Single molecule oligopeptide fingerprinting based on templated self-assembly of oligonucleotide structures

Project Summary Our goal is to enable massively parallel identification and quantification of single oligopeptide molecules in very small samples. The development of such method will be complementary to the mainstay technologies for large-scale protein sequencing and quantitation, such as mass spectrometry, and would enable routine analysis of small amounts of protein as well as variations in posttranslational modification (PTMs). In its first implementation, streamlined for quickly reaching the proof-of-concept, while at the same time generating physicochemical parameters required for further optimization, the new method will rely on conjugates of organic receptors with short oligonucleotides. These conjugates will undergo a self-assembly templated by target oligopeptides to provide specific fingerprints. Here, organic receptors, such as, ketoboronates forming imines with amines (lysines), cyclodextrins hosting hydrophobic side chains, 1,2- diketones recognizing guanidines (arginines), metal complex coordinating with imidazoles (histidines), and activated olefins undergoing reversible Michael additions with thiols (cysteines), will all provide weak side- chain-specific interactions that would stabilize otherwise reversible duplex formations by short complementary oligonucleotides. Each next-in-line conjugate would bind to an oligonucleotide epitope newly displayed by the previously bound receptor while being selected by the next, proximal, solvent- exposed amino-acid side chain. One by one, the template would select conjugates forming the most stable complex. Through ligation as the final step, each oligopeptide would effectively be reverse-translated into linear modified DNA sequences that would be readable by, for example, nanopore sequencing. Over the first two years of the project, relying on the single molecule spectroscopy method called DNA- PAINT, we will provide the proof-of-concept that fundamental steps of this process work as designed. We will first show, in Aim 1, that a peptide conjugated to a guide oligonucleotide can attract a specific oligonucleotide-organic receptor conjugate, with the organic receptor stabilizing duplex formation through interactions with the specific amino acid side chain. We will directly measure the impact that this receptor has on off rates of short oligonucleotides, positioning then these observations into the context of structure of oligopeptide templates, and assessing the resolution (specificity) of our approach. In Aim 2, we will expand these studies, using the same DNA-PAINT approach to study ternary complexes, which will be formed by the first conjugates attracting the second. In our final demonstration, different model peptides will template specific sequences, to be subsequently ligated through a click chemistry protocol. This progress will enable the next phase of our project, in which we will tackle self-assembly in the context of analysis of mixtures of peptides and proteins.