DNA catalysts as phosphatases and as phosphoserine lyases

Proteins and RNA are the only known biopolymers that have catalytic roles in nature, whereas DNA is primarily considered to store and transfer genetic information. However, artificial single-stranded DNA has been identified by in vitro selection to catalyze several chemical reactions and several of those are of biological relevance. For in vitro selection or directed evolution of proteins, direct amplification is not possible, and it essential to attach the genotype to the phenotype. For nucleic acids however, the functional biopolymer can be readily amplified. DNA has the advantage of being directly amplified by polymerases, whereas RNA requires an additional reverse transcription step. Moreover, DNA catalysts identified by in vitro selection processes have shown similar catalytic proficiency as RNA. DNA has added advantages of low cost of chemical synthesis and higher stability. Considering these factors combined, identification of artificial DNA catalysts (deoxyribozymes) for chemical reactions is a valuable endeavor with long-term implications. Protein post-translational modifications (PTMs) are highly important in biological processes involving cellular regulation. Additionally, PTMs serve as important intermediates or key motifs on natural products and bioactive peptides. The natural protein enzymes carrying out the essential modifications may have several shortcomings for biotechnological use. Identification of artificial DNA catalysts with ability to perform chemoselective post-translation chemical reaction would be highly useful in studying biological regulatory processes, performing synthesis and late-stage diversification of post-translationally modified peptides, as well as carrying out other important functions that natural proteins may not readily solve. My first effort was to identify DNA enzymes with peptide/protein phosphatase activity, more specifically dephosphorylation of peptide side chains. Without a catalyst, phosphomonoester hydrolysis reactions have exceedingly low spontaneous reaction rates. Nature utilizes proficient protein enzymes to perform this challenging reaction with great efficiency. Using a known DNA catalyst for the in vitro selection process, new DNA catalysts were identified with phosphatase activity. The phosphatase DNA catalysts exhibited multiple-turnover activity with phosphotyrosine-containing free peptides and were active even in the presence of externally added cell lysate or bovine serum albumin (BSA). Furthermore, the best DNA phosphatase functioned with a larger protein substrate. This established the fundamental ability of DNA to catalyze dephosphorylation of amino acid side chain residues. The study also suggested that phosphatase DNA catalysts could perform intracellular phosphatase activity. Hence, these deoxyribozymes were functionalized on gold nanoparticles and delivered inside live mammalian cells to investigate if they behave as functional protein analogues (or mimics) of recombinantly expressed Protein Tyrosine Phosphatase (PTP1B). Separately, efforts were directed towards the important goal of identifying sequence-selective phosphatase deoxyribozymes. Although three separate efforts were directed towards identifying sequence-selective phosphatases deoxyribozymes, we were unsuccessful in accomplishing this specific goal of selectivity in the context of peptide sequence discrimination. Dehydroalanine (Dha) is a non-proteinogenic electrophilic amino acid that serves as a synthetic intermediate or product in the biosynthesis of several bioactive cyclic peptides such as lantibiotics, thiopeptides and microcystins. DNA enzymes were identified to establish the fundamental catalytic ability to eliminate phosphate from phosphoserine (pSer) to form Dha, namely phosphoserine lyase activity. Furthermore, DhaDz1 was utilized to achieve chemo-enzymatic synthesis of a cyclic cystathionine-containing peptide. Based on this initial success, future efforts will be directed to achieve sequence-general phosphoserine and phosphotyrosine lyase activity. Separately, application of sequence-general lyases in the synthesis of complex lanthipeptides and enrichment of phosphopeptides/proteins in phosphoproteomics will be explored

Phosphoserine Lyase Deoxyribozymes: DNA-Catalyzed Formation of Dehydroalanine Residues in Peptides

Dehydroalanine (Dha) is a nonproteinogenic electrophilic amino acid that is a synthetic intermediate or product in the biosynthesis of several bioactive cyclic peptides such as lantibiotics, thiopeptides, and microcystins. Dha also enables labeling of proteins and synthesis of post-translationally modified proteins and their analogues. However, current chemical approaches to introducing Dha into peptides have substantial limitations. Using in vitro selection, here we show that DNA can catalyze Zn²⁺ or Zn²⁺/Mn²⁺-dependent formation of Dha from phosphoserine (pSer), i.e., exhibit pSer lyase activity, a fundamentally new DNA-catalyzed reaction. Two new pSer lyase deoxyribozymes, named Dha-forming deoxyribozymes 1 and 2 (DhaDz1 and DhaDz2), each function with multiple turnover on the model hexapeptide substrate that was used during selection. Using DhaDz1, we generated Dha from pSer within an unrelated linear 13-mer peptide. Subsequent base-promoted intramolecular cyclization of homocysteine into Dha formed a stable cystathionine (thioether) analogue of the complement inhibitor compstatin. These findings establish the fundamental catalytic ability of DNA to eliminate phosphate from pSer to form Dha and suggest that with further development, pSer lyase deoxyribozymes will have broad practical utility for site-specific enzymatic synthesis of Dha from pSer in peptide substrates