Single-molecule Assay Research Articles

A pH-sensitive switch activates virulence in Salmonella.

The transcriptional regulator SsrB acts as a switch between virulent and biofilm lifestyles of non-typhoidal Salmonella enterica serovar Typhimurium. During infection, phosphorylated SsrB activates genes on Salmonella Pathogenicity Island-2 (SPI-2) essential for survival and replication within the macrophage. Low pH inside the vacuole is a key inducer of expression and SsrB activation. Previous studies demonstrated an increase in SsrB protein levels and DNA-binding affinity at low pH; the molecular basis was unknown (Liew et al., 2019). This study elucidates its underlying mechanism and in vivo significance. Employing single-molecule and transcriptional assays, we report that the SsrB DNA-binding domain alone (SsrBc) is insufficient to induce acid pH-sensitivity. Instead, His12, a conserved residue in the receiver domain confers pH sensitivity to SsrB allosterically. Acid-dependent DNA binding was highly cooperative, suggesting a new configuration of SsrB oligomers at SPI-2-dependent promoters. His12 also plays a role in SsrB phosphorylation; substituting His12 reduced phosphorylation at neutral pH and abolished pH-dependent differences. Failure to flip the switch in SsrB renders Salmonella avirulent and represents a potential means of controlling virulence.

Single-molecule imaging reveals distinct elongation and frameshifting dynamics between frames of expanded RNA repeats in C9ORF72-ALS/FTD

C9ORF72 hexanucleotide repeat expansion is the most common genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). One pathogenic mechanism is the accumulation of toxic dipeptide repeat (DPR) proteins like poly-GA, GP and GR, produced by the noncanonical translation of the expanded RNA repeats. However, how different DPRs are synthesized remains elusive. Here, we use single-molecule imaging techniques to directly measure the translation dynamics of different DPRs. Besides initiation, translation elongation rates vary drastically between different frames, with GP slower than GA and GR the slowest. We directly visualize frameshift events using a two-color single-molecule translation assay. The repeat expansion enhances frameshifting, but the overall frequency is low. There is a higher chance of GR-to-GA shift than in the reversed direction. Finally, the ribosome-associated protein quality control (RQC) factors ZNF598 and Pelota modulate the translation dynamics, and the repeat RNA sequence is important for invoking the RQC pathway. This study reveals that multiple translation steps modulate the final DPR production. Understanding repeat RNA translation is critically important to decipher the DPR-mediated pathogenesis and identify potential therapeutic targets in C9ORF72-ALS/FTD.

Molecular regulatory mechanism of human myosin-7a

Myosin-7a is an actin-based motor protein essential for vision and hearing. Mutations of myosin-7a cause type 1 Usher syndrome, the most common and severe form of deafblindness in humans. The molecular mechanisms that govern its mechanochemistry remain poorly understood, primarily because of the difficulty of purifying stable intact protein. Here, we recombinantly produce the complete human myosin-7a holoenzyme in insect cells and characterize its biochemical and motile properties. Unlike the Drosophila ortholog that primarily associates with calmodulin (CaM), we found that human myosin-7a utilizes a unique combination of light chains including regulatory light chain, CaM, and CaM-like protein 4. Our results further reveal that CaM-like protein 4 does not function as a Ca2+ sensor but plays a crucial role in maintaining the lever arm's structural-functional integrity. Using our recombinant protein system, we purified two myosin-7a splicing isoforms that have been shown to be differentially expressed along the cochlear tonotopic axis. We show that they possess distinct mechanoenzymatic properties despite differing by only 11 amino acids at their N termini. Using single-molecule invitro motility assays, we demonstrate that human myosin-7a exists as an autoinhibited monomer and can move processively along actin when artificially dimerized or bound to cargo adaptor proteins. These results suggest that myosin-7a can serve multiple roles in sensory systems such as acting as a transporter or an anchor/force sensor. Furthermore, our research highlights that human myosin-7a has evolved unique regulatory elements that enable precise tuning of its mechanical properties suitable for mammalian auditory functions.

Serum neurofilament and glial fibrillary acidic protein in idiopathic and seropositive transverse myelitis

BackgroundSerum levels of neurofilament light chain (NfL) and glial fibrillary acidic protein (GFAP) reflect the disease activity and disability in central nervous system (CNS) demyelinating diseases. However, the clinical significance of NfL and GFAP in idiopathic transverse myelitis (iTM), an inflammatory spinal cord disease with unknown underlying causes, remains unclear. This study aimed to investigate NfL and GFAP levels in iTM and their association with the clinical parameters compared with those in TM with disease-specific antibodies such as anti-aquaporin 4 or myelin oligodendrocyte glycoprotein antibodies (sTM). MethodsWe collected serum and clinical data of 365 patients with CNS inflammatory diseases from 12 hospitals. The serum NfL and GFAP levels were measured in patients with iTM (n = 37) and sTM (n = 39) using ultrasensitive single-molecule array assays. Regression analysis was performed to investigate the associations between serum levels of NfL and GFAP and the clinical parameters such as higher EDSS scores (EDSS ≥ 4.0). ResultsMean NfL levels were not significantly different between iTM (50.29 pg/ml) and sTM (63.18 pg/ml) (p = 0.824). GFAP levels were significantly lower in iTM (112.34 pg/ml) than in sTM (3814.20 pg/ml) (p = 0.006). NfL levels correlated with expanded disability status scale (EDSS) scores in sTM (p = 0.001) but not in iTM (p = 0.824). Disease duration also correlated with higher EDSS scores in sTM (p = 0.017). ConclusionNfL levels and disease duration correlated with EDSS scores in sTM, and GFAP levels could be a promising biomarker to differentiate iTM from sTM.

Single-Molecule Bioelectronic Portable Array for Early-Diagnosis of Pancreatic Cancer Precursors.

A cohort of 47 patients is screened for pancreatic cancer-precursors with a portable 96-well bioelectronic sensing-array for single-molecule assay in cysts fluid and blood plasma deployable at point-of-care (POC). Pancreatic cancer precursors are mucinous cysts diagnosed with a sensitivity of at most 80% by state-of-the-art cytopathological molecular analyses (e.g., KRASmut DNA). Adding the simultaneous assay of proteins related to malignant transformation (e.g., MUC1 and CD55) is deemed essential to enhance diagnostic accuracy. The bioelectronic array here proposed, based on the SiMoT (Single-Molecule-with-a-large-Transistor) technology, can assay both nucleic acids and proteins at the single-molecule Limit-Of-Identification, LOI (<1% of false-positives and false-negatives). It comprises an ELISA-like 8×12-array organic-electronics disposable cartridge with an electrolyte-gated organic transistor sensor array, and a reusable reader, integrating a custom Si-IC chip, operating via software installed on a USB-connected smart device. The cartridge is complemented by a 3D-printed sensing gate cover plate. KRASmut , MUC1, and CD55 biomarkers either in plasma or cysts-fluid from 5-6 patients at a time, are multiplexed at single-molecule LOI in 1.5 hours. The pancreatic cancer precursors are classified via a machine-learning analysis resulting in at least 96% diagnostic-sensitivity and 100% diagnostic-specificity. This preliminary study opens the way to POC liquid-biopsy-based early diagnosis of pancreatic-cancer precursors in plasma. This article is protected by copyright. All rights reserved.

Ester Bond: Chemically Labile Yet Mechanically Stable.

Due to the dynamic nature of ester linkages, ester-bond-containing materials are well known for their outstanding degradability and stimuli responsiveness. However, whether ester hydrolysis is affected by mechanical forces remains unclear. Here, we develop a single-molecule assay to quantitatively study the force-dependent ester hydrolysis using an engineered circular permutant protein with a caged ester bond as a model. Our single-molecule force spectroscopy results show that the ester hydrolysis rate is surprisingly insensitive to forces, with a ∼7 s-1 dissociation rate that remains almost unchanged in the force range of 80 to 200 pN. Quantum calculations reveal that the ester hydrolysis involves an intermediate state formed by either H3O+- or OH--bonded tetrahedral intermediates. The measured ester-hydrolysis kinetics at the single-molecule level may primarily correspond to the rupture of these intermediate states. However, the rate-limiting step appears to be the formation of the tetrahedral intermediates, which cannot be quantitatively characterized in our experiments. Nonetheless, based on the quantum calculations, this step is also insensitive to applied forces. Altogether, our study suggests that the ester bond is chemically labile yet mechanically stable, serving as the basis for the design of responsive materials using ester bonds as mechanically inert units.

Highly sensitive digital detection of SARS-CoV-2 nucleocapsid protein through single-molecule counting.

Nucleocapsid protein (NP) is one of the structural proteins of SARS-CoV-2 which is stable, well-conserved, highly immunogenic, and abundantly expressed due to the host's adaptive immune response, making it a promising antigenic biomarker for the early and rapid identification and diagnosis of SARS-CoV-2. Traditional antigen analytical methods with NP as the detection marker often have insufficient sensitivity. To achieve rapid and highly sensitive detection of NP, we constructed a novel single-molecule (digital) fluorescence-linked immunosorbent assay (FLISA) based on streptavidin-modified transparent 96-well microplates. Streptavidin was immobilized on the microplate under optimized conditions with a 15mM carbonate buffer solution (pH 9.6) as the coating solution, biotinylated antibodies conjugated with streptavidin as capture probes, and carboxylated fluorescent microsphere-conjugated monoclonal antibodies (FMs-mAbs) as fluorescent probes. Individual sandwich immunolabeled complexes of the SARS-CoV-2 diagnostic marker NP were detected and counted though wide-field inverted fluorescence microscopy (1.1 × 1.4 mm2). FLISA had a linear detection range of 0.2pg/mL to 200ng/mL and a limit of detection (LOD) of 0.73fg/mL and 8fg/mL for NP in phosphate buffer saline and spiked nasal swab samples, respectively. The sensitivity was much higher than commercial antigen detection kits, providing wide detection prospects in future clinical diagnosis, environmental monitoring, and other fields.

A mutation-sensitive, multiplexed and amplification-free detection of nucleic acids by stretching single-molecule tandem hairpin probes.

The detection of nucleic acid sequences in parallel with the discrimination of single nucleotide variations (SNVs) is critical for research and clinical applications. A few limitations make the detection technically challenging, such as too small variation in probe-hybridization energy caused by SNVs, the non-specific amplification of false nucleic acid fragments and the few options of dyes limited by spectral overlaps. To circumvent these limitations, we developed a single-molecule nucleic acid detection assay without amplification or fluorescence termed THREF (hybridization-induced tandem DNA hairpin refolding failure) based on multiplexed magnetic tweezers. THREF can detect DNA and RNA sequences at femtomolar concentrations within 30 min, monitor multiple probes in parallel, quantify the expression level of miR-122 in patient tissues, discriminate SNVs including the hard-to-detect G-U or T-G wobble mutations and reuse the probes to save the cost. In our demonstrative detections using mock clinic samples, we profiled the let-7 family microRNAs in serum and genotyped SARS-CoV-2 strains in saliva. Overall, the THREF assay can discriminate SNVs with the advantages of high sensitivity, ultra-specificity, multiplexing, reusability, sample hands-free and robustness.

Lifting the curtain on loop extrusion barriers: Single-molecule insights into cohesin stalling

In this issue, Zhang etal.1 show that CTCF blocks cohesin-mediated loop extrusion in an orientation-dependent manner. Using single-molecule imaging assays, the authors find that dCas9 and R-loops can also stall extrusion.

The performance of plasma phosphorylated tau231 in detecting Alzheimer's disease: A systematic review with meta-analysis.

Cerebrospinal fluid (CSF) phosphorylated tau231 (P-tau231) is associated with neuropathological outcomes of Alzheimer's disease (AD). The invasive access of cerebrospinal fluid has greatly stimulated interest in the identification of blood-based P-tau231, and the recent advent of single-molecule array assay for the quantification of plasma P-tau231 may provide a turning point to evaluate the usefulness of P-tau231 as an AD-related biomarker. Yet, in the plasma P-tau231 literature, findings with regard to its diagnostic utility have been inconsistent, and thus, we aimed to statistically investigate the potential of plasma P-tau231 in the context of AD via meta-analysis. Publications on plasma P-tau231 were systematically retrieved from PubMed, EMBASE, the Cochrane library and Web of Science databases. A total of 10 studies covering 2007 participants were included, and we conducted random-effect or fixed-effect meta-analysis, sensitivity analysis and publication bias analysis using the STATA SE 14.0 software. According to our quantitative integration, plasma P-tau231 increased from cognitively unimpaired (CU) populations to mild cognitive impairment to AD and showed significant changes in pairwise comparisons of AD, mild cognitive impairment and CU. Plasma P-tau231 level was significantly higher in CU controls with positive amyloid-β (Aβ) status compared with Aβ-negative CU group. Additionally, the excellent diagnostic accuracy of plasma P-tau231 for asymptomatic Aβ pathology was verified by the pooled value of area under the receiver operating characteristic curves (standard mean difference [95% confidence interval]: .75 [.69, .81], P < 0.00001). Overall, the increased plasma P-tau231 concentrations were found in relation to the early development and progression of AD.

Microtubule-associated septin complexes modulate kinesin and dynein motility with differential specificities

Long-range membrane traffic is guided by microtubule-associated proteins and posttranslational modifications, which collectively comprise a traffic code. The regulatory principles of this code and how it orchestrates the motility of kinesin and dynein motors are largely unknown. Septins are a large family of GTP-binding proteins, which assemble into complexes that associate with microtubules. Using single-molecule invitro motility assays, we tested how the microtubule-associated SEPT2/6/7, SEPT2/6/7/9, and SEPT5/7/11 complexes affect the motilities of the constitutively active kinesins KIF5C and KIF1A and the dynein-dynactin-bicaudal D (DDB) motor complex. We found that microtubule-associated SEPT2/6/7 is a potent inhibitor of DDB and KIF5C, preventing mainly their association with microtubules. SEPT2/6/7 also inhibits KIF1A by obstructing stepping along microtubules. On SEPT2/6/7/9-coated microtubules, KIF1A inhibition is dampened by SEPT9, which alone enhances KIF1A, showing that individual septin subunits determine the regulatory properties of septin complexes. Strikingly, SEPT5/7/11 differs from SEPT2/6/7, in permitting the motility of KIF1A and immobilizing DDB to the microtubule lattice. In hippocampal neurons, filamentous SEPT5 colocalizes with somatodendritic microtubules that underlie Golgi membranes and lack SEPT6. Depletion of SEPT5 disrupts Golgi morphology and polarization of Golgi ribbons into the shaft of somato-proximal dendrites, which is consistent with the tethering of DDB to microtubules by SEPT5/7/11. Collectively, these results suggest that microtubule-associated complexes have differential specificities in the regulation of the motility and positioning of microtubule motors. We posit that septins are an integral part of the microtubule-based code that spatially controls membrane traffic.

Plasma Neurofilament Light Chain Is Elevated in Adaptor Protein Complex 4-Related Hereditary Spastic Paraplegia.

Adaptor protein complex 4-associated hereditary spastic paraplegia (AP-4-HSP) is caused by pathogenic biallelic variants in AP4B1, AP4M1, AP4E1, and AP4S1. The aim was to explore blood markers of neuroaxonal damage in AP-4-HSP. Plasma neurofilament light chain (pNfL) and glial fibrillary acidic protein (GFAP) levels were measured in samples from patients and age- and sex-matched controls (NfL: n = 46 vs. n = 46; GFAP: n = 14 vs. n = 21) using single-molecule array assays. Patients' phenotypes were systematically assessed using the AP-4-HSP natural history study questionnaires, the Spastic Paraplegia Rating Scale, and the SPATAX disability score. pNfL levels increased in AP-4-HSP patients, allowing differentiation from controls (Mann-Whitney U test: P = 3.0e-10; area under the curve = 0.87 with a 95% confidence interval of 0.80-0.94). Phenotypic cluster analyses revealed a subgroup of individuals with severe generalized-onset seizures and developmental stagnation, who showed differentially higher pNfL levels (Mann-Whitney U test between two identified clusters: P = 2.5e-6). Plasma GFAP levels were unchanged in patients with AP-4-HSP. pNfL is a potential disease marker in AP-4-HSP and can help differentiate between phenotypic subgroups. © 2023 International Parkinson and Movement Disorder Society.

Regulation of replication origin licensing by ORC phosphorylation reveals a two-step mechanism for Mcm2-7 ring closing

Eukaryotic DNA replication must occur exactly once per cell cycle to maintain cell ploidy. This outcome is ensured by temporally separating replicative helicase loading (G1 phase) and activation (S phase). In budding yeast, helicase loading is prevented outside of G1 by cyclin-dependent kinase (CDK) phosphorylation of three helicase-loading proteins: Cdc6, the Mcm2-7 helicase, and the origin recognition complex (ORC). CDK inhibition of Cdc6 and Mcm2-7 is well understood. Here we use single-molecule assays for multiple events during origin licensing to determine how CDK phosphorylation of ORC suppresses helicase loading. We find that phosphorylated ORC recruits a first Mcm2-7 to origins but prevents second Mcm2-7 recruitment. The phosphorylation of the Orc6, but not of the Orc2 subunit, increases the fraction of first Mcm2-7 recruitment events that are unsuccessful due to the rapid and simultaneous release of the helicase and its associated Cdt1 helicase-loading protein. Real-time monitoring of first Mcm2-7 ring closing reveals that either Orc2 or Orc6 phosphorylation prevents Mcm2-7 from stably encircling origin DNA. Consequently, we assessed formation of the MO complex, an intermediate that requires the closed-ring form of Mcm2-7. We found that ORC phosphorylation fully inhibits MO complex formation and we provide evidence that this event is required for stable closing of the first Mcm2-7. Our studies show that multiple steps of helicase loading are impacted by ORC phosphorylation and reveal that closing of the first Mcm2-7 ring is a two-step process started by Cdt1 release and completed by MO complex formation.

Direct Comparison of Lysine versus Site-Specific Protein Surface Immobilization in Single-Molecule Mechanical Assays.

Single-molecule force spectroscopy (SMFS) is powerful for studying folding states and mechanical properties of proteins, however, it requires protein immobilization onto force-transducing probes such as cantilevers or microbeads. A common immobilization method relies on coupling lysine residues to carboxylated surfaces using 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide and N-hydroxysuccinimide (EDC/NHS). Because proteins typically contain many lysine groups, this strategy results in a heterogeneous distribution of tether positions. Genetically encoded peptide tags (e.g., ybbR) provide alternative chemistries for achieving site-specific immobilization, but thus far a direct comparison of site-specific vs. lysine-based immobilization strategies to assess effects on the observed mechanical properties was lacking. Here, we compared lysine- vs. ybbR-based protein immobilization in SMFS assays using several model polyprotein systems. Our results show that lysine-based immobilization results in significant signal deterioration for monomeric streptavidin-biotin interactions, and loss of the ability to correctly classify unfolding pathways in a multipathway Cohesin-Dockerin system. We developed a mixed immobilization approach where a site-specifically tethered ligand was used to probe surface-bound proteins immobilized through lysine groups, and found partial recovery of specific signals. The mixed immobilization approach represents a viable alternative for mechanical assays on in vivo-derived samples or other proteins of interest where genetically encoded tags are not feasible.

Serum Neurofilament light chain (NfL) levels in children with and without neurologic diseases.

Serum neurofilament light chain (sNfL) is a specific biomarker of neuronal damage. Elevated sNfL levels have been reported in numerous neurologic diseases in adults, whereas data on sNfL in the pediatric population are incomplete. The aim of this study was to investigate sNfL levels in children with various acute and chronic neurologic disorders and describe the age dependence of sNfL from infancy to adolescence. The total study cohort of this prospective cross-sectional study consisted of 222 children aged from 0 to 17 years. Patients' clinical data were reviewed and patients were assigned to the following groups: 101 (45.5%) controls, 34 (15.3%) febrile controls, 23 (10.4%) acute neurologic conditions (meningitis, facial nerve palsy, traumatic brain injury, or shunt dysfunction in hydrocephalus), 37 (16.7%) febrile seizures, 6 (2.7%) epileptic seizures, 18 (8.1%) chronic neurologic conditions (autism, cerebral palsy, inborn mitochondrial disorder, intracranial hypertension, spina bifida, or chromosomal abnormalities), and 3 (1.4%) severe systemic disease. sNfL levels were measured using a sensitive single-molecule array assay. There were no significant differences in sNfL levels between controls, febrile controls, febrile seizures, epileptic seizures, acute neurologic conditions, and chronic neurologic conditions. In children with severe systemic disorders, by far the highest NfL levels were found with an sNfL of 429pg/ml in a patient with neuroblastoma, 126pg/ml in a patient with cranial nerve palsy and pharyngeal Burkitt's lymphoma, and 42pg/ml in a child with renal transplant rejection. The relationship between sNfL and age could be described by a second order polynomial with an R2 of 0.153 with a decrease of sNfL by 3.2% per year from birth to age 12 years and thereafter an increase by 2.7% per year until age 18 years. In this study cohort, sNfL levels were not elevated in children with febrile or epileptic seizures, or various other neurologic diseases. Strikingly high sNfL levels were detected in children with oncologic disease or transplant rejection. A biphasic sNfL age-dependency was documented, with highest levels in infancy and late adolescence and the lowest levels in middle school age.

Alteration of replication protein A binding mode on single-stranded DNA by NSMF potentiates RPA phosphorylation by ATR kinase.

Replication protein A (RPA), a eukaryotic single-stranded DNA (ssDNA) binding protein, dynamically interacts with ssDNA in different binding modes and plays essential roles in DNA metabolism such as replication, repair, and recombination. RPA accumulation on ssDNA due to replication stress triggers the DNA damage response (DDR) by activating the ataxia telangiectasia and RAD3-related (ATR) kinase, which phosphorylates itself and downstream DDR factors, including RPA. We recently reported that the N-methyl-D-aspartate receptor synaptonuclear signaling and neuronal migration factor (NSMF), a neuronal protein associated with Kallmann syndrome, promotes RPA32 phosphorylation via ATR upon replication stress. However, how NSMF enhances ATR-mediated RPA32 phosphorylation remains elusive. Here, we demonstrate that NSMF colocalizes and physically interacts with RPA at DNA damage sites in vivo and in vitro. Using purified RPA and NSMF in biochemical and single-molecule assays, we find that NSMF selectively displaces RPA in the more weakly bound 8- and 20-nucleotide binding modes from ssDNA, allowing the retention of more stable RPA molecules in the 30-nt binding mode. The 30-nt binding mode of RPA enhances RPA32 phosphorylation by ATR, and phosphorylated RPA becomes stabilized on ssDNA. Our findings provide new mechanistic insight into how NSMF facilitates the role of RPA in the ATR pathway.

Multiple RNA- and DNA-binding proteins exhibit direct transfer of polynucleotides with implications for target-site search

We previously demonstrated that the polycomb repressive complex 2 chromatin-modifying enzyme can directly transfer between RNA and DNA without a free-enzyme intermediate state. Simulations suggested that such a direct transfer mechanism may be generally necessary for RNA to recruit proteins to chromatin, but the prevalence of direct transfer capability is unknown. Herein, we used fluorescence polarization assays and observed direct transfer for several well-characterized nucleic acid-binding proteins: three-prime repair exonuclease 1, heterogeneous nuclear ribonucleoprotein U, Fem-3-binding factor 2, and MS2 bacteriophage coat protein. For TREX1, the direct transfer mechanism was additionally observed in single-molecule assays, and the data suggest that direct transfer occurs through an unstable ternary intermediate with partially associated polynucleotides. Generally, direct transfer could allow many DNA- and RNA-binding proteins to conduct a one-dimensional search for their target sites. Furthermore, proteins that bind both RNA and DNA might be capable of readily translocating between those ligands.

Integrated regulation of tubulin tyrosination and microtubule stability by human α-tubulin isotypes

Tubulin isotypes are critical for the functions of cellular microtubules, which exhibit different stability and harbor various post-translational modifications. However, how tubulin isotypes determine the activities of regulators for microtubule stability and modifications remains unknown. Here, we show that human α4A-tubulin, a conserved genetically detyrosinated α-tubulin isotype, is a poor substrate for enzymatic tyrosination. To examine the stability of microtubules reconstituted with defined tubulin compositions, we develop a strategy to site-specifically label recombinant human tubulin for single-molecule TIRF microscopy-based invitro assays. The incorporation of α4A-tubulin into the microtubule lattice stabilizes the polymers from passive and MCAK-stimulated depolymerization. Further characterization reveals that the compositions of α-tubulin isotypes and tyrosination/detyrosination states allow graded control for the microtubule binding and the depolymerization activities of MCAK. Together, our results uncover the tubulin isotype-dependent enzyme activity for an integrated regulation of α-tubulin tyrosination/detyrosination states and microtubule stability, two well-correlated features of cellular microtubules.

High-yield ligation-free assembly of DNA constructs with nucleosome positioning sequence repeats for single-molecule manipulation assays

Force and torque spectroscopy have provided unprecedented insights into the mechanical properties, conformational transitions, and dynamics of DNA and DNA-protein complexes, notably nucleosomes. Reliable single-molecule manipulation measurements require, however, specific and stable attachment chemistries to tether the molecules of interest. Here, we present a functionalization strategy for DNA that enables high-yield production of constructs for torsionally constrained and very stable attachment. The method is based on two subsequent PCRs: first ∼380bp long DNA strands are generated that contain multiple labels, which are used as "megaprimers" in a second PCR to generate ∼kbp long double-stranded DNA constructs with multiple labels at the respective ends. To achieve high-force stability, we use dibenzocyclooctyne-based click chemistry for covalent attachment to the surface and biotin-streptavidin coupling to the bead. The resulting tethers are torsionally constrained and extremely stable under load, with an average lifetime of 70± 3h at 45 pN. The high yield of the approach enables nucleosome reconstitution by salt dialysis on the functionalized DNA, and we demonstrate proof-of-concept measurements on nucleosome assembly statistics and inner turn unwrapping under force. We anticipate that our approach will facilitate a range of studies of DNA interactions and nucleoprotein complexes under forces and torques.

The CYP24A1 gene variant rs2762943 is associated with low serum 1,25-dihydroxyvitamin D levels in multiple sclerosis patients.

Vitamin D is considered to play a role in multiple sclerosis (MS) etiopathogenesis. A polymorphism in the CYP24A1 gene, rs2762943, was recently identified that was associated with an increased MS risk. CYP24A1 encodes a protein involved in the catabolism of the active form of vitamin D. The immunological effects of carrying the rs2762943 risk allele were investigated, as well as its role as genetic modifier. Serum levels of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D (1,25(OH)2 D) were measured in a cohort of 167 MS patients. In a subgroup of patients, expression levels of major histocompatibility complex class II and co-stimulatory molecules were determined by flow cytometry, and serum levels of pro-inflammatory (interferon gamma, granulocyte macrophage colony-stimulating factor, C-X-C motif chemokine ligand 13) and anti-inflammatory (interleukin 10) cytokines and neurofilament light chain were measured by single-molecule array assays. The effect of the rs2762943 polymorphism on disease activity and disability measures was evaluated in 340 MS patients. Compared to non-carriers, carriers of the rs2762943 risk allele were characterized by reduced levels of 1,25(OH)2 D (p = 0.0001) and elevated levels of interferon gamma (p = 0.03) and granulocyte macrophage colony-stimulating factor (p = 0.008), whereas no significant differences were observed for the other markers. The presence of the rs2762943 risk allele had no significant impact on disease activity and disability outcomes during follow-up. However, risk allele carriers were younger at disease onset (p = 0.04). These findings suggest that the CYP24A1 rs2762943 polymorphism plays a more important role in MS susceptibility than in disease prognosis and is associated with lower 1,25(OH)2 D levels and a heightened pro-inflammatory environment in MS patients.