RNase A Does Not Translocate the Alpha-Hemolysin Pore

Abstract

The application of nanopore sensing utilizing the α-hemolysin pore to probe proteins at single-molecule resolution has expanded rapidly. In some studies protein translocation through the α-hemolysin has been reported. However, there is no direct evidence, as yet, that proteins can translocate the α-hemolysin pore. The biggest challenge to obtaining direct evidence is the lack of a highly sensitive assay to detect very low numbers of protein molecules. Furthermore, if an activity based assay is applied then the proteins translocating by unfolding should refold back to an active confirmation for the assay technique to work. To overcome these challenges we selected a model enzyme, ribonuclease A, that readily refolds to an active conformation even after unfolding it with denaturants. In addition we have developed a highly sensitive reverse transcription polymerase chain reaction based activity assay for ribonuclease A. Initially, ribonuclease A, a protein with a positive net charge and dimensions larger than the smallest diameter of the pore, was subjected to nanopore analysis under different experimental conditions. Surprisingly, although the protein was added to the cis chamber (grounded) and a positive potential was applied, the interaction of ribonuclease A with α-hemolysin pore induced small and large blockade events in the presence and the absence of a reducing and/or denaturing agent. Upon measuring the zeta potential, it was found that the protein undergoes a charge reversal under the experimental conditions used for nanopore sensing. From the investigation of the effect of voltage on the interaction of ribonuclease A with the α-hemolysin pore, it was impossible to conclude if the events observed were translocations. However, upon testing for ribonuclease A activity on the trans chamber it was found that ribonuclease A does not translocate the α-hemolysin pore.

Modelling of electrokinetic phenomena for capture of PEGylated ribonuclease A in a microdevice with insulating structures

“…Furthermore, since in our experiments RNase A was suspended in a slightly basic buffer (pH ¼ 8), the selection of a negative f p value is supported by previous reports. 26 Note that when EP is excluded (f p ¼ 0), the EK velocity is still higher (by $3 times) in comparison to the EK velocity response with f p ¼ À10 mV (Figure 6(c)). A lower EK velocity is expected to reduce the drag force on the protein molecules, contributing to improve the dielectrophoretic trapping.…”

“…It has been reported that RNase A f p can vary from À0.010 to 0.010 V approximately under different conditions of ionic strength and pH. 26 So far, there is lack of literature reporting conductivity of proteins, especially for RNase A, although there are some reported values available for other proteins that vary in a very wide range. 22,[28][29][30] As we experimentally observed streaming or trapping DEP for both proteins (native and PEGylated) in positive mode, we set r p ¼ 1000 lS/cm, one order of magnitude higher than r m , to guarantee positive DEP theoretical predictions.…”

“…26,41 To the best of our knowledge, to the time of writing of this paper, no mathematical model that calculates f p as a function of pH is available in the literature. Therefore, we performed a parametric sweep study to find out what values of f p favored our model by reducing the resultant EK velocity.…”

“…The range of values for f p (between À10 and 10 mV) fed to the model was selected according to a previous investigation where it was demonstrated that the zeta-potential of native RNase A takes on positive values in acidic buffers or negative values in neutral or basic buffers. 26 Computational predictions of the impact of f p on the EK velocity are shown in Figure 6. The highest EK velocity, with a magnitude of 0.1 m/s, was obtained for a f p ¼ 10 mV at the narrowest regions of the microfluidic channel (at the tips of the diamond-shape posts).…”

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

“…Furthermore, owing to the lack of a PCR-like technique for amplifying proteins, it is very difficult to even acquire direct evidences to prove protein translocation. 12 Recently, Akeson and his coworkers demonstrated that a recombinant ubiquitin-like protein Smt3 bearing a polyanionic peptide at its C-terminus was unfolded and pulled through a α-hemolysin (α-HL) nanopore by the AAA+ unfoldase ClpX. 13 Almost at the same time, Bayley's team reported that a thioredoxin protein tethered to a negatively charged oligonucleotide could also be unfolded and translocated through the α-HL nanopore by an applied voltage.…”