Ferrobotic swarms enable accessible and adaptable automated viral testing.

Expanding our global testing capacity is critical to preventing and containing pandemics1-9. Accordingly, accessible and adaptable automated platforms that in decentralized settings perform nucleic acid amplification tests resource-efficiently are required10-14. Pooled testing can be extremely efficient if the pooling strategy is based on local viral prevalence15-20; however, it requires automation, small sample volume handling and feedback not available in current bulky, capital-intensive liquid handling technologies21-29. Here we use a swarm of millimetre-sized magnets as mobile robotic agents ('ferrobots') for precise and robust handling of magnetized sample droplets and high-fidelity delivery of flexible workflows based on nucleic acid amplification tests to overcome these limitations. Within a palm-sized printed circuit board-based programmable platform, we demonstrated the myriad of laboratory-equivalent operations involved in pooled testing. These operations were guided by an introduced square matrix pooled testing algorithm to identify the samples from infected patients, while maximizing the testing efficiency. We applied this automated technology for the loop-mediated isothermal amplification and detection of the SARS-CoV-2 virus in clinical samples, in which the test results completely matched those obtained off-chip. This technology is easily manufacturable and distributable, and its adoption for viral testing could lead to a 10-300-fold reduction in reagent costs (depending on the viral prevalence) and three orders of magnitude reduction in instrumentation cost. Therefore, it is a promising solution to expand our testing capacity for pandemic preparedness and to reimagine the automated clinical laboratory of the future.

TIMAHAC: Streamlined Tandem IMAC-HILIC Workflow for Simultaneous and High-Throughput Plant Phosphoproteomics and N-glycoproteomics.

Protein post-translational modifications (PTMs) are crucial in plant cellular processes, particularly in protein folding and signal transduction. N-glycosylation and phosphorylation are notably significant PTMs, playing essential roles in regulating plant responses to environmental stimuli. However, current sequential enrichment methods for simultaneous analysis of phosphoproteome and N-glycoproteome are labor-intensive and time-consuming, limiting their throughput. Addressing this challenge, this study introduces a novel tandem S-Trap-IMAC-HILIC (S-Trap: suspension trapping; IMAC: immobilized metal ion affinity chromatography; HILIC: hydrophilic interaction chromatography) strategy, termed TIMAHAC, for simultaneous analysis of plant phosphoproteomics and N-glycoproteomics. This approach integrates IMAC and HILIC into a tandem tip format, streamlining the enrichment process of phosphopeptides and N-glycopeptides. The key innovation lies in the use of a unified buffer system and an optimized enrichment sequence to enhance efficiency and reproducibility. The applicability of TIMAHAC was demonstrated by analyzing the Arabidopsis phosphoproteome and N-glycoproteome in response to abscisic acid (ABA) treatment. Up to 1954 N-glycopeptides and 11,255 phosphopeptides were identified from Arabidopsis, indicating its scalability for plant tissues. Notably, distinct perturbation patterns were observed in the phosphoproteome and N-glycoproteome, suggesting their unique contributions to ABA response. Our results reveal that TIMAHAC offers a comprehensive approach to studying complex regulatory mechanisms and PTM interplay in plant biology, paving the way for in-depth investigations into plant signaling networks.

A touch-based multimodal and cryptographic bio-human-machine interface.

The awareness of individuals' biological status is critical for creating interactive and adaptive environments that can actively assist the users to achieve optimal outcomes. Accordingly, specialized human­machine interfaces­equipped with bioperception and interpretation capabilities­are required. To this end, we devised a multimodal cryptographic bio-human­machine interface (CB-HMI), which seamlessly translates the user's touch-based entries into encrypted biochemical, biophysical, and biometric indices. As its central component, the CB-HMI features thin hydrogel-coated chemical sensors and inference algorithms to noninvasively and inconspicuously acquire biochemical indices such as circulating molecules that partition onto the skin (here, ethanol and acetaminophen). Additionally, the CB-HMI hosts physical sensors and associated algorithms to simultaneously acquire the user's heart rate, blood oxygen level, and fingerprint minutiae pattern. Supported by human subject studies, we demonstrated the CB-HMI's capability in terms of acquiring physiologically relevant readouts of target bioindices, as well as user-identifying and biometrically encrypting/decrypting these indices in situ (leveraging the fingerprint feature). By upgrading the common surrounding objects with the CB-HMI, we created interactive solutions for driving safety and medication use. Specifically, we demonstrated a vehicle-activation system and a medication-dispensing system, where the integrated CB-HMI uniquely enabled user bioauthentication (on the basis of the user's biological state and identity) prior to rendering the intended services. Harnessing the levels of bioperception achieved by the CB-HMI and other intelligent HMIs, we can equip our surroundings with a comprehensive and deep awareness of individuals' psychophysiological state and needs.

Synthesis and Mechanism of 1D ZnO Based on Alkaline Dissolution-Conversion Strategy of High Stable and Soluble ɛ-Zn(OH)2.

A high soluble and stable ɛ-Zn(OH)2 precursor is synthesized at below room temperature to efficiently prepare ZnO whiskers. The experimental results indicate that the formation of ZnO whiskers is carried out mainly via two steps: the formation of ZnO seeds from ɛ-Zn(OH)2 via the in situ solid conversion, and the following growth of whiskers via dissolution-precipitation route. The decrease of temperature from 25 to 5 °C promotes the formation of ɛ-Zn(OH)2 with higher solubility and stability, which balances the conversion and dissolution rates of precursor. The Rietveld refinement, DFT calculations and MD simulations reveal that the primary reason for these characteristics is the expansion of ɛ-Zn(OH)2 lattice due to temperature, causing difficulties in the dehydration of adjacent ─OH. Simultaneously, the larger specific surface area favors the dissolution of ɛ-Zn(OH)2 . Based on this precursor, well-dispersed ZnO whiskers with 9.82 µm in length, 242.38 nm in diameter, and an average aspect ratio of 41 are successfully synthesized through a SDSN-assisted hydrothermal process at 80 °C. The process has an extremely high solid content of 2.5% (mass ratio of ZnO to solution) and an overall yield of 92%, which offers a new approach for the scaled synthesis of high aspect ratio ZnO whiskers by liquid-phase method.

Coupled vibration of a radially polarized piezoelectric cylinder with a co-axial elastic cylinder of varying height.

The cylindrical piezoelectric transducer has the advantages of large radiation area, high electromechanical coupled coefficient, and omni-direction radiation along the radius. In this paper, a piezoelectric transducer consisting of a radially polarized piezoelectric cylinder and an outer metal cylinder of varying height is presented. The metal cylinder of varying height is approximated as the radial superposition of multiple uniform height metal cylinders, and the equivalent impedance of the transducer's coupled vibration is obtained by using the impedance matrix method, and then the resonance frequency, anti-resonance frequency, effective electromechanical coupled coefficient, and displacement amplification coefficient are obtained. In this paper, the relationship between the vibration characteristics of the cylindrical piezoelectric transducer and its geometric dimensions is studied. An experimental sample of the transducer is fabricated and assembled, and its electrical impedance curve is measured. The measured results are in good agreement with the simulation results and the theoretical calculation results. The displacement distribution of the radiation surface of the transducer at resonance frequency is measured, which verifies that the two coupled vibration modes of the transducer can be effectively excited.

Cu(II) Coordination Polymers Containing Mixed Ligands with Different Flexibilities: Structural Diversity and Iodine Adsorption.

Reactions of N,N'-bis(3-methylpyridyl)oxalamide (L1), N,N'-bis(3-methylpyridyl)adipoamide (L2) and N,N'-bis(3-methylpyridyl)sebacoamide (L3) with tricarboxylic acids and Cu(II) salts afforded {[Cu(L1)(1,3,5-HBTC)]·H2O}n (1,3,5-H3BTC = 1,3,5-benzenetricarboxylic acid), 1, {[Cu1.5(L2)1.5(1,3,5-BTC)(H2O)2]·6.5H2O}n, 2, [Cu(L2)0.5(1,3,5-HBTB)]n (1,3,5-H3BTB = 1,3,5-tri(4-carboxyphenyl)benzene), 3, [Cu4(L3)(OH)2(1,3,5-BTC)2]n, 4, {[Cu3(L3)2(1,3,5-BTB)2]·2.5MeOH·2H2O}n, 5, and {[Cu3(L3)2(1,3,5-BTB)2 ]·DMF·2H2O}n, 6, which have been structurally characterized by using single crystal X-ray crystallography. Complexes 1-4 form a 2D layer with the {44.62}-sql topology, a 2D layer with the (4.62)2(42.62.82)-bex topology, a three-fold interpenetrated 3D net with the (412·63)-pcu topology and a 3D framework with the (410·632·83)(42·6)2(43·63) topology, respectively, whereas 5 and 6 are 3D frameworks with the (63)2(64·82)(68·85·102) topology. Complex 5 shows a better iodine adsorption factor of 290.0 mg g-1 at 60 °C for 360 min than the other ones, revealing that the flexibility of the spacer ligand governs the structural diversity and the adsorption capacity.

Suspension Trapping-Based Sample Preparation Workflow for In-Depth Plant Phosphoproteomics.

Plant phosphoproteomics provides a global view of phosphorylation-mediated signaling in plants; however, it demands high-throughput methods with sensitive detection and accurate quantification. Despite the widespread use of protein precipitation for removing contaminants and improving sample purity, it limits the sensitivity and throughput of plant phosphoproteomic analysis. The multiple handling steps involved in protein precipitation lead to sample loss and process variability. Herein, we developed an approach based on suspension trapping (S-Trap), termed tandem S-Trap-IMAC (immobilized metal ion affinity chromatography), by integrating an S-Trap micro-column with a Fe-IMAC tip. Compared with a precipitation-based workflow, the tandem S-Trap-IMAC method deepened the coverage of the Arabidopsis (Arabidopsis thaliana) phosphoproteome by more than 30%, with improved number of multiply phosphorylated peptides, quantification accuracy, and short sample processing time. We applied the tandem S-Trap-IMAC method for studying abscisic acid (ABA) signaling in Arabidopsis seedlings. We thus discovered that a significant proportion of the phosphopeptides induced by ABA are multiply phosphorylated peptides, indicating their importance in early ABA signaling and quantified several key phosphorylation sites on core ABA signaling components across four time points. Our results show that the optimized workflow aids high-throughput phosphoproteome profiling of low-input plant samples.

Characterizing Alcohol Expectancies in the ABCD Study: Associations with Sociodemographic Factors, the Immediate Social Environment, and Genetic Propensities.

Alcohol expectancies (AEs) are associated with likelihood of alcohol initiation and subsequent alcohol use disorders. It is unclear whether genetic predisposition to alcohol use and/or related traits contributes to shaping how one expects to feel when drinking alcohol. We used the Adolescent Brain Cognitive Development study to examine associations between genetic propensities (i.e., polygenic risk for problematic alcohol use, depression, risk-taking), sociodemographic factors (i.e., parent income), and the immediate social environment (i.e., peer use and disapproval toward alcohol) and positive and negative AEs in alcohol-naïve children (max analytic N = 5,352). Mixed-effect regression models showed that age, parental education, importance of the child's religious beliefs, adverse childhood experiences, and peer disapproval of alcohol use were associated with positive and/or negative AEs, to varying degrees. Overall, our results suggest several familial and psychosocial predictors of AEs but little evidence of contributions from polygenic liability to problematic alcohol use or related phenotypes.

Noninvasive wearable electroactive pharmaceutical monitoring for personalized therapeutics.

To achieve the mission of personalized medicine, centering on delivering the right drug to the right patient at the right dose, therapeutic drug monitoring solutions are necessary. In that regard, wearable biosensing technologies, capable of tracking drug pharmacokinetics in noninvasively retrievable biofluids (e.g., sweat), play a critical role, because they can be deployed at a large scale to monitor the individuals' drug transcourse profiles (semi)continuously and longitudinally. To this end, voltammetry-based sensing modalities are suitable, as in principle they can detect and quantify electroactive drugs on the basis of the target's redox signature. However, the target's redox signature in complex biofluid matrices can be confounded by the immediate biofouling effects and distorted/buried by the interfering voltammetric responses of endogenous electroactive species. Here, we devise a wearable voltammetric sensor development strategy-centering on engineering the molecule-surface interactions-to simultaneously mitigate biofouling and create an "undistorted potential window" within which the target drug's voltammetric response is dominant and interference is eliminated. To inform its clinical utility, our strategy was adopted to track the temporal profile of circulating acetaminophen (a widely used analgesic and antipyretic) in saliva and sweat, using a surface-modified boron-doped diamond sensing interface (cross-validated with laboratory-based assays, R2 ∼ 0.94). Through integration of the engineered sensing interface within a custom-developed smartwatch, and augmentation with a dedicated analytical framework (for redox peak extraction), we realized a wearable solution to seamlessly render drug readouts with minute-level temporal resolution. Leveraging this solution, we demonstrated the pharmacokinetic correlation and significance of sweat readings.

Systematic design and experimental realization of a radially cascaded spherical piezoelectric transducer.

As a critical component of ultrasonic vibration systems, piezoelectric transducers play an essential role in various practical application scenarios. Recent advances in spherical transducers have been widely used in underwater sound and structural health monitoring, while the cascaded spherical piezoelectric transducer with arbitrary piezoceramic shell thickness has not been investigated. Here, we propose a radially cascaded spherical piezoelectric transducer (RCSPT) and derive its electromechanical equivalent circuit with mechanical losses, dielectric losses, and load mechanical impedances. The resulting device is composed of three concentric spherical metal shells and two radially polarized spherical piezoceramic shells. The underlying physical mechanism is the inverse piezoelectric effect, which converts electrical signals into mechanical vibrations. The effects of the spherical piezoceramic shell's thickness and location on the RCSPT are studied. We also analyze the effects of mechanical losses, dielectric losses, and load mechanical impedances on the modulus of input electric impedance of the cascaded spherical transducer. The experiments are conducted to verify the electromechanical characteristics of the resulting device, which are in good agreement with the simulated results and theoretical predictions. Our methodology will offer new possibilities for designing RCSPTs and may promote applications in various fields, such as underwater acoustic detection and structural health monitoring.

Acetylation-Mimic Mutation of TRIM28-Lys304 to Gln Attenuates the Interaction with KRAB-Zinc-Finger Proteins and Affects Gene Expression in Leukemic K562 Cells.

TRIM28/KAP1/TIF1ß is a crucial epigenetic modifier. Genetic ablation of trim28 is embryonic lethal, although RNAi-mediated knockdown in somatic cells yields viable cells. Reduction in TRIM28 abundance at the cellular or organismal level results in polyphenism. Posttranslational modifications such as phosphorylation and sumoylation have been shown to regulate TRIM28 activity. Moreover, several lysine residues of TRIM28 are subject to acetylation, but how acetylation of TRIM28 affects its functions remains poorly understood. Here, we report that, compared with wild-type TRIM28, the acetylation-mimic mutant TRIM28-K304Q has an altered interaction with Krüppel-associated box zinc-finger proteins (KRAB-ZNFs). The TRIM28-K304Q knock-in cells were created in K562 erythroleukemia cells by CRISPR-Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein nuclease 9) gene editing method. Transcriptome analysis revealed that TRIM28-K304Q and TRIM28 knockout K562 cells had similar global gene expression profiles, yet the profiles differed considerably from wild-type K562 cells. The expression levels of embryonic-related globin gene and a platelet cell marker integrin-beta 3 were increased in TRIM28-K304Q mutant cells, indicating the induction of differentiation. In addition to the differentiation-related genes, many zinc-finger-proteins genes and imprinting genes were activated in TRIM28-K304Q cells; they were inhibited by wild-type TRIM28 via binding with KRAB-ZNFs. These results suggest that acetylation/deacetylation of K304 in TRIM28 constitutes a switch for regulating its interaction with KRAB-ZNFs and alters the gene regulation as demonstrated by the acetylation mimic TRIM28-K304Q.

Selective and antagonist-dependent µ-opioid receptor activation by the combination of 2-{[2-(6-chloro-3,4-dihydro-1(2H)-quinolinyl)-2-oxoethyl]sulfanyl}-5-phenyl-4,6-(1H,5H)-pyrimidinedione and naloxone/naltrexone.

We identified, via high-throughput screening using a FLIPR® calcium assay, compound 1, which incorporated a dihydroquinolinyl-2-oxoethylsulfanyl-(1H,5H)-pyrimidinedione core and activated the µ-opioid receptor (MOR) in the presence of naloxone or naltrexone. A structure-activity relationship study of the analogs of 1 led to the design of compound 21, which activated MOR in the presence of naloxone with an EC50 of 3.3 ± 0.2 µM. MOR activation by the compound 21-antagonist pair was antagonist-dependent. Compound 21 did not affect the potency of the orthosteric agonist, morphine, toward MOR, indicating that it affected the function of MOR antagonists rather than that of the agonists. Computer modeling of the compound 21-MOR-naloxone complex revealed major interactions between compound 21 and MOR, including hydrogen bonding with Ser196, π-π stacking with Tyr149, and sulfur-aromatic interaction with Trp192. This study may pave the way for developing agents capable of safe and effective MOR modulation.

K33-Linked Polyubiquitination of Coronin 7 by Cul3-KLHL20 Ubiquitin E3 Ligase Regulates Protein Trafficking.

Ubiquitin chains are formed as structurally distinct polymers via different linkages, and several chain types including K33-linkage remain uncharacterized. Here, we describe a role for K33-polyubiquitination in protein trafficking. We show that the Cullin 3 (Cul3) substrate adaptor KLHL20 is localized to the trans-Golgi network (TGN) and is important for post-Golgi trafficking by promoting the biogenesis of TGN-derived transport carriers. The Cul3-KLHL20 ubiquitin E3 ligase catalyzes a nondegradable, K33-linked polyubiquitination on coronin 7 (Crn7), which facilitates Crn7 targeting to TGN through a ubiquitin-dependent interaction with Eps15. Blockage of K33-chain formation, Crn7 ubiquitination, or disruption of Crn7-Eps15 interaction impairs TGN-pool F-actin assembly, a process essential for generating transport carriers. Enforced targeting of Crn7 to TGN bypasses the requirement of K33-ubiquitination for TGN-pool F-actin assembly and post-Golgi trafficking. Our study reveals a role of KLHL20-mediated K33-ubiquitination of Crn7 in post-Golgi transport and identifies a cellular recognition mechanism for this ubiquitin chain type.

Sugar starvation-regulated MYBS2 and 14-3-3 protein interactions enhance plant growth, stress tolerance, and grain weight in rice.

Autotrophic plants have evolved distinctive mechanisms for maintaining a range of homeostatic states for sugars. The on/off switch of reversible gene expression by sugar starvation/provision represents one of the major mechanisms by which sugar levels are maintained, but the details remain unclear. α-Amylase (αAmy) is the key enzyme for hydrolyzing starch into sugars for plant growth, and it is induced by sugar starvation and repressed by sugar provision. αAmy can also be induced by various other stresses, but the physiological significance is unclear. Here, we reveal that the on/off switch of αAmy expression is regulated by 2 MYB transcription factors competing for the same promoter element. MYBS1 promotes αAmy expression under sugar starvation, whereas MYBS2 represses it. Sugar starvation promotes nuclear import of MYBS1 and nuclear export of MYBS2, whereas sugar provision has the opposite effects. Phosphorylation of MYBS2 at distinct serine residues plays important roles in regulating its sugar-dependent nucleocytoplasmic shuttling and maintenance in cytoplasm by 14-3-3 proteins. Moreover, dehydration, heat, and osmotic stress repress MYBS2 expression, thereby inducing αAmy3 Importantly, activation of αAmy3 and suppression of MYBS2 enhances plant growth, stress tolerance, and total grain weight per plant in rice. Our findings reveal insights into a unique regulatory mechanism for an on/off switch of reversible gene expression in maintaining sugar homeostatic states, which tightly regulates plant growth and development, and also highlight MYBS2 and αAmy3 as potential targets for crop improvement.

Broadband ventilated meta-barrier based on the synergy of mode superposition and consecutive Fano resonances.

Sound insulation under ventilation conditions is an important issue in acoustic fields that has significant applications in various practical scenarios. The emergence of acoustic metasurfaces breaks the limitation of manipulating large-scale waves at subwavelength scales and enables a better ventilating capability, while there is still a problem that the bandwidth of previous studies is usually smaller than half an octave. Here, we design and experimentally implement a ventilated meta-barrier with subwavelength thickness capable of realizing broadband sound insulation while maintaining efficient ventilation. The underlying mechanism is the synergy of the consecutive Fano resonances and superposition of equal-strength monopolar mode of the gradient helical structure and dipolar mode of the central orifice, leading to an efficient blocking of approximately 90% of sound waves coming from various directions in the range from 1145 to 1815 Hz while preserving high-efficiency ventilation. The experiments are conducted to verify the effectiveness of the resulting device, which is in good agreement with the simulated results and theoretical predictions. Our design with functionality and flexibility opens up possibilities for the design of broadband ventilated acoustic devices and may find important application prospects in diverse fields such as noise control and architectural acoustics.

Spherical piezoelectric transducers of functionally graded materials.

In this work, a functionally graded spherical piezoelectric transducer (FG-sPET) is proposed and an accurate theoretical model is constructed, mainly composed of a three-port electromechanical equivalent circuit model (EECM). The EECM of FG-sPET can be connected to that of other vibration systems according to the boundary conditions (force and vibration velocity), making it easier to evaluate the whole mechanical vibration system. The validity of the EECM for FG-sPET is verified by comparison with other literature. The effects of geometric dimensions and non-uniform coefficients on the vibration characteristics (resonance/anti-resonance frequencies and effective electromechanical coupling coefficient) of FG-sPET are also studied, contributing to systematically evaluating the key factors determining the vibration characteristics of FG-sPET. The proposed analytical system is of excellent guidance for the structural optimization design of functionally graded piezoelectric devices.

Multi-mode coupled vibration performance analysis of a radial-longitudinal (R-L) ultrasonic transducer.

A radial-longitudinal (R-L) ultrasonic transducer is designed by compounding a piezoelectric ceramic and an outer metal ring on the central coupling cylinder of a longitudinal cascade transducer. This design is used to realize multi-mode vibration and increase the radiation range. By applying longitudinal and radial double excitation, three coupled vibration modes of the transducer are generated in the frequency range of 15-65 kHz. The coupled vibration dominated by radial vibration is regarded as the best vibration mode of this transducer. The electromechanical equivalent circuit and the resonance frequency equation of the transducer's coupled vibration are derived by using the equivalent elastic method and one-dimensional vibration theory and verified by the finite element method and experimental method. The results show that the electrical impedance frequency curves of the transducer from the three methods are consistent. The transducer is expected to be used in ultrasonic cleaning and liquid processing applications.

Novel Stable Capacitive Electrocardiogram Measurement System.

This study presents a noncontact electrocardiogram (ECG) measurement system to replace conventional ECG electrode pads during ECG measurement. The proposed noncontact electrode design comprises a surface guard ring, the optimal input resistance, a ground guard ring, and an optimal voltage divider feedback. The surface and ground guard rings are used to reduce environmental noise. The optimal input resistor mitigates distortion caused by the input bias current, and the optimal voltage divider feedback increases the gain. Simulated gain analysis was subsequently performed to determine the most suitable parameters for the design, and the system was combined with a capacitive driven right leg circuit to reduce common-mode interference. The present study simulated actual environments in which interference is present in capacitive ECG signal measurement. Both in the case of environmental interference and motion artifact interference, relative to capacitive ECG electrodes, the proposed electrodes measured ECG signals with greater stability. In terms of R-R intervals, the measured ECG signals exhibited a 98.6% similarity to ECGs measured using contact ECG systems. The proposed noncontact ECG measurement system based on capacitive sensing is applicable for use in everyday life.

Revealing USP7 Deubiquitinase Substrate Specificity by Unbiased Synthesis of Ubiquitin Tagged SUMO2.

Ubiquitination and SUMOylation of protein are crucial for various biological responses. The recent unraveling of cross-talk between SUMO and ubiquitin (Ub) has shown the pressing needs to develop the platform for the synthesis of Ub tagged SUMO2 dimers to decipher its biological functions. Still, the platforms for facile synthesis of dimers under native condition are less explored and remain major challenges. Here, we have developed the platform that can expeditiously synthesize all eight Ub tagged SUMO2 and SUMOylated proteins under native condition. Expanding genetic code (EGC) method was employed to incorporate Se-alkylselenocysteine at lysine positions. Oxidative selenoxide elimination generates the electrophilic center, dehydroalanine, which upon Michael addition with C-terminal modified ubiquitin, a nucleophile, yield Ub tagged SUMO2. The dimers were further interrogated with USP7, a SUMO2 deubiquitinase, which is involved in DNA repair, to understand specificity toward the Ub tagged SUMO2 dimer. Our results have shown that the C-terminal domain of USP7 is crucial for USP7 efficiency and selectivity for the Ub tagged SUMO2 dimer.

Deriving a sub-nanomolar affinity peptide from TAP to enable smFRET analysis of RNA polymerase II complexes.

Our capability to visualize protein complexes such as RNA polymerase II (pol II) by single-molecule imaging techniques has largely been hampered by the absence of a simple bio-orthogonal approach for selective labeling with a fluorescent probe. Here, we modify the existing calmodulin-binding peptide (CBP) in the widely used Tandem Affinity Purification (TAP) tag to endow it with a high affinity for calmodulin (CaM) and use dye-CaM to conduct site-specific labeling of pol II. To demonstrate the single molecule applicability of this approach, we labeled the C-terminus of the Rpb9 subunit of pol II with donor-CaM and a site in TFIIF with an acceptor to generate a FRET (fluorescence resonance energy transfer) pair in the pol II-TFIIF complex. We then used total internal reflection fluorescence microscopy (TIRF) with alternating excitation to measure the single molecule FRET (smFRET) efficiency between these two sites in pol II-TFIIF. We found they exhibited a proximity consistent with that observed in the transcription pre-initiation complex by cryo-electron microscopy (cryo-EM). We further compared our non-covalent labeling approach with an enzyme-enabled covalent labeling method. The virtually indistinguishable results validate our smFRET approach and show that the observed proximity between the two sites represents a hallmark of the pol II-TFIIF complex. Taken together, we present a simple and versatile bio-orthogonal method derived from TAP to enable selective labeling of a protein complex. This method is suitable for analyzing dynamic relationships among proteins involved in transcription and it can be readily extended to many other biological processes.