PROJECT SUMMARY/ABSTRACT
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.
