Biomaterials
SINGLE MOLECULE OLIGOPEPTIDE FINGERPRINTING BASED ON TEMPLATED SELF-ASSEMBLY OF OLIGONUCLEOTIDE STRUCTURES
Caitlin Therien, BS Chemical Engineering
PhD Student
Columbia University
New York, New York, United States
Henry Hess
Professor of Biomedical Engineering
Columbia University Biomedical Engineering, United States
Steven Taylor
Researcher
Columbia University Medical Campus, United States
Sergei Rudchenko
Researcher
Columbia University Medical Campus, United States
Milan Stojanovic
Professor of Systems Biology
Columbia University Medical Campus, United States
Our objective is to enable massively parallel identification and quantification of single oligopeptide molecules in 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 proof-of-concept implementation, this new method relies on the conjugation of short oligonucleotides with organic receptors. As an example, the otherwise reversible interaction between the two strands of 10 base-pair DNA will be stabilized by the subsequent interaction between the ketoboronate (the organic ion) and amine groups (located on the sidechains of basic amino acids, like lysine). This stabilization allows us to locally identify where the amine group resides with respect to the the peptide residues, which in turn provides insight on the identity of each amino acid. 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.
Four DNA oligonucleotides were ordered from IDT Technologies: (1) A 10-mer (ATACATCTAG) biotinylated at the 3’ end; (2) A complementary 10-mer (CTAGATGTAT) fluorescently labeled with an ATTO647 dye at the 5’ end; (3) A 10-mer (ATACATCTAG) biotinylated at the 3’ end and thiolated at the 5’ end for the purposes of conjugating an oligopeptide or organic ion; (4) A complementary 10-mer (CTAGATGTAT) fluorescently labeled with an ATTO647 dye at the 5’ end and thiolated at the 3’ end.
Strands (3) and (4) were passed through a reduction column to reduce the thiol group and attach a bismaleimide linker. Then, strand (3) is further modified with a peptide (GGGKC) and strand (4) with a ketoboronate ion.
Single molecule fluorescence imaging experiments using TIRF illumination on surfaces coated with biotinylated BSA and Streptavidin were conducted to observe the binding of strand (2) to strand (1) immobilized via the biotin-streptavidin linkage on the surface. Typically, images were acquired every 500 ms for 10 min.
Label-free ensemble binding experiments were conducted using the Sartorius Octet platform relying on a biolayer interferometric technique. Streptavidin-coated sensors were immersed in PBS for 15 min, immersed in a solution containing 250 nM of strand (1) for 5 min, washed with PBS for 2 min, immersed in a solution containing strand (2) in concentrations ranging from 250 nM to 2mM for 10 min, and regenerated in PBS with 50 mM NaCl for 12 min.
We measured in single molecule experiments the kinetic rates for binding between a 10-mer oligonucleotide and its complement (strands 1 and 2) and reproduced the literature values from Jungmann et al. (Nano Letters 10, 4756, 2010). We will also discuss the binding rates between strand (3) and strand (4). We will also present ensemble experiments with the Octet (Bio-Layer Interferometry) instrument, where we measured the off rates for binding between strand (1) and strand (2) as well as binding between strand (3) and strand (4). Future experiments will involve further development of both single molecule and ensemble methods to obtain kinetic binding rates for every possible interaction, and the evaluation of longer peptides.