Synthetic protein-conductive membrane nanopores built with DNA

Abstract

Nanopores are key in portable sequencing and research given their ability to transport elongated DNA or small bioactive molecules through narrow transmembrane channels. Transport of folded proteins could lead to similar scientific and technological benefits. Yet this has not been realised due to the shortage of wide and structurally defined natural pores. Here we report that a synthetic nanopore designed via DNA nanotechnology can accommodate folded proteins. Transport of fluorescent proteins through single pores is kinetically analysed using massively parallel optical readout with transparent silicon-on-insulator cavity chips vs. electrical recordings to reveal an at least 20-fold higher speed for the electrically driven movement. Pores nevertheless allow a high diffusive flux of more than 66 molecules per second that can also be directed beyond equillibria. The pores may be exploited to sense diagnostically relevant proteins with portable analysis technology, to create molecular gates for drug delivery, or to build synthetic cells.

Lysenin Channels as Sensors for Ions and Molecules

“…The gel electrophoresis analysis (Figure 18) of the From Figure 15 and the distribution of the current blockage (I D ) and dwell time (T d ) ( Figure 16), one may easily observe that the current drops are relatively uniform for the recorded events (~56 pA); however, some short and reduced-magnitude spikes are observed in both the current trace ( Figure 16b) and I D histogram (Figure 17a). This type of noise is common in translocation experiments, and it is considered a consequence of molecules colliding with the mouth of the pore without being captured by the electric field and translocated [70,80,82,83]. As opposed to the relatively uniform changes in the ionic current, the dwell time seems to be not only unusually long for some events but also extremely variable compared with other ssDNA translocation experiments (Figure 17b).…”

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

“…[26,53,77,[82][83][84][85][86][87][88] Notably, the recent description of synthetic membrane-spanning nanopores constructed with DNA nanotechnology, holds the game-changing potential in many biotechnology and biomedicine applications. [89] As a brief comparison on their particular benefits, solid-state nanopores, although nontransferrable to lipid-based membranes, are more stable than biological ones in extreme buffer conditions (e.g., urea, sodium dodecyl sulfate (SDS)) and can be used in sensing protocols that require strong denaturating agent. [27,29,90] On the other hand, biological nanopores with known crystal structure are amenable to atomic-level engineering and functionalization, as their surface can be finely tuned through site mutation on individual single amino acid groups.…”

The Fusion of Lipid and DNA Nanotechnology

“…The second layer of the funnel was docked onto the surface of the membrane via 19 cholesterol groups. Very recently, a funnel-shaped scaffolded nanopore with a duplexed square lattice was assembled that could accommodate folded proteins at high flux and be driven beyond equilibrium [113].…”

“…Lastly, the rational design of DNA nanopores is a promising avenue for development in resistive pulse sensing, where the passage of an analyte through a narrow aperture is detected by a transient drop in voltage [148]. Translocation of DNA [19], proteins [113], and various small molecules [92,111] has been achieved, demonstrating the potential versatility of the technology in the sensing of biological molecules.…”