Buyst, Dieter

Gheerardijn, Vicky

Madder, Annemieke

Martins, José

Van Gasse, Björn

Ghent University Academic Bibliography

	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  Dieter	  Buyst1,2*,	  Bjorn	  Van	  Gasse1,	  Vicky	  Gheerardijn2,	  Annemieke	  Madder2	  &	  José	  C.	  MarAns1	  	  1	  NMRSTR,	  Department	  of	  Organic	  Chemistry,	  University	  of	  Ghent,	  Krijgslaan	  281	  S4,	  B-­‐9000,	  	  Ghent,	  Belgium	  2	  OBCR,	  Department	  of	  Organic	  Chemistry,	  University	  of	  Ghent,	  Krijgslaan	  281	  S4,	  B-­‐9000,	  	  Ghent,	  Belgium	  	  (*e-­‐mail:	  Dieter.Buyst@UGent.be)	  In	  the	  development	  of	  new	  types	  of	  synthe'c	  enzymes,	  the	  DNA	  scaﬀold	  has	  recently	  been	  shown	  to	  be	  a	  suitable	  scaﬀold	  for	  the	  acceleraAon	  of	  a	  range	  of	  reacAons	  by	  providing	  a	  hydrophobic	  and	  chiral	  environment.	  One	  of	  the	  most	  aMracAve	  features	  of	  a	  DNA	   sequence	   is	   the	  mutual	   recogni'on	   of	   two	   complementary	   strands	   with	   the	   formaAon	   of	   a	   double	   helix	   structure	   in	   a	  predictable	   and	   programmed	   manner.	   Contrary	   to	   the	   use	   of	   intercalated	   ligands	   or	   in	   vitro	   selecAon	   procedures,	   recent	  developments	   within	   OBCR	   have	   made	   the	   introducAon	   of	   amino-­‐acid	   like	   side-­‐chain	   func'onali'es	   on	   the	   thymine	   base	  possible.	   In	   addiAon,	   it	   was	   demonstrated	   both	   by	   NMR	   and	   UV-­‐melAng	   experiments	   that	   their	   incorporaAon	   leaves	   duplex	  stability	   unaltered	   or	   even	   enhanced,	   therefore	   opening	   new	  possibiliAes	   for	   the	   development	   of	   two	   types	   of	   new	   syntheAc	  enzymes:	  serine	  protease	  and	  	  esterase	  like	  DNAzymes.	  The	  aim	  of	  the	  ﬁrst	  system	  is	  to	  successfully	  mimic	  the	  catalyAc	  triad	  of	  α-­‐chymotrypsin	  while	  the	  second	  type	  of	  systems	  is	  inspired	  by	  the	  syntheAc	  enzyme	  developed	  by	  Baltzer	  and	  co-­‐workers1.	  Systems	  of	  Interest	  α-­‐chymotrypsin	  and	  it’s	  acSve	  site	  	  (Asp102,	  His	  57	  and	  Ser195;	  PDB	  ID	  1AFQ)	  Acknowledgements	  The	  700MHz	  is	  part	  of	  the	  INMRF	  jointly	  operated	   by	   UGent,	   UA	   and	   VUB.	   The	  FWO	   WOG	   MULTIMAR	   is	   thanked	   for	  covering	  travel	  expenses.	  References	  [1]	  Broo,	  K.S.,	  et	  al.,	  J.	  Am.	  Chem.	  Soc.,	  1997,	  119(47),	  11362-­‐11372	  [2]	  Wüthrich,	  K.,	  NMR	  of	  Proteins	  And	  Nucleic	  Acids,	  1986	  	  	  [3]	  Vranken,	  W.F.,	  E.D.	  Laue,	  Proteins,	  2005,	  59,	  687-­‐696	  [4]	  Rieping,	  W.,	  M.	  Nilges,	  BioinformaAcs,	  2007,	  23,	  381-­‐382	  5’-GACCATTTGCAGCG-3’ 3’-CTGGTAATCGTCGC-5’  1  2  3  4  5  6  7  8 9 10 11 12 13 14 28 27 26 25 24 23 22 21 20 19 18 17 16 15 Wild	  type	  5’-GACCATTTHisGCAGCG-3’ 3’-CTGGTAAT  CGTCGC-5’  1  2  3  4  5  6  7  8       9 10 11 12 13 14 28 27 26 25 24 23 22 21      20 19 18 17 16 15 T8His	  Sequence	   Tm	  (°C,	  UV-­‐VIS)	  Wild	  type	   48,6	  ±	  0,3	  T6His	   50,5	  ±	  0,2	  T7His	   50,1±	  0,3	  T8His	   54,6±	  0,1	  T21His	   53,3±	  0,3	  •  MelAng	   temperatures	   (Tm)	   determined	   by	  NMR	  and	   UV-­‐VIS	   independently	   show	   a	   noAceable	  increase	  in	  stability	  of	  T8His	  and	  T21His	  	  •  Increase	  in	  pKa	  of	  modiﬁed	  nucleoAde	  inserted	  in	  duplex:	   stabilizaAon	   of	   protonated	   His-­‐like	  modiﬁcaAon	   through	   electrostaAc	   interacAon	  with	  negaAvely	  charged	  DNA	  backbone	  ?	  	  •  Increase	  in	  pKa	  is	  far	  higher	  for	  T8His	  	  	  	  	  	  	  è	  AddiAonal,	  speciﬁc	  electrostaAc	  interacAon	  T7His	  T6His	   T8His	  Building	  block	  T21His	  pH-­‐'tra'on	  study	  on	  THis	  modiﬁed	  systems	  Assignment	  Wild	  Type	  Assignments	  were	  performed	  mainly	  by	  nonlabile	  H1’-­‐H6/H8	  and	  H2’/H2”-­‐H6/H8	  sugar-­‐base	   through	  space	  contacts.	  H5-­‐H6,	   H6-­‐CH3	   and	   sugar	   protons	  were	   assigned	   using	   TOCSY/COSY	   spectra,	   granAng	   addiAonal	   conﬁrmaAon	   of	   each	   H1’	  and	   H2’/H2”	   belonging	   to	   the	   same	   deoxyribose	   spin	  system2.	  NHOOCH3NOOPOOO-HNNNNNH2OOPOOO-HNNH2OOOPOOO-HHNC	  A	  T	  3’	  5’	  2’2”	  2’2”	  2’2”	  1’	  1’	  1’	  Pictorial	  presentaAon	  of	  short	  1H-­‐1H	  distances	  di(6,8;2’)	  and	  ds(2”;6,8)	  (solid	  arrows),	  di(6,8;1’)	  and	  ds(1’;6,8)	  (dashed	  arrows)	  T-­‐T	  mismatch	  5’-GACCATTTGCAGCG-3’ 3’-CTGGTAATCGTCGC-5’  1  2  3  4  5  6  7  8 9 10 11 12 13 14 28 27 26 25 24 23 22 21 20 19 18 17 16 15 Chain	  A	  Chain	  B	  SchemaAc	  representaAon	  assignment	  Wild	  Type;	  All	  residues	  are	  located	  in	  standard	  1H	  chemical	  shit	  regions	  for	  DNA/RNA	  except	  H2’/H2”	  and	  H6	  protons	  of	  the	  T-­‐T	  mismatch	  H8/H6	  H5/CH3	  H1’	  H2’	  H2”	  H3’	  Assignment	  T8His	  T8	  T21	  C20	  G19	  T18	  δ2	  ε1	  Preliminary	  proposal	  posiAon	  of	  T8His	  modiﬁed	  funcAonality,	  this	  orientaAon	  could	  permit	  a	  H-­‐bond	  with	  T18	  or	  G19	  explaining	  the	  higher	  increase	  in	  pKa	  Special	   aMenAon	   is	   given	   to	   the	   assignment	   of	   the	   THis	  funcAonality	  and	   its	  posiAon.	  Diﬀerences	   in	  chemical	  shit	  δ	  as	  well	   as	   the	   nOe-­‐contacts	   present,	   suggest	   the	   linker	   is	  posiAoned	   inside	   the	  major	  groove,	  with	   the	   imidazole	  moiety	  situated	  between	  T18	  and	  G19.	  ε1	  ε1	  –	  H8	  G19	  	  ε1	  –	  δ2	  T8His	  	  ε1	  –	  H1’	  T18	  ε1	  –	  H2”	  T18	  ε1	  –	  H2’	  T18	  ε1	  –	  CH3	  T18	  2D	  NOESY	  tm	  250ms,	  pH	  5,0	  @	  700MHz,	  25°C	  1H	  shits	  T8His	  nucleoAde	  (ppm)	  4,82 1,84; 2,385,793,052,847,329,07,05OOONNHOOONHNHNH10,60,000	  0,005	  0,010	  0,015	  0,020	  0,025	  0,030	  0,035	  0	   10	   20	   30	   40	   50	   60	  T 2*-­‐'me	  (s)	  Temperature	  (°C)	  T2*	  in	  func'on	  of	  temperature	  T8	  wild	  type	  T21	  wild	  type	  T-­‐T	  mismatch	  =	  wobble	  type	  base	  pairing	  Wé	  and	  Wê	  in	  fast	  exchange	  ~	  5x1010	  Hz	  	  	  Average	  T-­‐T	  mismatch	  from	  nOe-­‐restrained	  based	  modeling	  (T8-­‐T21)	  superposiAon	  of	  ﬁve	  structures	  with	  the	  lowest	  energy3,4	  Since	  both	  types	  of	  the	  wobble	  type	  of	  base	   pairing	   are	   already	   in	   fast	  exchange	  at	  25°C,	  the	  nOe-­‐contacts	  of	  this	  base	  pair	  are	  averaged	  over	  both	  conformaAons.	   The	   T2*	   proﬁle	  indicates	   this	   as	  well	   as	   the	   inﬂuence	  of	  exchange	  with	  water	  at	  ±	  45°C.	  As	   shown	   above,	   valuable	   informaAon	   regarding	   assignment	   and	   determinaAon	   of	   the	   precise	   state	   of	   the	  modiﬁcaAons	   introduced	  inside	   the	   scaﬀold	   is	   available	   via	   NMR.	   In	   later	   stages,	   the	   T21His	   system	   as	   well	   as	   the	   addiAvity	   of	   the	   other	  modiﬁcaAons	   will	   be	  invesAgated	  while	  the	  cooperaAvity	  between	  the	  various	  funcAonal	  groups	  may	  also	  be	  obtained,	  providing	  valuable	  informaAon	  on	  the	  catalyAc	  site.	  Restrained	  based	  modeling	  on	  the	  T8His	  system	  will	  be	  used	  to	  visualize	  the	  posiAon	  of	  the	  modiﬁcaAon	  and	  conﬁrm	  the	  hypothesis	  of	  H-­‐bond	  formaAon	  of	  the	  imidazole	  funcAonality	  with	  the	  T18	  or	  G19	  base	  on	  the	  opposite	  strand	  of	  the	  DNA	  duplex.	  hMp://nmrstr.ugent.be	  δ1	  

Investigation of structure and dynamics of modified deoxyoligonucleotides via NMR spectroscopy

https://biblio.ugent.be/publication/4108011/file/4108019

Investigation of structure and dynamics of modified deoxyoligonucleotides via NMR spectroscopy

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Ghent University Academic Bibliography