •Shape complementarity. O W-Shaped Nucleic Analog (WNA) 1.A short spacer O O O O 5' O O O H N N H N O H 3' N N N N 3' O O a H Aromatic ring O O 3' N Bicyclic ring 5' 5' O 3. Stacking unit 2. Fixed conformation O H N N H N O 3' H N N N N 3' O H N N H N O H N N N 3' Diversity of WNA Structure SubstitutedBenzene Naphthalene Thiophene Furane Pyrrole etc Aromatic part D NA O D NA O O O Recognition part H guanine thymine adenine cytosine 5-methylcytosine pyridone imidazole etc Synthesis of WNA R=TB DPS HO 1)Acetone, H+ O HO O H 2)Ac2O, Pyr OH 3) piperidine Ac O O O OH 1) PCC O 2) PhLi RO O O D-ribose O H 1) TBDPSCl O RO 2) Allyltrimethylsilane ZnBr2, CH3NO2 O O O (α-Allyl: β-Allyl= 7 : 6) RO O O H OsO4, NaIO4, Pyr O O 5%H2SO4, THF, 60 Ž RO O HO O HH O H Ac O, Pyr 2 RO O H AcO O OA c Syntheses of WNA Analogs Et3SiH TMSOTf CH2Cl2 O RO O R'O OA c R : TBDPS R' : Ac RO R'O iBu-G BSA TMSOTf CH3CN Bicyclic intermediate O O WNA-H: 72% iBuNH N RO O R'O N O WNA-bT: 42% + aT: 37% Bz-C BSA SnCl4 CH3CN Bz-mC Thymine HMDS TMSCl SnCl4 CH3CN Bz-A BSA TMSOTf CH3CN BSA SnCl4 CH3CN RO O R'O NH O N N O WNA-9bG: 38% +9aG:15%, 7bG: 25%, 7aG: 10% O NHBz NHBz NH N N RO O O R'O N O WNA-bmC: 53% + amC: 14% O RO O R'O N O O WNA-bC: 53% + aC: 40% N RO O R'O O N N N WNA-9bA: 53% + 9aA: 40% NHBz Formation of the Stable Triplex Having a TA Interrupting Site 3' GGA AGG A Z G GAG GGA GGA -32P TFO(10nM) 5' GGG AGG GAG GGA AGG A X G GAG GGA GGA AGC (Pu) duplex 3' CCC TCC CTC CCT TCC T Y C CTC CCT CCT TCG (Py) Z=WNA-bT DN A O XY GC AT 0 4 8 10 40 100 0 4 8 10 40 100 O O D NA O duplex concentration thymine O N b O XY nM duplex concentration 0 Triplex Triplex 32P-TFO 32P-TFO Ks (M-1x109) 0.082 < 0.001 NH CG 4 8 10 40 100 0.015 TA 0 4 8 10 40 100 nM 0.30 Ks= [triplex] [TFO] x [duplex] Gel shift assay for determination of triplex formation. Triplex formation was done for 12 hours at 22 °C in the buffer containing 5 mM MgCl2, 20 mM Tris-HCl, 2.5 mM spermidine and 10 % sucrose at pH 7.5. Electrophoresis was done at 10 °C with 20 % non-denatured polyacrylamide gel. Recognition of all four base pairs by WNA analogs 3’ GGAAGG AZG GAGGAGGGA-32P 5’ Target 5’ GGGAGGGAGGGAAGG AXG GAGGAGGGAAGC 3’ Duplex 3’ CCCTCCCTCCCTTCC TYC CTCCTCCCTTCG 5’ TFO NH2 O DNA DNA NH O N O O b O O N O (Thymine) O b O WNA-bT DNA N O Selective to the TA pair DNA O (Cytosine) WNA-bC Selective to the CG pair Stability constants (Ks, 109M-1) XY Z TA AT CG GC dG 0.004 0.008 0.008 0.086 G / GC dA <0.001 0.074 <0.001 0.047 A / AT WNA-bT 0.300 <0.001 0.015 0.082 WNA-bT / TA WNA-bC <0.001 0.025 0.115 0.047 WNA-bC / CG *5mM MgCl2 , Ks = [Triplex] [TFO][Duplex] S. Sasaki, et al., J. Am. Chem. Soc., 126, 516-528 (2004). The selectivity of the WNA depends on the flanking bases TFO Target Duplex 3’ GGAAGG NZN GAGGAGGGA-32P 5’ 5’ GGGAGGGAGGGAAGG NXN GAGGAGGGAAGC 3’ 3’ CCCTCCCTCCCTTCC NYN CTCCTCCCTTCG 5’ NH2 O DNA DNA NH O N O O b O DNA O N O N O (Thymine) O b O WNA-bT DNA O (Cytosine) WNA-bC Stability constants (Ks, 109M-1) Z /XY 3’-AZG-5’ 3’-GZG-5’ 3’-AZA-5’ 3’-GZA-5’ AXG GXG AXA GXA TYC CYC TYT CYT WNA-bT/TA 0.300 0.130 <0.001 <0.001 WNA-bC/CG 0.115 0.002 <0.001 0.004 Recognition ability of WNA depends of flanking base pairs Suitable WNA Analogs for a TA Site 10 nM TFO 3’-GGAAGG Target 5’-GGGAGGGAGGGAAGG Duplex 3’-CCCTCCCTCCCTTCC 3’ Ks (109 M-1) 0.3 AZG AXG TYC NZN GAGGAGGGA NXN GAGGAGGGAAGC NYN CTCCTCCCTTCG 5’ Ks (109 M-1) 0.25 0.12 Br O 0.2 O O 0.15 NH O O 0.05 GC AT CG 0 pCN- pBr- mBr- oBr- WNA-b T dG M-1) 0.06 0.05 3’ AZA AXA TYT 0.06 0.04 0 O 5’ O pCN- pBr- mBr- oBr- WNA-bT dG TA NH O N O O Ks (109 M-1) 0.12 mBr-WNA-bT 3’ GZA GXA CYT 5’ 0.1 CN 0.08 O 0.03 0.02 O 0.01 GC AT CG 0 pBr- GC AT CG 0.02 TA 0.04 pCN- 5’ 0.08 Br O Ks GZG GXG CYC 0.1 N oBr-WNA-bT 0.1 (109 0.14 O 3’ mBr- oBr- WNA-b T dG TA N H 0.04 O O 0.06 N O pCN-WNA-bT O 0.02 GC AT CG 0 pCN- pBr- mBr- oBr- WNA-b T dG TA 1) Y. Taniguchi, et al. Tetrahedron, 64, 7164–7170 (2008), 2) Y. Taniguchi, et al. J. Org. Chem., 71, 2115–2122 (2006), 3) Y. Taniguchi, et al. Nucleosides, Nucleotides and Nucleic Acids, 24, 823–827 (2005) An Application of TFO for Antiproliferative Effects to A549 Cells Blc2 TFO(bT) Survivin TFO(nat) Survivin TFO(bT) Random Cell viability (%) Bcl2 TFO(nat) (mM) The A549 cell was treated with the complex of TFOs and OligofectamineTM and PLUS Reagent in DMEM medium containing 10%FBS, incubated for 72 hr under 5% CO2 at 37 oC. The cell proliferation was checked by CellTiter 96® (PROMEGA). (n = 3) Bcl2 Survivin 3’ ---------AGGGG GTGGTGGAGGAAGAGGGGTG GGGAG- ------5’ . 3’ ---------GTGAC GGAAGAAGGAGGGAGTGAAGAG TGGAC- --5’ 5’ ---------TCCCC CACCACCTCCTTCTCCCCAC CCCTC-------3’ 5’ ---------CACTG CCTTCTTCCTCCCTCACTTCTC ACCTG- --3’ Bcl2 TFO(bT) 5’ GbTGGbTGGUGGUUGUGGGGbTG-amino 3’ Survivin TFO(bT) 5’ GGUUGUUGGUGGGUGbTGUUGUG-amino 3’ Bcl2 TFO(nat) 5’ GTGGTGGUGGUUGUGGGGTG-amino 3’ Survivin TFO(nat) 5’ GGUUGUUGGUGGGUGTGUUGUG-amino 3’ Random 5’ UUGTGGUGGGUGGUGGUGUGUU-amino 3’ O HO O HO N O NH O WNA-bT Taniguchi Y and Sasaki S.,,Org. Biomol. Chem., 10(41),8336-8341 (2012). Desigen of N 2-modified Isocytidine Derivatives for Selective CG Base Pair Recognition Dr. Hidenori Okamura, Associate Prof. Yosuke Okamura Non-natural nucleoside TFO Duplex DNA Triplex DNA N = A, G,T, C GuanidinoisodC Thymidine C G Weak and low-selective interaction C G AP-isodC C G H. Okamura, Y. Taniguchi and S. Sasaki, Org. Biomol. Chem., 2013 H. Okamura, Y. Taniguchi and S. Sasaki, ChemBioChem, 2014 Confirmaiton of Isocytidine Derivatives for Selective CG Base Pair Recognition TFO Duplex DNA Z= 3’-GGAAGGG Z AGAGGAGGGA 5’-GAGGGAAGGG X AGAGGAGGGAAGC 3’-CTCCCTTCCC Y TCTCCTCCCTTCG-FAM XY = TFO (nM) Triplex DNA Duplex DNA (100 nM) GC 0 10 50 100 500 1000 Conditions : 20 mM Tris-HCl buffer, 20 mM MgCl2, 5 mM spermidine, pH 7.5 Ks (10-6 M-1) = [Triplex] / ([TFO][Duplex]) n.d.: not determined CG 0 10 50 100 500 1000 AT 0 10 50 100 500 1000 TA 0 10 50 100 500 1000 Ks = 6.9 Ks = 6.7 Ks = 39.2 Ks = 0.5 Ks = 0.2 Ks = 0.6 n.d. n.d. n.d. Ks = 12.09 n.d. n.d. Triplex DNA Duplex DNA (100 nM) Triplex DNA Duplex DNA (100 nM) Iso-dC derivatives selectively recognize CG base pair But affinity needs to be improved Challenge to In Vivo Application of Triplex DNA and Selective Chemical Modification of RNA Triplex DNA Covalent Modification Delivery System Release Antisense Delivery Penetration Chemical Modification Nucleus Antigene Efficient Inhibition Editing mRNA mRNA Triplex DNA DNA 17