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Industry Experts Question Claims of Sequencing Study Published in Nature Nanotechnology

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Industry experts are questioning the scientific claims of a study published recently in Nature Nanotechnology that details a single-molecule sequencing technology.

The study describes a method of single-molecule sequencing that makes use of a protein transistor composed of immunoglobulin G and two gold nanoparticles bound to source and drain electrodes. Voltage is applied across the 10-nanometer gap between the electrodes, driving DNA polymerase phi 29 to incorporate nucleotides. According to the study, each base incorporated generates a distinct electrical signature, which is detected by the protein transistor.

Since its publication online earlier this month, however, several experts have found issues with many of the principles behind the study and have submitted those concerns to the journal.

"There are a number of claims made in the paper that need to be substantiated, because otherwise this would constitute completely new physics and chemistry," Meni Wanunu, an assistant professor in the departments of physics and chemistry as well as chemical biology at Northeastern University, told In Sequence.

Stuart Lindsay, director of the Center for Single Molecule Biophysics at Arizona State University's Biodesign Institute, said he and a number of other researchers in advanced sequencing technology and biomolecular electronics contacted the corresponding author of the article with a number of questions about the study and how it was done. After the author responded that he could not answer the questions due to intellectual property issues, Lindsay and the group sent their concerns to the journal editor.

Steven Huang, senior author of the study and a professor in materials science and engineering at National Chiao Tung University in Taiwan, also declined to comment on the paper to IS.

The group questioning the paper has issues with components of the physics, enzymology, and chemistry behind the study.

One issue, said Lindsay, is that the study describes using superconducting materials at the interface of the protein transistor and probes to reduce signal decay. However, he said, "none of us know of a superconducting material that works at the same temperature as a polymerase."

Superconducting materials operate at well below freezing with even so-called high temperature superconductors operating below -135 degrees Celsius. Polymerases, on the other hand, generally operate around room temperature

Another issue relates to the bioelectronics and chemistry. The study's authors say that they applied a voltage across the electrodes of up to 9.0 volts. However, at that high of a voltage, water would become hydrolyzed, generating hydrogen and oxygen gases. If that happened, said Wanunu, it would be difficult to measure signals that were a property of the enzyme.

In order to not hydrolyze water, he said the applied voltage would have to be below around 1.5 volts.

Finally, the scientists had concerns with the enzymology described in the paper.

Polymerase incorporation of nucleotides depends on diffusion of the nucleotide into the appropriate binding site, explained Lindsay. This diffusion is stochastic. However, the authors describe incorporation events occurring at very regular intervals. "None of us understand how this would be achieved," Lindsay said.

"There are things in the paper that physicists, physical chemists, and enzymologists would be very surprised at and would require the development of new science and technology of which none of us are aware," Lindsay added. And, while "that might be a reflection of our ignorance, it gives the group of us great concern."

Wanunu said that the publication builds upon a previous study by the same group, also published in Nature Nanotechnology, that describes the same protein transistor the authors used in the recent study.

He added that the reaction of the group of scientists to submit a letter to the journal editor is not surprising, and not an overreaction. "The claims in this paper are extremely important, and our skepticism is an essential component of progress in science."

Nature Nanotechnology senior editor Owain Vaughan told IS that "some significant concerns have been raised about the paper and we are currently looking into it," but declined to provide more information about the process or timeline.

According to the publication's policies, "an inherent principle of publication is that others should be able to replicate and build upon the authors' published claims." As such, authors are required to make "materials, data, and associated protocols promptly available to readers without undue qualifications."

If the author refuses to make the information available, as was the case with Huang's group, the reader should submit its queries to the journal, as Lindsay's group did. Lindsay said the group is now waiting to see how the journal responds.

He added that he is surprised that the same questions he and others are now asking were not asked during the peer-review process. "Anyone in physical sciences would have asked the same questions," he said.

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