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Korean Team Pursues Sequencing-by-Synthesis Scheme Based on Atomic Force Microscopy

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NEW YORK (GenomeWeb) – A study published online earlier this month in the Journal of the American Chemical Society provided details on a proposed single-molecule sequencing approach that brings together sequencing-by-synthesis with atomic force microscopy (AFM).

The method, which is being pursued by Korean investigators based at the Pohang University of Science and Technology, the LG Electronics Advanced Research Institute, and Chonnam National University, involves tethering a DNA polymerase enzyme to an AFM probe, or "tip," during the process of AFM force spectroscopy. That enzymatically enhanced AFM tip is then used to capture a strand of primed template DNA.

By sequentially dipping the template and AFM tip into separate deoxyribonucleotide (dNTP) pools immobilized to the surface of a glass slide, the team explained, it becomes possible to track complementary base incorporation — and, consequently, the sequence of the initial template — by tracing applied force "ruptures" at the AFM tip as each complementary nucleotide interacts with the polymerase active site.

"[I]nstead of optical signals from fluorescence labels or electrical signals through channels, atomic force microscopy measures the mechanical rupture force between [DNA polymerase] and dNTPs immobilized on surface," the study's first author Youngkyu Kim, and senior author Joon Won Park, both chemistry researchers at Pohang University of Science and Technology, told In Sequence in an email message.

"For the current sequencing-by-synthesis platforms, the fluorescence signals generated at the incorporation step are monitored, and the information allows [us] to read the sequence," they explained. "In contrast, in our approach, the complementary base is recognized by observing a specific rupture force on a corresponding dNTP spot prior to the incorporation."

At the moment, the AFM-based sequencing-by-synthesis method can read roughly 10 sequential bases at around 10 minutes a base, though the team intends to pursue methods to improve the speed, throughput, and read lengths associated with the approach.

Kim, Park, and colleagues are not the first to consider applying AFM to the sequencing realm. Rather, their scheme is the latest in a series of attempts over the past two and a half decades to harness the force manipulation method as a means of reading the nucleotide sequence in a strand of DNA.

In the past, teams have taken a crack at using AFM in DNA sequencing schemes that measure signals associated with everything from labeled complementary nucleotides to friction from diminutive rings moving along single-stranded molecules of DNA, they noted, though it has remained difficult to distinguish individual bases.

For their new publication, the researchers turned to a form of AFM known as force spectroscopy, which characterizes molecules based on their response to force exerted by a tiny microscopic tip.

In this case, the team coats NanoInk silicon nitride probes with either DNA polymerase enzyme alone or DNA polymerase in combination with primer DNA. After grabbing a given strand of primer-associated template DNA, the probe is then exposed to one pool of dNTPs after another.

When a dNTP representing the complementary base at each position is encountered on the chip, the researchers explained, their force-mapping experiments see this event as a "rupture" at the polymerase-coated probe, which is recorded prior to incorporating the complementary base and moving on to the next template position to repeat the process.

"Upon the examination of a surface-conjugated dNTP spot, the complementary dNTP is bound within the active site of [the DNA polymerase]," Kim and Park explained. "Then, the rupture force of the complex is detected by [a] laser-photodiode system monitoring the bending of a cantilever."

For their proof-of-principle study, the researchers used versions of each dNTP that were tagged with biotin via the nucleotides' gamma phosphates — which allowed anchoring to streptavidin-coated slides. A linker carbon chain was also included to bolster the bases' flexibility. Beyond that, Kim and Park noted, neither the pools of complementary bases nor the template DNA were modified during sample preparation.

For their experiments, they chose a form of the DNA polymerase enzyme called Therminator gamma that's known for rapid interactions with bases modified in this manner.

After demonstrating that recordable rupture force events were far more likely to occur when an AFM tip-polymerase duo encountered a complementary dNTP, the study's authors went on to consider some of the other parameters needed to achieve strand sequencing, such as polymerase processivity and stability of polymerase interactions with the DNA template.

For instance, their results indicated that the complex may come apart during sequencing due to relatively low processivity for the Therminator enzyme under the initial experimental conditions used.

To remedy that, the team explored strategies for securing not only the DNA polymerase, but also primer DNA to the AFM tip. After attaching the tip to the primer, which is complementary to the template DNA, the targeted DNA can be covalently zipped to the AFM tip, Kim and Park said.

That approach showed promise for recognizing DNA bases, though the researchers noted that further optimization is still needed, since just 20 percent of the tips designed for DNA polymerase and primer immobilization produced accurate rupture curves.

Base incorporation conditions also had to be tweaked in the team's experiments to avoid incorporating two consecutive bases in the same AFM tip immersion, which would be read as just one base rather than two, and to prevent the tip from becoming damaged — an event that appeared to lead to termination of sequencing after just a handful of bases.

Results so far hint that an AFM-based sequencing method would not be prone to substitution errors, the study's authors reported, though deletion errors are possible if the concentration of some complementary bases become too high.

"Because the polymerase is exposed to the identified dNTP only at each step," Park and Kim explained, "we can avoid error of the incorporation step. Therefore, enhanced accuracy is an expected advantage."

Provided the tip remains undamaged, it should be possible to wash out a given template DNA after sequencing and replace it with another, the researchers noted, and they added that the method is expected to be suitable for targeted gene sequencing.

The researchers also said additional research is required to automate base recognition by the polymerase-AFM probe system, speed up the AFM scanning and analysis processes, boost read lengths, and bump up the throughput associated with the method.

"In our proof-of-principle study, it took approximately 10 minutes to identify a base and 15 minutes to incorporate," Kim and Park said. "If the use of high-speed AFM and on-chip incorporation is realized, it would take less than a minute to read a base."

They noted that "it will be possible to sequence up to 10 DNAs simultaneously with an AFM equipped with the multiple-cantilevers monitoring system."

In pursuit of a full sequencing system, for example, the team believes it will be necessary to optimize the tip preparation process, automate the base incorporation step, and identify a more efficient DNA polymerase. It also hopes to apply the approach in a higher-speed AFM system with multi-cantilevers to allow for parallel measurement of more than one DNA strand.

Those involved in the study have filed for Korean and international patents related to the apparatus and method used to accomplish AFM-based sequencing approach and said they are open to the possibility of collaborating with other groups to commercialize the method.

They noted that it should be feasible to accomplish RNA sequencing with a similar strategy that employs a reverse transcriptase enzyme rather than a DNA polymerase at the AFM tip.

The team is also keen to explore strategies for using the AFM-based sequencing-by-synthesis approach to detect base modification such as cytosine methylation.

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