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SUMMARY:Sequence-specific labeling of DNA with methyltransferases and synt
 hetic cofactor analogues - Prof Elmar Weinhold
DTSTART:20160923T160000Z
DTEND:20160923T170000Z
UID:TALK67579@talks.cam.ac.uk
CONTACT:Dr. Ulrich Keyser
DESCRIPTION:Sequence-specific labeling of long DNA is of major interest fo
 r functional studies of DNA and DNA-modifying enzymes as well as for DNA m
 apping\, gene delivery and (nano)-biotechnology. We employ DNA methyltrans
 ferases (MTases) which transfer the activated methyl group of the ubiquito
 us cofactor S-adenosyl-L-methionine (AdoMet or SAM) to adenine-N6\, cytosi
 ne-N4 or cytosine-C5 within specific double-stranded DNA sequences ranging
  from two to eight base pairs. Since the methyl group is not an attractive
  reporter group\, we have synthesized two classes AdoMet analogues which s
 erve as new cofactors for sequence-specific DNA functionalization and labe
 ling by DNA MTases.\nIn the first class of cofactor analogues the amino ac
 id side chain of AdoMet is replaced with an aziridinyl residue and reporte
 r groups are attached to the adenine ring. DNA MTase-catalyzed nucleophili
 c attack and aziridine ring opening leads to sequence-specific coupling of
  the whole aziridine cofactor with DNA and hence to covalent DNA labeling 
 (Sequence-specific Methyltransferase-Induced Labeling of DNA\, SMILing DNA
 ). In the second class of cofactor analogues the methyl group of AdoMet is
  replaced with extended carbon chains carrying an unsaturated bond in β-p
 osition to the sulfonium center (double-activated AdoMet analogues). Enzym
 atic transfer of extended chains containing chemical reporters followed by
  chemo-selective ligation of reporter groups was used for sequence-specifi
 c labeling of plasmid DNA (methyltransferase-directed Transfer of Activate
 d Groups\, mTAG). Although such a two-step procedure offers the flexibilit
 y of using one AdoMet analogue for labeling with different reporter groups
 \, it adds an extra chemical step which is difficult to quantify on the DN
 A and can lead to partial DNA labeling.\nTo overcome this limitation we ha
 ve synthesized double-activated AdoMet analogues with over fiftyfold exten
 ded methyl group replacements\, carrying different reporter or affinity gr
 oups in the activated side chain\, and used them to sequence-specifically 
 label plasmid and genomic DNA with DNA MTases in one step. These enzymatic
  reactions are applied for genome barcoding and DNA mapping. In addition\,
  we have developed a method to produce covalent DNA-protein conjugates by 
 SNAP-tag technology. AdoMet analogues are used to enzymatically transfer b
 enzylguanine residues to specific sites in DNA and the modified DNA is the
 n reacted with SNAP-tag fusion proteins to specifically attach proteins to
  DNA. We have used this method to produce covalent plasmid DNA-antibody co
 njugates for targeted cell delivery.\n
LOCATION:Rayleigh seminar room\, 2nd floor\, Maxwell Building\, Cavendish 
 laboratory
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