DNA‐Catalyzed Lysine Side Chain Modification

Abstract

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

DNAzymes for amine and peptide lysine acylation

“…With success in identifying DNAzymes that acylate the simple amine nucleophile DNA-C3-NH2, especially at the lower pH of 7.5, we shifted our attention to the substrate that presents a peptide Lys nucleophile, DNA-HEG-AAAKAA, where our ultimate goal is DNAzyme-catalyzed acylation of peptide and protein Lys residues. Unsurprisingly based on our previous report with DNA-catalyzed Lys phosphoramidate formation, 66 none of the above-described DNAzymes identified using the DNA-C3-NH2 substrate had any detectable activity (<0.2%) when tested with DNA-HEG-AAAKAA (data not shown).…”

“…(A) 5-Phosphorimidazolide (5-Imp) DNA, for which we previously found DNAzymes that catalyze Lysphosphoramidite formation. 66 (B) 5-Thioester DNA, for which here we were unable to identify any amine acylation DNAzymes. (C) 5-Aryl ester DNA, for which here we describe new DNAzymes that catalyze amine acylation, including with Lys peptides.…”

“…Toward this goal, we previously reported the first DNAzymes that catalyze Lys modification of any kind. 66 We used 5-phosphorimidazolide (5-Imp) DNA as the electrophile, resulting in the formation of a Lys-phosphoramidite bond ( Figure 1A). From that study, a key lesson was the need for a suitably reactive electrophile to react with the amine nucleophile, a consideration that outweighed the value of highly preorganizing the two substrates.…”

Pursuing DNA Catalysts for Protein Modification

“…By providing a more reactive 5′-phosphorimidazolide (Imp) electrophile in place of the 5′-triphosphate (ppp), successful DNA catalysis with lysine was observed, even when the 3HJ architecture was abandoned in favor of the less preorganized arrangement (Figure 11). 48 This study was valuable for revealing the greater importance, at least in this context, of substrate reactivity relative to preorganization. These findings especially inform our ongoing efforts toward DNA-catalyzed amine (e.g., lysine) acylation reactions, in which we are evaluating a variety of reactive acyl donors.…”

Catalytic DNA: Scope, Applications, and Biochemistry of Deoxyribozymes

“…Some of this work was recently reviewed [22]; selected highlights are provided here (Figure 3B). Deoxyribozymes have been found for conjugation of oligonucleotides to tyrosine [68–70], tyrosine phosphorylation [71,72], phosphotyrosine (pTyr) and phosphoserine (pSer) dephosphorylation [73], formation of dehydroalanine (Dha) by elimination of phosphate from pSer [74], and lysine modification [75]. The case of tyrosine phosphorylation illustrates that DNA catalysts can distinguish among various peptide substrate sequences [72], which is important for sequence-specific modification.…”