Subangstrom Measurements of Enzyme Function Using a Biological Nanopore, SPRNT

Abstract

Nanopores are emerging as new single-molecule tools in the study of enzymes. Based on progress in nanopore sequencing of DNA, a tool called Single-molecule Picometer Resolution Nanopore Tweezers (SPRNT) was developed to measure the movement of enzymes along DNA in real time. In this new method, an enzyme is loaded onto a DNA (or RNA) molecule. A single stranded DNA end of this complex is drawn into a nanopore by an electrostatic potential that is applied across the pore. The single-stranded DNA passes through the pore’s constriction until the enzyme comes into contact with the pore. Further progression of the DNA through the pore is then controlled by the enzyme. An ion current that flows through the pore’s constriction is modulated by the DNA in the constriction. Analysis of ion current changes reveals the advance of the DNA with high spatiotemporal precision, thereby providing a real-time record of the enzyme’s activity. Using an engineered version of the protein nanopore MspA, SPRNT has spatial resolution as small as 40 pm at milliseconds time scales, while simultaneously providing the DNA’s sequence within the enzyme. In this chapter SPRNT is introduced and its extraordinary potential is exemplified using the helicase Hel308. Two distinct sub-states are observed for each one-nucleotide advance; one of these about half-nucleotide long steps is ATP-dependent and the other is ATP-independent. The spatiotemporal resolution of this low-cost single-molecule technique lifts the study of enzymes to a new level of precision, enabling exploration of hitherto unobservable enzyme dynamics in real-time.

Helicase SPRNTing through the nanopore

“…In nanopore-based sequencing, a pore formed by a protein channel embedded into a lipid membrane forms an electrical connection between two salt solutions; electrostatic potential applied across the nanopore drives individual ssDNA molecules through the nanopore aperture; the helicase bound to the DNA controls the ssDNA movement by either pulling the DNA out of the nanopore or by feeding it into the nanopore one nucleotide at a time. The ion current through the nanopore reveals the DNA sequence, while changes in this current reflect the ssDNA movement within the helicase (2,3). Choosing the right enzyme and utilizing its full potential in the nanoporebased sequencing require a detailed understanding of the helicase's mechanochemical cycle.…”

“…Since the discovery that charged molecules, such as ssDNA, could be electrophoresed through an ion channel in a lipid bilayer (5), nanopore technology has developed into a robust method of nucleic acid sequencing (2,3). Nanopore sequencing utilizes a pore placed in a phospholipid bilayer separating two electrolyte solutions.…”

“…In an elegant study in PNAS, Craig et al (4) applied singlemolecule picometer resolution nanopore tweezers (SPRNT), a high-resolution, nanopore-based single-molecule approach (3) to study the ssDNA translocation of HEL308 DNA helicase from Thermococcus gammatolerans (Fig. 1).…”

Modeling of Ion and Water Transport in the Biological Nanopore ClyA

“…7-10 Such detectors have applications ranging from the analysis of biopolymers such as DNA [11][12][13][14][15][16][17][18] or proteins, [19][20][21][22][23] to the detection and quantification of biomarkers, [24][25][26][27][28][29][30] to the fundamental study of chemical or enzymatic reactions at the single molecular level. 22,[31][32][33][34] Nanopores are typically operated in the resistive-pulse mode, where the fluctuations of their ionic conductance are monitored over time. [7][8][9]35 Experimentally, this is achieved by placing the nanopore between two electrolyte compartments and applying a constant DC (or AC) voltage across them.…”

Nanopore device-based fingerprinting of RNA oligos and microRNAs enhanced with an Osmium tag

“…30 Using an engineered version of MspA a tool was developed to measure the movement of enzymes along DNA in real time. 31 Noteworthy using an experimental platform is a complex process. Specifically, the monomeric unit of the nanopore protein needs to be manufactured, purified, assembled as a pore and inserted in a premade stable lipid bilayer, followed by addition of the nucleic acid in a small reaction vessel without disrupting the formation of the nanopore; all this before experimentation can begin.…”