VILNIUS UNIVERSITY INSTITUTE OF BIOTECHNOLOGY ...

Specific aims:1. To chemically synthesize a series of activated AdoMet analogs with extended propargylic sidechains replacing the methyl group.2. To investigate chemical stability of the extended AdoMet analogs and assess their activity in DNAmethyltransferase-catalyzed transalkylation reactions.3. To engineer the cofactor binding center in the HhaI MTase for efficient transfer of extendedgroups from the AdoMet analogs.4. To demonstrate sequence-specific functionalization and labeling of natural DNA molecules usingthe AdoMet analogs and engineered DNA methyltransferases.Scientific novelty:The specificity of an enzymatic reaction has been successfully redesigned via syntheticmodification the AdoMet cofactor and rational engineering of the cofactor binding center in a DNAcytosine-5 methyltransferase. It was shown for the first time that sterically-impaired linear side chainscan be activated for enzymatic S N 2 reactions by introducing unsaturated carbon-carbon bonds next tothe transferable carbon atom. We also demonstrate for the first time an efficient site-specific transfer oflinear carbon chains containing primary aliphatic amino groups, followed by amino-specific covalentcoupling of reporter groups onto DNA.Practical value:A synthetic strategy has been developed for obtaining novel analogs of the ubiquitous cofactor ofAdoMet-dependent methyltransferases that contain functional or reporter groups within extendedtransferable side chains. The newly developed mTAG technology (methyltransferase-directed Transferof Activated Groups) was shown to be effective for sequence-specific functionalization and labeling ofplasmid DNA, which can be further applied for analysis of genomic DNA methylation. These findingspave the way for targeted labeling of other biopolymers such as RNA and proteins using a myriad ofhighly specific AdoMet-dependent methyltransferases known in nature. These new molecular toolsenvision numerous potential applications ranging from probes for genetic screening technologies tomolecular building blocks in DNA-based nanobiotechnology.Findings presented for defense:1. S-Adenosyl-L-methionine (AdoMet) analogs with extended propargylic side chains have beenobtained by direct chemoselective S-alkylation of S-adenosyl-L-homocysteine.2. AdoMet analogs bearing unsaturated bonds next to the reactive carbon are more active in S N 2and A N reactions than corresponding saturated compounds.3. Adenine-N6, cytosine-N4 and cytosine-C5 DNA methyltransferases catalyze the transfer ofextended groups onto DNA with full retention of their target specificity.4. Steric engineering of the cofactor binding center in a DNA cytosine-C5 methyltransferase,M.HhaI, leads to an enhanced transfer of extended side chains from synthetic AdoMet analogs.5. AdoMet analogs containing a primary aliphatic amino group in the extended side chain can beused for methyltransferase-directed sequence-specific labeling of natural DNA.7