A framework for individualized splice-switching oligonucleotide therapy.

Splice-switching antisense oligonucleotides (ASOs) could be used to treat a subset of individuals with genetic diseases1, but the systematic identification of such individuals remains a challenge. Here we performed whole-genome sequencing analyses to characterize genetic variation in 235 individuals (from 209 families) with ataxia-telangiectasia, a severely debilitating and life-threatening recessive genetic disorder2,3, yielding a complete molecular diagnosis in almost all individuals. We developed a predictive taxonomy to assess the amenability of each individual to splice-switching ASO intervention; 9% and 6% of the individuals had variants that were 'probably' or 'possibly' amenable to ASO splice modulation, respectively. Most amenable variants were in deep intronic regions that are inaccessible to exon-targeted sequencing. We developed ASOs that successfully rescued mis-splicing and ATM cellular signalling in patient fibroblasts for two recurrent variants. In a pilot clinical study, one of these ASOs was used to treat a child who had been diagnosed with ataxia-telangiectasia soon after birth, and showed good tolerability without serious adverse events for three years. Our study provides a framework for the prospective identification of individuals with genetic diseases who might benefit from a therapeutic approach involving splice-switching ASOs.

Strong In-Plane Optoelectronic Anisotropy and Polarization Sensitivity in Low-Symmetry 2D Violet Phosphorus.

Anisotropic optoelectronics based on low-symmetry two-dimensional (2D) materials hold immense potential for enabling multidimensional visual perception with improved miniaturization and integration capabilities, which has attracted extensive interest in optical communication, high-gain photoswitching circuits, and polarization imaging fields. However, the reported in-plane anisotropic photocurrent and polarized dichroic ratios are limited, hindering the achievement of high-performance anisotropic optoelectronics. In this study, we introduce novel low-symmetry violet phosphorus (VP) with a unique tubular cross-linked structure into this realm, and the corresponding anisotropic optical and optoelectronic properties are investigated both experimentally and theoretically for the first time. Remarkably, our prepared VP-based van der Waals phototransistor exhibits significant optoelectronic anisotropies with a giant in-plane anisotropic photocurrent ratio exceeding 10 and a comparable polarized dichroic ratio of 2.16, which is superior to those of most reported 2D counterparts. Our findings establish VP as an exceptional candidate for anisotropic optoelectronics, paving the way for future multifunctional applications.

Deep learning for precise axial localization of trapped microspheres in reflective optical systems.

High-precision axial localization measurement is an important part of micro-nanometer optical measurement, but there have been issues such as low calibration efficiency, poor accuracy, and cumbersome measurement, especially in reflected light illumination systems, where the lack of clarity of imaging details leads to the low accuracy of commonly used methods. Herein, we develop a trained residual neural network coupled with a convenient data acquisition strategy to address this challenge. Our method improves the axial localization precision of microspheres in both reflective illumination systems and transmission illumination systems. Using this new localization method, the reference position of the trapped microsphere can be extracted from the identification results, namely the "positioning point" among the experimental groups. This point relies on the unique signal characteristics of each sample measurement, eliminates systematic repeatability errors when performing identification across samples, and improves the localization precision of different samples. This method has been verified on both transmission and reflected illumination optical tweezers platforms. We will bring greater convenience to measurements in solution environments and will provide higher-order guarantees for force spectroscopy measurements in scenarios such as microsphere-based super-resolution microscopy and the surface mechanical properties of adherent flexible materials and cells.

Microsphere probe: combining microsphere-assisted microscopy with AFM.

In recent years, microsphere-assisted microscopy (MAM) and atomic force microscope (AFM) have been rapidly developed to meet the measurement needs of microstructures. However, the positioning of microspheres, the inability of AFM to touch the underlying sample through the transparent insulating layer, and the challenge of AFM fast positioning limit their use in practical measurements. In this paper, we propose a method that combines MAM with AFM by adhering the microsphere to the cantilever. This method allows MAM and AFM to work in parallel, and their imaging positions can correspond with each other. We use this method to measure memory devices, and the results show that MAM and AFM yield complementary advantages. This approach provides a new tool for analyzing complex structures in devices and has potential for wide application.

Mechanistic Insights into the Folding Mechanism of Region V in Ice-Binding Protein Secreted by Marinomonas primoryensis Revealed by Single-Molecule Force Spectroscopy.

The Gram-negative bacteria Marinomonas primoryensis secrete an ice-binding protein (MpIBP), which is a vital bacterial adhesin facilitating the adaptation and survival of the bacteria in the harsh Antarctic environment. The C-terminal region of MpIBP, known as region V (RV), is the first domain to be exported into the Ca2+-rich extracellular environment and acts as a folding nucleus for the entire adhesin. However, the mechanisms underlying the secretion and folding of RV remain poorly understood. Here, we used optical tweezers (OT) to investigate the secretion and folding mechanisms of RV at the single-molecule level. In the absence of Ca2+, apo-RV remains unstructured, while Ca2+-bound RV folds into a mechanically stable structure. The folding of RV could occur via the formation of an intermediate state. Even though this folding intermediate is "hidden" during the folding process of wild type RV in vitro, it likely forms in vivo and plays an important role in facilitating protein secretion. Additionally, our results revealed that the N-terminal part of the RV can significantly stabilize its C-terminal structure. Our study paves the way for further investigations into the structure and functions of MpIBP that help bacteria survive in challenging environments.

Patient-Customized Oligonucleotide Therapy for a Rare Genetic Disease.

Genome sequencing is often pivotal in the diagnosis of rare diseases, but many of these conditions lack specific treatments. We describe how molecular diagnosis of a rare, fatal neurodegenerative condition led to the rational design, testing, and manufacture of milasen, a splice-modulating antisense oligonucleotide drug tailored to a particular patient. Proof-of-concept experiments in cell lines from the patient served as the basis for launching an "N-of-1" study of milasen within 1 year after first contact with the patient. There were no serious adverse events, and treatment was associated with objective reduction in seizures (determined by electroencephalography and parental reporting). This study offers a possible template for the rapid development of patient-customized treatments. (Funded by Mila's Miracle Foundation and others.).

Addressing the imaging limitations of a microsphere-assisted nanoscope.

In the past decade, microsphere-assisted nanoscopy has been developed rapidly to overcome the diffraction limit. However, due to the limited size and high surface curvature of microspheres, the magnified imaging still suffers from problems like limited view scope, imaging distortion, and low contrast. In this paper, we specialize in the imaging mechanism of microspheres and find irradiance as the key factor for microsphere imaging quality. Utilizing a modified optical tweezer system, we achieve precise manipulation of microspheres and further propose a high-quality large-field magnified imaging scheme. The results show that the imaging area of 5 µm microspheres can reach 16×12 µm2 with the minimum identifiable feature of 137 nm. This scheme provides a new solution for extending the measuring scope of microsphere-assisted nanoscope, and will certainly promote the application of this technology in practice.

Learning-based event locating for single-molecule force spectroscopy.

Acquiring events massively from single-molecule force spectroscopy (SMFS) experiments, which is crucial for revealing important biophysical information, is usually not straightforward. A significant amount of human labor is usually required to identify events in the measured force spectrum during measuring or before performing further data analysis. This prevents the experiment from being done in a fully-automated manner or scaling with the throughput of the measuring setup. In this work, we attempt to tackle this problem with a deep learning approach. A deep neural network model is developed to infer the occurrence of the events using the data stream from the measuring setup. We demonstrated that the proposed method could achieve high accuracy with force spectrums of a variety of samples from both optical tweezers and AFMs by learning from user-given samples instead of complicated manual algorithm designing or parameter tuning. Furthermore, we found that the trained model can be used to perform event detection on datasets measured from a different optical tweezer setup, showing the potential of being leveraged in more complex deep learning schemes.

Birefringence and Dichroism in Quasi-1D Transition Metal Trichalcogenides: Direct Experimental Investigation.

Birefringence and dichroism are very important properties in optical anisotropy. Understanding the intrinsic birefringence and dichroism of a material can provide great help to utilize its optical anisotropy. But the direct experimental investigation of birefringence in nanoscale materials is rarely reported. As typical anisotropic transition metals trichalcogenides (TMTCs) materials with quasi-1D structure, TiS3 and ZrS3 have attracted extensive attention due to their special crystal structure and optical anisotropy characteristics. Here, the optical anisotropy properties such as birefringence and dichroism of two kinds of quasi-1D TMTCs, TiS3 and ZrS3 , are theoretically and experimentally studied. In experimental results, the anisotropic refraction and anisotropic reflection of TiS3 and ZrS3 are studied by polarization-resolved optical microscopy and azimuth-dependent reflectance difference microscopy, respectively. In addition, the birefringence and dichroism of ZrS3 nanoribbon in experiment are directly measured by spectrometric ellipsometry measurements, and a reasonable result is obtained. This work provides the basic optical anisotropy information of TiS3 and ZrS3 . It lays a foundation for the further study of the optical anisotropy of these two materials and provides a feasible method for the study of birefringence and dichroism of other nanomaterials in the future.

Synergistic Additive-Assisted Growth of 2D Ternary In2 SnS4 with Giant Gate-Tunable Polarization-Sensitive Photoresponse.

2D ternary materials exhibit great promise in the field of polarization-sensitive photodetectors due to the low-symmetry crystal structure. However, the realization of ternary material growth is still a huge challenge because of the complex reaction process. Here, for the first time, 2D ternary In2 SnS4 flakes are obtained via synergistic additive of salt and molecular sieve-assisted chemical vapor deposition. Raman vibration mode of In2 SnS4 flakes exhibits polarization-dependent properties. The polarization-resolved absorption spectroscopy and azimuth-dependent reflectance difference microscopy further confirm its anisotropy of in-plane optical absorption and reflection. Besides, the In2 SnS4 flake based device on mica shows ultrafast rising and decay rates of ≈20 and 20 µs. Impressively, In2 SnS4 flake based phototransistor demonstrates giant gate-tunable polarization-sensitive photoresponse: the dichroic ratio can be adjusted in the range of 1.13-1.70 with gate voltage varying from -35-35 V. This work provides an effective means for modulating the polarization-sensitive photoresponse, which may significantly promote the research progress of polarization-sensitive photodetectors.

Measuring the multilayer silicon based microstructure using differential reflectance spectroscopy.

The yield of a large-area ultra-thin display panel depends on the realization of designed thickness of multilayer films of all pixels. Measuring the thicknesses of multilayer films of a single pixel is crucial to the accurate manufacture. However, the thinnest layer is reaching the sub-20nm level, and different layers feature remarkable divergence in thickness with similar optical constants. This turns to a key obstruction to the thickness characterization by optical spectroscopy. Based on the tiny differences in absorptivity, a fast method for measuring the film thickness in a single pixel is proposed which combines the layer number reducing model and micro-area differential reflectance spectroscopy. The lower layers can be considered as semi-infinite in the corresponding spectral range whose thickness is infinite in the fitting algorithm. Hence, the thickness of the upper layer is fitted in a simplified layer structure. For demonstration, a multilayer silicon microstructure in a single pixel, p-Si/a-Si/n-Si (10nm/950nm/50nm) on complex substrate, is measured. The light spot diameter is about 60 microns with measuring-time in 2 seconds. The measurement deviation is 3% compared by a commercial ellipsometer. To conclude, the proposed method realizes the layer number reduction for fitting multilayer thickness with large thickness difference and similar optical constants, which provides a powerful approach for multilayer microstructure characterizations.

Symmetry-Reduction Enhanced Polarization-Sensitive Photodetection in Core-Shell SbI3 /Sb2 O3 van der Waals Heterostructure.

Structural symmetry is a simple way to quantify the anisotropic properties of materials toward unique device applications including anisotropic transportation and polarization-sensitive photodetection. The enhancement of anisotropy can be achieved by artificial symmetry-reduction design. A core-shell SbI3 /Sb2 O3 nanowire, a heterostructure bonded by van der Waals forces, is introduced as an example of enhancing the performance of polarization-sensitive photodetectors via symmetry reduction. The structural, vibrational, and optical anisotropies of such core-shell nanostructures are systematically investigated. It is found that the anisotropic absorbance of a core-shell nanowire is obviously higher than that of two single compounds from both theoretical and experimental investigations. Anisotropic photocurrents of the polarization-sensitive photodetectors based on these core-shell SbI3 /Sb2 O3 van der Waals nanowires are measured ranging from ultraviolet (UV) to visible light (360-532 nm). Compared with other van der Waals 1D materials, low anisotropy ratio (Imax /Imin ) is measured based on SbI3 but a device based on this core-shell nanowire possesses a relatively high anisotropy ratio of ≈3.14 under 450 nm polarized light. This work shows that the low-symmetrical core-shell van der Waals heterostructure has large potential to be applied in wide range polarization-sensitive photodetectors.

Imaging layer thickness of large-area graphene using reference-aided optical differential reflection technique.

Transparent layers are critical for enhancing optical contrast of graphene on a substrate. However, once the substrate is fully covered by large-area graphene, there are no accurate transparent layer and reference for optical contrast calculations. The thickness uncertainty of the transparent layer reduces the analytical accuracy of graphene. Thus, in this Letter, we propose a reference-aided differential reflection (DR) method with a dual-light path. The accurate thickness of the transparent layer is obtained by improving the DR spectrum sensitivity using a designable reference. Hence, the analytical accuracy of graphene thickness is guaranteed. To demonstrate this concept, a centimeter-scale chemical-vapor-deposition-synthesized graphene was measured on a SiO2/Si substrate. The thickness of underlying SiO2 was first identified with the 1 nm resolution by the DR spectrum. Then, the thickness distribution of graphene was directly deduced from a DR map with submonolayer resolution at a preferred wavelength. The results were also confirmed by ellipsometry and atomic force microscopy. As a result, this new method provides an extra degree of freedom for the DR method to accurately measure the thickness of large-area two-dimensional materials.

Initial stage of MBE growth of MoSe2 monolayer.

An atomically thin MoSe2 layer has been synthesized on mica using molecular beam epitaxy (MBE). The polymorphous of the MoSe2 layer depends on the coverage and the growth temperature. At low coverages and low growth temperature, 1T-MoSe2 forms in addition to a comparable quantity of 2H-MoSe2. The metastable 1T-MoSe2 transfers gradually to the stable 2H-MoSe2 before the completion of the first monolayer. The current result sheds some light on the complexity of the nucleation and growth of transition metal dichalcogenide (TMDC) monolayers and implies a possible route for a phase selective synthesis using MBE.

Mechanical Unfolding and Folding of a Complex Slipknot Protein Probed by Using Optical Tweezers.

Knotted and slipknotted proteins are topologically complex. Understanding their folding and unfolding mechanism has attracted considerable interest. Here we combined protein engineering, single-molecule optical tweezers, and steered molecular dynamics (SMD) simulations to investigate the mechanical unfolding and folding of a slipknotted protein pyruvoyl-dependent arginine decarboxylase (PADC). In its slipknotted structure, PADC contains a long threaded loop (85 residues), which is almost twice the size of the knotting loop. When stretched from its N- and C-termini, the majority of PADC can be readily unfolded in a two-state manner, and the slipknotted structure was untied. A small percentage of PADC unfolded following a three-state pathway involving the formation of an unfolding intermediate state. These unfolding intermediate states showed a broad distribution of contour length increments, suggesting that they did not have a well-defined specific structure. SMD simulations revealed the main free energy barrier to the unfolding of PADC and suggested that the unfolding intermediate states may originate from the frication of polypeptide chain sliding during the process of pulling the threaded loop out of the knotting loop. Upon relaxation, a small percentage of the unfolded and untied PADC polypeptide chain can refold back to its native slipknotted conformation, but a large fraction can only reach a misfolded state. Our results revealed the complexity of the mechanical unfolding and refolding of a slipknotted protein with a long threaded loop.

Explanation of the distorted spectrum in a spectral hole burning RF spectrum analyzer using Bloch equations.

A readout using a frequency-chirped laser is investigated in a spectrum analyzer with spectral-hole-burning (SHB). An analysis based on the Bloch equations is presented for the spectral distortion due to a fast readout, and a recovery algorithm is developed for the distortion. The experiment of a SHB spectrum analyzer is executed to demonstrate the optical spectral distortion due to fast readout. The experimental spectral distortion is recovered by the recovery algorithm developed from the Bloch equations.

Wavelength tunable polarizer based on layered black phosphorus on Si/SiO2 substrate.

We report on both the theoretical and experimental design of a black phosphorus (BP)-based reflective linear polarizer on Si/SiO2 substrate in visible range using the Fabry-Perot cavities method. Thanks to the optical anisotropy of BP, polarization wavelength regulation and a high extinction ratio are achievable via optimizing the thickness of BP. Using azimuth-dependent reflectance difference microscopy, we directly measured a huge optical anisotropy of 1.58, corresponding to an extinction ratio of ∼9 dB, from a 96 nm BP on a silicon substrate capped by 260 nm thermally oxidized silicon at a wavelength of 690 nm for the first time, to the best of our knowledge. Our results not only provide a new route to designing nanoscale polarizers based on anisotropic two-dimensional (2D) materials, promoting the application of 2D materials in integrated optoelectronics and system-on-chip, but also suggest a modulation technique for optical anisotropy by integrating the BP film with cavity structures.

Real-time identification of the singleness of a trapped bead in optical tweezers.

Beads trapped in optical tweezers are aligned along the optical propagation direction, which makes it difficult to determine the number of beads with bright-field microscopy. This problem also dramatically influences the measurement of the optical trapping based single-molecule force spectroscopy. Here, we propose a video processing approach to count the number of trapped micro-objects in real time. The approach uses a normalized cross-correlation algorithm and image enhancement techniques to amplify a slight change of the image induced by the entry of an exotic object. As tested, this method introduces a ∼10% change per bead to the image similarity, and up to four beads, one-by-one falling into the trap, are identified. Moreover, the feasibility of the above analysis in a moving trap is investigated. A movement of the trap leads to a fluctuation of less than 2% for the similarity signal and can be ignored in most cases. The experimental results prove that image similarity measurement is a sensitive way to monitor the interruption, which is very useful, especially during experiments. In addition, the approach is easy to apply to an existing optical tweezers system.

Reversible Unfolding and Folding of the Metalloprotein Ferredoxin Revealed by Single-Molecule Atomic Force Microscopy.

Plant type [2Fe-2S] ferredoxins function primarily as electron transfer proteins in photosynthesis. Studying the unfolding-folding of ferredoxins in vitro is challenging, because the unfolding of ferredoxin is often irreversible due to the loss or disintegration of the iron-sulfur cluster. Additionally, the in vivo folding of holo-ferredoxin requires ferredoxin biogenesis proteins. Here, we employed atomic force microscopy-based single-molecule force microscopy and protein engineering techniques to directly study the mechanical unfolding and refolding of a plant type [2Fe-2S] ferredoxin from cyanobacteria Anabaena. Our results indicate that upon stretching, ferredoxin unfolds in a three-state mechanism. The first step is the unfolding of the protein sequence that is outside and not sequestered by the [2Fe-2S] center, and the second one relates to the force-induced rupture of the [2Fe-2S] metal center and subsequent unraveling of the protein structure shielded by the [2Fe-2S] center. During repeated stretching and relaxation of a single polyprotein, we observed that the completely unfolded ferredoxin can refold to its native holo-form with a fully reconstituted [2Fe-2S] center. These results demonstrate that the unfolding-refolding of individual ferredoxin is reversible at the single-molecule level, enabling new avenues of studying both folding-unfolding mechanisms, as well as the reactivity of the metal center of metalloproteins in vitro.

Real-time monitoring of 2D semiconductor film growth with optical spectroscopy.

Real-time monitoring of the growth is essential for synthesizing high quality two dimensional (2D) transition-metal dichalcogenides with precisely controlled thickness. Here, we report the first real time in situ optical spectroscopic study on the molecular beam epitaxy of atomically thin molybdenum diselenide (MoSe2) films on sapphire substrates using differential reflectance spectroscopy. The characteristic optical spectrum of MoSe2 monolayer is clearly distinct from that of bilayer allowing a precise control of the film thickness during the growth. Furthermore, the evolution of the characteristic differential reflectance spectrum of the MoSe2 thin film as a function of the thickness sheds light on the details of the growth process. Our result demonstrates the importance and the great potential of the real time in situ optical spectroscopy for the realization of controlled growth of 2D semiconductor materials.