Nature has developed the use of proteins and RNA as enzymes, while DNA is used for the storage and transfer of genetic information. Proteins and RNA are biopolymers that can fold into specific secondary and tertiary structures to enable catalysis. Considering the structural similarity to RNA, single-stranded DNA should also be able to form complex structures capable of catalyzing reactions. DNA catalysts have not been identified in nature, but in vitro selection has led to the identification of DNA catalysts for a variety of chemical reactions. Identification of new catalysts favors the use of DNA for multiple reasons. Amplification of functional DNA sequences is directly possible using natural polymerases, whereas amplification of RNA requires an additional reverse transcription step and amplification of proteins is not possible. The total number of possible sequences is smaller for nucleic acids (4n, where n is the number of residues) than for proteins (20n). Within this sequence space a large number of random nucleic acid sequences will fold into secondary and tertiary structures unlike proteins which require specific amino acid sequences to form complex structures. Therefore, in vitro selection experiments to identify DNA catalysts will cover a large portion of sequence space, and a large fraction of the covered space will contain structured DNA sequences with the potential to be catalytically active. The ease of synthesis and stability of DNA compared to RNA or proteins also provides an advantage for its use as a catalyst.
Natural post-translational modifications (PTMs) are important in biological systems. PTMs modulate protein activity resulting in rapid changes to cellular processes. Studying the role of specific PTMs is often limited to the ability to generate site-specific post-translationally modified proteins of interest. Phosphorylation of amino acid side chains is an abundant natural PTM that is essential for cellular function. Protein kinases, which catalyze phosphorylation, are often motif specific. Engineering these natural kinases to change motif requirements is challenging and often results in decreased substrate specificity. To identify new catalysts for the site-specific phosphorylation of a desired protein the use of DNA as a catalytic biomolecule is advantageous because an initially random population of DNA sequences does not have substrate biases, and DNA is a large biopolymer with the possibility to interact specifically with the substrates.
Both ribozymes and deoxyribozymes have been identified to catalyze the phosphorylation of oligonucleotides. However, previous efforts to identify kinase deoxyribozymes to catalyze the phosphorylation of amino acid side chains were unsuccessful because the -thiophosphoryl donor used was not stable in the selection conditions. As described herein, a new in vitro selection method was developed using a previously identified deoxyribozyme to separate the active deoxyribozymes from the inactive DNA sequences. This method led to the identification of the first kinase deoxyribozymes capable of phosphorylating tyrosine residues within a tethered peptide substrate using a bound 5′-triphosphorylated RNA oligonucleotide as the phosphoryl donor. Separate selection experiments were performed using 1 mM GTP as the phosphoryl donor. The identified DNA catalysts are able to phosphorylate tyrosine within a peptide substrate and require only low micromolar concentrations of GTP.
Site-specific modification of proteins is often desired. Most deoxyribozymes identified to modify peptide substrates have been identified using peptide substrates containing the reactive residue flanked by alanine residues. Peptide sequences derived from natural proteins contain a variety of amino acid residues with diverse functional groups that could be a point of interaction between the peptide substrate and DNA catalyst. Selection experiments were performed with biologically derived peptide sequences to identify tyrosine kinase deoxyribozymes with the ability to phosphorylate peptides sequence-specifically. Of the three peptide substrates evaluated the use of one led to deoxyribozymes that are peptide motif-specific, the second peptide led to deoxyribozymes with partial peptide sequence-selectivity, and the third did not lead to the identification of deoxyribozymes. The identification of peptide motif-specific deoxyribozymes demonstrates that DNA catalysts can interact specifically with peptide substrates, and individual DNA enzymes can interact with the same peptide substrate in a different manner.
The ability to phosphorylate substrates that are free in solution is desired. However, previously identified kinase deoxyribozymes are unable to phosphorylate untethered peptide substrates. Original efforts increased the length of the tether between the peptide substrate and DNA anchor to mimic a peptide free in solution. These selection experiments did not lead to deoxyribozymes, and further analysis of other deoxyribozymes with untethered peptide reactivity suggests the long tethers may interfere with catalysis. Further efforts have focused on the incorporation of hydrophobic modifications into the DNA catalysts to improve peptide binding. DNA aptamers containing hydrophobic modifications have improved protein binding. Increased binding affinity between the peptide substrate and DNA catalyst may enable untethered peptide reactivity.
While initial efforts focused on tyrosine phosphorylation, serine phosphorylation is also abundant in nature. Serine kinase deoxyribozymes have been identified to phosphorylate serine within tightly tethered peptide substrates using 5′-triphosphorylated RNA as the phosphoryl donor. Subsequent efforts to increase the tether length or use ATP as the phosphoryl donor were unsuccessful. Efforts to improve DNA catalysts with the ability to phosphorylate serine include using biologically derived peptide sequences to increase interactions between the deoxyribozyme and peptide substrate, and incorporating catalytically participatory modifications into the DNA enzymes
