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 Zn2+ and Mn2+ 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 (44) 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 N40 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.

Catalysis of covalent modification of aliphatic amine groups, such as the lysine (Lys) side chain, by nucleic acids has been challenging to achieve. Such catalysis will be valuable, e.g., for practical preparation of Lys-modified proteins. We previously reported DNA-catalyzed modification of the tyrosine and serine hydroxyl side chains, but Lys modification has been elusive. In this study, we show that increasing the reactivity of the electrophilic reaction partner by using 5′-phosphorimidazolide (5′-Imp) rather than 5′-triphosphate (5′-ppp) enables DNA-catalyzed modification of Lys in a DNA-anchored peptide substrate. DNA-catalyzed reaction of Lys + 5′-Imp is observed in an architecture in which the nucleophile and electrophile are not preorganized, whereas catalysis was not observed in our prior efforts that used Lys + 5′-ppp 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. Keywords deoxyribozymes; DNA; in vitro selection; peptides; lysine modification Deoxyribozymes are specific DNA sequences that have catalytic activity. [1] We have focused on expanding deoxyribozyme catalysis to include reactions of peptide side chains, [2] with the longer-term goal of achieving DNA-catalyzed covalent modification of large proteins. Our initial report demonstrated robust DNA catalysis (>70% yield in 1 h) of nucleopeptide formation between the nucleophilic tyrosine (Tyr) phenolic OH side chain and an electrophilic 5′-triphosphate RNA (5′-pppRNA; Fig. 1a). [2a] In parallel, however, catalysis by separate new deoxyribozymes involving the serine (Ser) aliphatic hydroxyl side chain was extremely poor (only ∼0.2% yield), and reactivity of the lysine (Lys) amine side chain was not observed. That initial study presented each single amino acid residue in a highly preorganized three-helix-junction (3HJ) architecture, in which the nucleophilic side

Catalysis of covalent modification of aliphatic amine groups, such as the lysine (Lys) side chain, by nucleic acids has been challenging to achieve. Such catalysis will be valuable, e.g., for practical preparation of Lys-modified proteins. We previously reported DNA-catalyzed modification of the tyrosine and serine hydroxyl side chains, but Lys modification has been elusive. In this study, we show that increasing the reactivity of the electrophilic reaction partner by using 5′-phosphorimidazolide (5′-Imp) rather than 5′-triphosphate (5′-ppp) enables DNA-catalyzed modification of Lys in a DNA-anchored peptide substrate. DNA-catalyzed reaction of Lys + 5′-Imp is observed in an architecture in which the nucleophile and electrophile are not preorganized, whereas catalysis was not observed in our prior efforts that used Lys + 5′-ppp 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.