Using peptide barcodes for simultaneous profiling of protein expression from mRNA.

RATIONALE: mRNA technology has begun to play a significant role in the areas of therapeutic intervention and vaccine development. However, optimizing the mRNA sequence that influences protein expression levels is a resource-intensive and time-consuming process. This study introduces a new method to accelerate the selection of sequences of mRNA for optimal protein expression. METHODS: We designed the mRNA sequences in such a way that a unique peptide barcode, corresponding to each mRNA sequence, is attached to the expressed protein. These barcodes, cleaved off by a protease and simultaneously quantified by mass spectrometry, reflect the protein expression, enabling a parallel analysis. We validated this method using two mRNAs, each with different untranslated regions (UTRs) but encoding enhanced green fluorescence protein (eGFP), and investigated whether the peptide barcodes could analyze the differential eGFP expression levels. RESULTS: The fluorescence intensity of eGFP, a marker of its expression level, has shown noticeable changes between the two UTR sequences in mRNA-transfected cells when measured using flow cytometry. This suggests alterations in the expression level of eGFP due to the influence of different UTR sequences. Furthermore, the quantified amount of peptide barcodes that were released from eGFP showed consistent patterns with these changes. CONCLUSIONS: The experimental findings suggest that peptide barcodes serve as a valuable tool for assessing protein expression levels. The process of mRNA sequence selection, aimed at maximizing protein expression, can be enhanced by the parallel analysis of peptide barcodes using mass spectrometry.

Dwell Time Prolongation and Identification of Single Nucleotides Passing through a Solid-State Nanopore by Using Ammonium Sulfate Aqueous Solution.

The ionic current blockades when poly(dT)60 or dNTPs passed through SiN nanopores in an aqueous solution containing (NH4)2SO4 were investigated. The dwell time of poly(dT)60 in the nanopores in an aqueous solution containing (NH4)2SO4 was significantly longer compared to that in an aqueous solution that did not contain (NH4)2SO4. This dwell time prolongation effect due to the aqueous solution containing (NH4)2SO4 was also confirmed when dCTP passed through the nanopores. In addition, when the nanopores were fabricated via dielectric breakdown in the aqueous solution containing (NH4)2SO4, the dwell time prolongation effect for dCTP still occurred even after the aqueous solution was displaced with the aqueous solution without (NH4)2SO4. Furthermore, we measured the ionic current blockades when the four types of dNTPs passed through the same nanopore, and the four types of dNTPs could be statistically identified according to their current blockade values.

Low-frequency noise induced by cation exchange fluctuation on the wall of silicon nitride nanopore.

Nanopore-based biosensors have attracted attention as highly sensitive microscopes for detecting single molecules in aqueous solutions. However, the ionic current noise through a nanopore degrades the measurement accuracy. In this study, the magnitude of the low-frequency noise in the ionic current through a silicon nitride nanopore was found to change depending on the metal ion species in the aqueous solution. The order of the low-frequency noise magnitudes of the alkali metal ionic current was consistent with the order of the adsorption affinities of the metal ions for the silanol surface of the nanopore (Li

Solid-state nanopores towards single-molecule DNA sequencing.

Nanopore DNA sequencing offers a new paradigm owing to its extensive potential for long-read, high-throughput detection of nucleotide modification and direct RNA sequencing. Given the remarkable advances in protein nanopore sequencing technology, there is still a strong enthusiasm in exploring alternative nanopore-sequencing techniques, particularly those based on a solid-state nanopore using a semiconductor material. Since solid-state nanopores provide superior material robustness and large-scale integrability with on-chip electronics, they have the potential to surpass the limitations of their biological counterparts. However, there are key technical challenges to be addressed: the creation of an ultrasmall nanopore, fabrication of an ultrathin membrane, control of the ultrafast DNA speed and detection of four nucleotides. Extensive research efforts have been devoted to resolving these issues over the past two decades. In this review, we briefly introduce recent updates regarding solid-state nanopore technologies towards DNA sequencing. It can be envisioned that emerging technologies will offer a brand new future in DNA-sequencing technology.

Stable fabrication of a large nanopore by controlled dielectric breakdown in a high-pH solution for the detection of various-sized molecules.

For nanopore sensing of various-sized molecules with high sensitivity, the size of the nanopore should be adjusted according to the size of each target molecule. For solid-state nanopores, a simple and inexpensive nanopore fabrication method utilizing dielectric breakdown of a membrane is widely used. This method is suitable for fabricating a small nanopore. However, it suffers two serious problems when attempting to fabricate a large nanopore: the generation of multiple nanopores and the non-opening failure of a nanopore. In this study, we found that nanopore fabrication by dielectric breakdown of a SiN membrane under high-pH conditions (pH ≥ 11.3) could overcome these two problems and enabled the formation of a single large nanopore up to 40 nm in diameter within one minute. Moreover, the ionic-current blockades derived from streptavidin-labelled and non-labelled DNA passing through the fabricated nanopore were clearly distinguished. The current blockades caused by streptavidin-labelled DNA could be identified even when its concentration is 1% of the total DNA.

Challenges of Single-Molecule DNA Sequencing with Solid-State Nanopores.

A powerful DNA sequencing tool with high accuracy, long read length and high-throughput would be required more and more for decoding the complicated genetic code. Solid-state nanopore has attracted many researchers for its promising future as a next-generation DNA sequencing platform due to the processability, the robustness and the large-scale integratability. While the diverse materials have been widely explored for a solid-state nanopore, silicon nitride (Si3N4) is especially preferable from the viewpoint of mass production based on semiconductor fabrication process. Here, as a nanopore sensing mechanism, we focused on the ionic blockade current method which is the most developed technique. We also highlight the main challenges of Si3N4 nanopore-based DNA sequencer that should be addressed: the fabrication of ultra-small nanopore and ultra-thin membrane, the modulation of DNA translocation speed and the detection of base-specific signals. In this chapter, we discuss the recent progress relating to solid-state nanopore DNA sequencing, which helps to provide a comprehensive information about the current technical situation.

Two-step breakdown of a SiN membrane for nanopore fabrication: Formation of thin portion and penetration.

For the nanopore sensing of various large molecules, such as probe-labelled DNA and antigen-antibody complexes, the nanopore size has to be customized for each target molecule. The recently developed nanopore fabrication method utilizing dielectric breakdown of a membrane is simple and quite inexpensive, but it is somewhat unsuitable for the stable fabrication of a single large nanopore due to the risk of generating multiple nanopores. To overcome this bottleneck, we propose a new technique called "two-step breakdown" (TSB). In the first step of TSB, a local conductive thin portion (not a nanopore) is formed in the membrane by dielectric breakdown. In the second step, the created thin portion is penetrated by voltage pulses whose polarity is opposite to the polarity of the voltage used in the first step. By applying TSB to a 20-nm-thick SiN membrane, a single nanopore with a diameter of 21-26 nm could be fabricated with a high yield of 83%.

Discrimination of three types of homopolymers in single-stranded DNA with solid-state nanopores through external control of the DNA motion.

To achieve DNA sequencing with solid-state nanopores, the speed of the DNA in the nanopore must be controlled to obtain sequence-specific signals. In this study, we fabricated a nanopore-sensing system equipped with a DNA motion controller. DNA strands were immobilized on a Si probe, and approach of this probe to the nanopore vicinity could be controlled using a piezo actuator and stepper motor. The area of the Si probe was larger than the area of the membrane, which meant that the immobilized DNA could enter the nanopore without the need for the probe to scan to determine the location of the nanopore in the membrane. We demonstrated that a single-stranded DNA could be inserted into and removed from a nanopore in our experimental system. The number of different ionic-current levels observed while DNA remained in the nanopore corresponded to the number of different types of homopolymers in the DNA.

Slowing the translocation of single-stranded DNA by using nano-cylindrical passage self-assembled by amphiphilic block copolymers.

We report a novel approach to slow the translocation of single-stranded DNA (ssDNA) by employing polyethylene oxide (PEO) filled nano-cylindrical domains as transportation channels. DNA strands were demonstrated to electrophoretically translocate through PEO filled cylindrical domains with diameters of 2 and 9 nm, which were self-assembled by amphiphilic liquid crystalline block copolymers. The average translocation rate of ssDNA strands was effectively reduced to an order of 10 µs per nucleotide, which was 1-2 orders slower than that attained by utilizing conventional solid-state nanopore devices.

Multichannel detection of ionic currents through two nanopores fabricated on integrated Si3N4 membranes.

Integration of solid-state nanopores and multichannel detection of signals from each nanopore are effective measures for realizing high-throughput nanopore sensors. In the present study, we demonstrated fabrication of Si3N4 membrane arrays and the simultaneous measurement of ionic currents through two nanopores formed in two adjacent membranes. Membranes with thicknesses as low as 6.4 nm and small nanopores with diameters of less than 2 nm could be fabricated using the poly-Si sacrificial-layer process and multilevel pulse-voltage injection. Using the fabricated nanopore membranes, we successfully achieved simultaneous detection of clear ionic-current blockades when single-stranded short homopolymers (poly(dA)60) passed through two nanopores. In addition, we investigated the signal crosstalk and leakage current among separated chambers. When two nanopores were isolated on the front surface of the membrane, there was no signal crosstalk or leakage current between the chambers. However, when two nanopores were isolated on the backside of the Si substrate, signal crosstalk and leakage current were observed owing to high-capacitance coupling between the chambers and electrolysis of water on the surface of the Si substrate. The signal crosstalk and leakage current could be suppressed by oxidizing the exposed Si surface in the membrane chip. Finally, the observed ionic-current blockade when poly(dA)60 passed through the nanopore in the oxidized chip was approximately half of that observed in the non-oxidized chip.

Side-gated ultrathin-channel nanopore FET sensors.

A side-gated, ultrathin-channel nanopore FET (SGNAFET) is proposed for fast and label-free DNA sequencing. The concept of the SGNAFET comprises the detection of changes in the channel current during DNA translocation through a nanopore and identifying the four types of nucleotides as a result of these changes. To achieve this goal, both p- and n-type SGNAFETs with a channel thicknesses of 2 or 4 nm were fabricated, and the stable transistor operation of both SGNAFETs in air, water, and a KCl buffer solution were confirmed. In addition, synchronized current changes were observed between the ionic current through the nanopore and the SGNAFET's drain current during DNA translocation through the nanopore.

Slowing single-stranded DNA translocation through a solid-state nanopore by decreasing the nanopore diameter.

To slow the translocation of single-stranded DNA (ssDNA) through a solid-state nanopore, a nanopore was narrowed, and the effect of the narrowing on the DNA translocation speed was investigated. In order to accurately measure the speed, long (5.3 kb) ssDNA (namely, ss-poly(dA)) with uniform length (±0.4 kb) was synthesized. The diameters of nanopores fabricated by a transmission electron microscope were controlled by atomic-layer deposition. Reducing the nanopore diameter from 4.5 to 2.3 nm slowed down the translocation of ssDNA by more than 16 times (to 0.18 µs base(-1)) when 300 mV was applied across the nanopore. It is speculated that the interaction between the nanopore and the ssDNA dominates the translocation speed. Unexpectedly, the translocation speed of ssDNA through the 4.5 nm nanopore is more than two orders of magnitude higher than that of double-stranded DNA (dsDNA) through a nanopore of almost the same size. The cause of such a faster translocation of ssDNA can be explained by the weaker drag force inside the nanopore. Moreover, the measured translocation speeds of ssDNA and dsDNA agree well with those calculated by molecular-dynamics (MD) simulation. The MD simulation predicted that reducing the nanopore diameter to almost the same as that of ssDNA (i.e. 1.4 nm) decreases the translocation speed (to 1.4 µs base(-1)). Narrowing the nanopore is thus an effective approach for accomplishing nanopore DNA sequencing.

Fabricating nanopores with diameters of sub-1 nm to 3†nm using multilevel pulse-voltage injection.

To date, solid-state nanopores have been fabricated primarily through a focused-electronic beam via TEM. For mass production, however, a TEM beam is not suitable and an alternative fabrication method is required. Recently, a simple method for fabricating solid-state nanopores was reported by Kwok, H. et al. and used to fabricate a nanopore (down to 2 nm in size) in a membrane via dielectric breakdown. In the present study, to fabricate smaller nanopores stably--specifically with a diameter of 1 to 2 nm (which is an essential size for identifying each nucleotide)--via dielectric breakdown, a technique called "multilevel pulse-voltage injection" (MPVI) is proposed and evaluated. MPVI can generate nanopores with diameters of sub-1 nm in a 10-nm-thick Si3N4 membrane with a probability of 90%. The generated nanopores can be widened to the desired size (as high as 3 nm in diameter) with sub-nanometre precision, and the mean effective thickness of the fabricated nanopores was 3.7 nm.

Improving zero-mode waveguide structure for enhancing signal-to-noise ratio of real-time single-molecule fluorescence imaging: a computational study.

We investigated the signal-to-noise ratio (S/N) of real-time single-molecule fluorescence imaging (SMFI) using zero-mode waveguides (ZMWs). The excitation light and the fluorescence propagating from a molecule in the ZMW were analyzed by computational optics simulation. The dependence of the S/N on the ZMW structure was investigated with the diameter and etching depth as the simulation parameters. We found that the SMFI using a conventional ZMW was near the critical level for detecting binding and dissociation events. We show that etching the glass surface of the ZMW by 60 nm enhances the S/N six times the conventional nonetched ZMWs. The enhanced S/N improves the temporal resolution of the SMFI at physiological concentrations.

Chemical recognition and binding kinetics in a functionalized tunnel junction.

4(5)-(2-mercaptoethyl)-1H-imidazole-2-carboxamide is a molecule that has multiple hydrogen bonding sites and a short flexible linker. When tethered to a pair of electrodes, it traps target molecules in a tunnel junction. Surprisingly large recognition-tunneling signals are generated for all naturally occurring DNA bases A, C, G, T and 5-methyl-cytosine. Tunnel current spikes are stochastic and broadly distributed, but characteristic enough so that individual bases can be identified as a tunneling probe is scanned over DNA oligomers. Each base yields a recognizable burst of signal, the duration of which is controlled entirely by the probe speed, down to speeds of 1 nm s -1, implying a maximum off-rate of 3 s -1 for the recognition complex. The same measurements yield a lower bound on the on-rate of 1 M -1 s -1. Despite the stochastic nature of the signals, an optimized multiparameter fit allows base calling from a single signal peak with an accuracy that can exceed 80% when a single type of nucleotide is present in the junction, meaning that recognition-tunneling is capable of true single-molecule analysis. The accuracy increases to 95% when multiple spikes in a signal cluster are analyzed.

Real-time imaging of single-molecule fluorescence with a zero-mode waveguide for the analysis of protein-protein interaction.

Real-time imaging of single-molecule fluorescence with a zero-mode waveguide (ZMW) was achieved. With modification of the ZMW geometry, the signal-to-background ratio is twice that obtainable with a conventional ZMW. The improved signal-to-background ratio makes it possible to visualize individual binding-release events between chaperonin GroEL and cochaperonin GroES at a concentration of 5 microM. Two rate constants representing two-timer kinetics in the release of GroES from GroEL were measured with the ZMW, and the measurements agreed well with those made with a total internal reflection fluorescence microscopy. These results indicate that the novel ZMW makes feasible the direct observation of protein-protein interaction at an intracellular concentration in real time.

Effect of the C-terminal truncation on the functional cycle of chaperonin GroEL: implication that the C-terminal region facilitates the transition from the folding-arrested to the folding-competent state.

To elucidate the exact role of the C-terminal region of GroEL in its functional cycle, the C-terminal 20-amino acid truncated mutant of GroEL was constructed. The steady-state ATPase rate and duration of GroES binding showed that the functional cycle of the truncated GroEL is extended by approximately 2 s in comparison with that of the wild type, without interfering with the basic functions of GroEL. We have proposed a model for the functional cycle of GroEL, which consists of two rate-limiting steps of approximately 3- and approximately 5-s duration (Ueno, T., Taguchi, H., Tadakuma, H., Yoshida, M., and Funatsu, T. (2004) Mol. Cell 14, 423-434 g). According to the model, detailed kinetic studies were performed. We found that a 20-residue truncation of the C terminus extends the time until inorganic phosphate is generated and the time for arresting protein folding in the central cavity, i.e. the lifetime of the first rate-limiting step in the functional cycle, to an approximately 5-s duration. These results suggest that the integrity of the C-terminal region facilitates the transition from the first to the second rate-limiting state.