Click Addition of a DNA Thread to the N-Termini of Peptides for Their Translocation through Solid-State Nanopores

Abstract

Foremost among the challenges facing single molecule sequencing of proteins by nanopores is the lack of a universal method for driving proteins or peptides into nanopores. In contrast to nucleic acids, the backbones of which are uniformly negatively charged nucleotides, proteins carry positive, negative and neutral side chains that are randomly distributed. Recombinant proteins carrying a negatively charged oligonucleotide or polypeptide at the Ctermini can be translocated through a α-hemolysin (α-HL) nanopore, but the required genetic engineering limits the generality of these approaches. In this present study, we have developed a chemical approach for addition of a charged oligomer to peptides so that they can be translocated through nanopores. As an example, an oligonucleotide PolyT20 was tethered to peptides through first selectively functionalizing their N-termini with azide followed by a click reaction. The data show that the peptide-PolyT20 conjugates translocated through nanopores whereas the unmodified peptides did not. Surprisingly, the conjugates with their peptides tethered at the 5′-end of PolyT20 passed the nanopores more rapidly than the PolyT20 alone. The PolyT20 also yielded a wider distribution of blockade currents. The same broad distribution was found for a conjugate with its peptide tethered at the 3′-end of PolyT20, suggesting that the larger blockades (and longer translocation times) are associated with events in which the 5′-end of the PolyT20 enters the pore first.

Nanopore‐Based Protein Sequencing Using Biopores: Current Achievements and Open Challenges

“…If combined with click addition of positive and negative tails to the termini of a short peptide, this approach for residues identification can be applied to any peptide construct, regardless of its charge. In this scenario, the click addition of a single strand DNA tail to a peptide terminal introduced by Biswas et al [ 130 ] can potentially foster the use of the nanopore tweezer method for other nanopore protein sensing applications.…”

“…One possibility is to add a charged tail to one protein terminal. [ 130,142,143,144 ] In this approach, the charged tail enters the nanopore by electrophoresis and it induces the translocation of the whole peptide chain, supplementary assisted in certain cases by a molecular motor. [ 143 ] A second strategy is to employ SDS, an anionic compound that in combination with heat and reducing agents is able to denature the protein and create a negative charged shell around it.…”

Paving the way to single-molecule protein sequencing

“…An alternative approach to control the direction of translocation is to attach an oligonucleotide strand to the N-or C-terminus of a protein. The negative charge carried by this lead sequence drags the polypeptide in the direction of the electrophoretic force [91][92][93][94][95] . This principle was first used by Bayley and colleagues to study the translocation of thioredoxin through α-hemolysin 91,92 .…”

“…Other groups have also recently used this approach. Lindsay's group developed a simple and effective click chemistry to facilitate the tagging reaction, while Pelta and colleagues used a DNA lead in a protein to present a direct proof of protein translocation using amplification by PCR 94,95 .…”

Graphene nanoelectrodes for biomolecular sensing

“…These issues may be overcome by the functionalization of neutral peptides with short and charged DNA oligomers by click chemistry. 90 Negatively charged oligomers can act as a molecular thread and exert a dragging force on the protein forcing it to translocate through the tunnel junction. The biggest challenge in protein sequencing however, is in the expected range of variability in the sequencing signal.…”