Functional nucleic acids are DNA and RNA aptamers that bind targets, or they are deoxyribozymes and ribozymes that have catalytic activity. These functional DNA and RNA sequences can be identified from random-sequence pools by in vitro selection, which requires choosing the length of the random region. Shorter random regions allow more complete coverage of sequence space but may not permit the structural complexity necessary for binding or catalysis. In contrast, longer random regions are sampled incompletely but may allow adoption of more complicated structures that enable function. In this study, we systematically examined random region length (N20 through N60) for two particular deoxyribozyme catalytic activities, DNA cleavage and tyrosine-RNA nucleopeptide linkage formation. For both activities, we previously identified deoxyribozymes using only N40 regions. In the case of DNA cleavage, here we found that shorter N20 and N30 regions allowed robust catalytic function, either by DNA hydrolysis or by DNA deglycosylation and strand scission via β-elimination, whereas longer N50 and N60 regions did not lead to catalytically active DNA sequences. Follow-up selections with N20, N30, and N40 regions revealed an interesting interplay of metal ion cofactors and random region length. Separately, for Tyr-RNA linkage formation, N30 and N60 regions provided catalytically active sequences, whereas N20 was unsuccessful, and the N40 deoxyribozymes were functionally superior (in terms of rate and yield) to N30 and N60. Collectively, the results indicate that with future in vitro selection experiments for DNA and RNA catalysts, and by extension for aptamers, random region length should be an important experimental variable.

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.