DNA-catalyzed lysine side chain modification.

Catalyzing the covalent modification of aliphatic amino groups, such as the lysine (Lys) side chain, by nucleic acids has been challenging to achieve. Such catalysis will be valuable, for example, for the practical preparation of Lys-modified proteins. We previously reported the DNA-catalyzed modification of the tyrosine and serine hydroxy side chains, but Lys modification has been elusive. Herein, we show that increasing the reactivity of the electrophilic reaction partner by using 5'-phosphorimidazolide (5'-Imp) rather than 5'-triphosphate (5'-ppp) enables the DNA-catalyzed modification of Lys in a DNA-anchored peptide substrate. The DNA-catalyzed reaction of Lys with 5'-Imp is observed in an architecture in which the nucleophile and electrophile are not preorganized. In contrast, previous efforts showed that catalysis was not observed when Lys and 5'-ppp were used in a preorganized arrangement. Therefore, substrate reactivity is more important than preorganization in this context. These findings will assist ongoing efforts to identify DNA catalysts for reactions of protein substrates at lysine side chains.

Establishing broad generality of DNA catalysts for site-specific hydrolysis of single-stranded DNA.

We recently reported that a DNA catalyst (deoxyribozyme) can site-specifically hydrolyze DNA on the minutes time scale. Sequence specificity is provided by Watson-Crick base pairing between the DNA substrate and two oligonucleotide binding arms that flank the 40-nt catalytic region of the deoxyribozyme. The DNA catalyst from our recent in vitro selection effort, 10MD5, can cleave a single-stranded DNA substrate sequence with the aid of Zn(2+) and Mn(2+) cofactors, as long as the substrate cleavage site encompasses the four particular nucleotides ATG^T. Thus, 10MD5 can cleave only 1 out of every 256 (4(4)) arbitrarily chosen DNA sites, which is rather poor substrate sequence tolerance. In this study, we demonstrated substantially broader generality of deoxyribozymes for site-specific DNA hydrolysis. New selection experiments were performed, revealing the optimality of presenting only one or two unpaired DNA substrate nucleotides to the N(40) DNA catalytic region. Comprehensive selections were then performed, including in some cases a key selection pressure to cleave the substrate at a predetermined site. These efforts led to identification of numerous new DNA-hydrolyzing deoxyribozymes, many of which require merely two particular nucleotide identities at the cleavage site (e.g. T^G), while retaining Watson-Crick sequence generality beyond those nucleotides along with useful cleavage rates. These findings establish experimentally that broadly sequence-tolerant and site-specific deoxyribozymes are readily identified for hydrolysis of single-stranded DNA.