Improving the Selectivity and Reducing the Leakage of DNA Strand Displacement Systems

Because of the elegance of Watson-Crick base pairing and the programmability of toehold-mediated strand displacement, DNA is a model material for designing, building, and testing molecular assemblies. DNA assemblies are categorized as structural when they are at thermodynamic equilibrium and dynamic when they are not. Through programmed perturbations, metastable assemblies perform physical, chemical, and computational work. When integrated into a diagnostic package, disease-specific nucleic acid sequences can be identified, amplified, and analyzed via standard DNA nanotechnology rules. In order for these rules to make an impact, two critical challenges in the field have been undertaken in this dissertation. First, the selectivity to distinguish an on-target sequence from off-target sequences, with a resolution of a single-nucleotide mutation, has been explored by site-specifically integrating locked nucleic acids into DNA sequences. Locked nucleic acids are RNA analogues that have higher thermal and hence mechanical stability than RNA and DNA. Second, the initiation of metastable chemical reaction networks, in the absence of on-target sequences, has been explored to suppress network leakage; which is the single greatest problem in dynamic DNA nanotechnology. To address this challenge, original catalytic substrates were designed, built, and tested to increase the energy barrier of the leakage reactions without sacrificing the performance of the favorable catalytic reactions. The experimental results showed that site-specific integration of LNA into DNA sequences improved the sequence selectivity by over 2 orders of magnitude. They also showed that network leakage could be suppressed by 2 orders of magnitude by decoupling the leakage pathway from the catalytic pathway in the original catalytic substrates. When combined, these results constitute a substantial contribution to the field of dynamic DNA nanotechnology and represent important steps towards the creation of low-cost, early-stage diagnostic tools for difficult to detect diseases such as lung, breast, and pancreatic cancers

Bridging Pico-to-Nanonewtons with a Ratiometric Force Probe for Monitoring Nanoscale Polymer Physics Before Damage

Understanding the transmission of nanoscale forces in the pico-to-nanonewton range is important in polymer physics. While physical approaches have limitations in analyzing the local force distribution in condensed environments, chemical analysis using force probes is promising. However, there are stringent requirements for probing the local forces generated before structural damage. The magnitude of those forces corresponds to the range below covalent bond scission (from 200 pN to several nN) and above thermal fluctuation (several pN). Here, we report a conformationally flexible dual-fluorescence force probe with a theoretically estimated threshold of approximately 100 pN. This probe enables ratiometric analysis of the distribution of local forces in a stretched polymer chain network. Without changing the intrinsic properties of the polymer, the force distribution was reversibly monitored in real time. Chemical control of the probe location demonstrated that the local stress concentration is twice as biased at crosslinkers than at main chains, particularly in a strain-hardening region. Due to the high sensitivity, the percentage of stressed force probes was estimated to be more than 1000 times higher than the activation rate of a conventional mechanophore.Comment: 21 pages and 5 figures in the main text, and 73 pages and 68 figures in the supplementary material

Bridging pico-to-nanonewtons with a ratiometric force probe for monitoring nanoscale polymer physics before damage

ピンと張られた分子鎖を定量する「羽ばたき型蛍光Force Probe」の開発 --高分子材料の中で力のかかった分子鎖の比率を蛍光イメージングで計測する--. 京都大学プレスリリース. 2022-01-14.Understanding the transmission of nanoscale forces in the pico-to-nanonewton range is important in polymer physics. While physical approaches have limitations in analyzing the local force distribution in condensed environments, chemical analysis using force probes is promising. However, there are stringent requirements for probing the local forces generated before structural damage. The magnitude of those forces corresponds to the range below covalent bond scission (from 200 pN to several nN) and above thermal fluctuation (several pN). Here, we report a conformationally flexible dual-fluorescence force probe with a theoretically estimated threshold of approximately 100 pN. This probe enables ratiometric analysis of the distribution of local forces in a stretched polymer chain network. Without changing the intrinsic properties of the polymer, the force distribution was reversibly monitored in real time. Chemical control of the probe location demonstrated that the local stress concentration is twice as biased at crosslinkers than at main chains, particularly in a strain-hardening region. Due to the high sensitivity, the percentage of the stressed force probes was estimated to be more than 1000 times higher than the activation rate of a conventional mechanophore

Availability: A Metric for Nucleic Acid Strand Displacement Systems

DNA strand displacement systems have transformative potential in synthetic biology. While powerful examples have been reported in DNA nanotechnology, such systems are plagued by leakage, which limits network stability, sensitivity, and scalability. An approach to mitigate leakage in DNA nanotechnology, which is applicable to synthetic biology, is to introduce mismatches to complementary fuel sequences at key locations. However, this method overlooks nuances in the secondary structure of the fuel and substrate that impact the leakage reaction kinetics in strand displacement systems. In an effort to quantify the impact of secondary structure on leakage, we introduce the concepts of availability and mutual availability and demonstrate their utility for network analysis. Our approach exposes vulnerable locations on the substrate and quantifies the secondary structure of fuel strands. Using these concepts, a 4-fold reduction in leakage has been achieved. The result is a rational design process that efficiently suppresses leakage and provides new insight into dynamic nucleic acid networks

Accuracy of Pedicle Screw Placement in Scoliosis Surgery: A Comparison between Conventional Computed Tomography-Based and O-Arm-Based Navigation Techniques

Study DesignRetrospective study.PurposeWe compared the accuracy of O-arm-based navigation with computed tomography (CT)-based navigation in scoliotic surgery.Overview of LiteratureNo previous reports comparing the results of O-arm-based navigation with conventional CT-based navigation in scoliotic surgery have been published.MethodsA total of 222 pedicle screws were implanted in 29 patients using CT-based navigation (group C) and 416 screws were implanted in 32 patients using O-arm-based navigation (group O). Postoperative CT was performed to assess the screw accuracy, using the established Neo classification (grade 0: no perforation, grade 1: perforation <2 mm, grade 2: perforation ≥2 and <4, and grade 3: perforation ≥4 mm).ResultsIn group C, 188 (84.7%) of the 222 pedicle screw placements were categorized as grade 0, 23 (10.4%) were grade 1, 11 (5.0%) were grade 2, and 0 were grade 3. In group O, 351 (84.4%) of the 416 pedicle screw placements were categorized as grade 0, 52 (12.5%) were grade 1, 13 (3.1%) were grade 2, and 0 were grade 3. Statistical analysis showed no significant difference in the prevalence of grade 2.3 perforations between groups C and O. The time to position one screw, including registration, was 10.9±3.2 minutes in group C, but was significantly decreased to 5.4±1.1 minutes in group O.ConclusionsO-arm-based navigation facilitates pedicle screw insertion as accurately as conventional CT-based navigation. The use of O-arm-based navigation successfully reduced the time, demonstrating advantages in the safety and accuracy of pedicle screw placement for scoliotic surgery

Assessment of the Initial Diagnostic Accuracy of a Fragility Fracture of the Sacrum: A Study of 56 Patients

Study Design Retrospective study. Purpose To investigate the clinical manifestations of a fragility fracture of the sacrum (FFS) and the factors that may contribute to a misdiagnosis. Overview of Literature The number of patients diagnosed with FFS has increased because of extended life expectancy and osteoporosis. Patients with FFS may report nonspecific symptoms, such as back, buttock, groin, and/or leg pain, leading to a misdiagnosis and a delay in definitive diagnosis. Methods Fifty-six patients (13 males and 43 females) with an average age of 80.2±9.2 years admitted to the hospital for FFS between 2006 and 2021 were analyzed retrospectively. The following patient data were collected using medical records: pain regions, a history of trauma, initial diagnoses, and rates of fracture detection using radiography, computed tomography (CT), and magnetic resonance imaging (MRI). Results Forty-one patients presented with low back and/or buttock pain, nine presented with groin pain, and 17 presented with thigh or leg pain. There was no history of trauma in 18 patients (32%). At the initial visit, 27 patients (48%) were diagnosed with sacral or pelvic fragility fractures. In contrast, 29 patients (52%) were initially misdiagnosed with lumbar spine disease (23 patients), hip joint diseases (three patients), and buttock bruises (three patients). Fracture detection rates for FFS were 2% using radiography, 71% using CT, and 93% using MRI. FFS was diagnosed definitively using an MRI with a coronal short tau inversion recovery (STIR) sequence. Conclusions Some patients with FFS have leg pain with no history of trauma and are initially misdiagnosed as having lumbar spine disease, hip joint disease, or simple bruises. When these clinical symptoms are reported, we recommend considering FFS as one of the differential diagnoses and performing lumbar or pelvic MRIs, particularly coronal STIR images, to rule out FFS

A Functional SNP in BNC2 Is Associated with Adolescent Idiopathic Scoliosis

Adolescent idiopathic scoliosis (AIS) is the most common spinal deformity. We previously conducted a genome-wide association study (GWAS) and detected two loci associated with AIS. To identify additional loci, we extended our GWAS by increasing the number of cohorts (2,109 affected subjects and 11,140 control subjects in total) and conducting a whole-genome imputation. Through the extended GWAS and replication studies using independent Japanese and Chinese populations, we identified a susceptibility locus on chromosome 9p22.2 (p = 2.46 × 10−13; odds ratio = 1.21). The most significantly associated SNPs were in intron 3 of BNC2, which encodes a zinc finger transcription factor, basonuclin-2. Expression quantitative trait loci data suggested that the associated SNPs have the potential to regulate the BNC2 transcriptional activity and that the susceptibility alleles increase BNC2 expression. We identified a functional SNP, rs10738445 in BNC2, whose susceptibility allele showed both higher binding to a transcription factor, YY1 (yin and yang 1), and higher BNC2 enhancer activity than the non-susceptibility allele. BNC2 overexpression produced body curvature in developing zebrafish in a gene-dosage-dependent manner. Our results suggest that increased BNC2 expression is implicated in the etiology of AIS

Evidence of causality of low body mass index on risk of adolescent idiopathic scoliosis: a Mendelian randomization study

IntroductionAdolescent idiopathic scoliosis (AIS) is a disorder with a three-dimensional spinal deformity and is a common disease affecting 1-5% of adolescents. AIS is also known as a complex disease involved in environmental and genetic factors. A relation between AIS and body mass index (BMI) has been epidemiologically and genetically suggested. However, the causal relationship between AIS and BMI remains to be elucidated.Material and methodsMendelian randomization (MR) analysis was performed using summary statistics from genome-wide association studies (GWASs) of AIS (Japanese cohort, 5,327 cases, 73,884 controls; US cohort: 1,468 cases, 20,158 controls) and BMI (Biobank Japan: 173430 individual; meta-analysis of genetic investigation of anthropometric traits and UK Biobank: 806334 individuals; European Children cohort: 39620 individuals; Population Architecture using Genomics and Epidemiology: 49335 individuals). In MR analyses evaluating the effect of BMI on AIS, the association between BMI and AIS summary statistics was evaluated using the inverse-variance weighted (IVW) method, weighted median method, and Egger regression (MR-Egger) methods in Japanese.ResultsSignificant causality of genetically decreased BMI on risk of AIS was estimated: IVW method (Estimate (beta) [SE] = -0.56 [0.16], p = 1.8 × 10-3), weighted median method (beta = -0.56 [0.18], p = 8.5 × 10-3) and MR-Egger method (beta = -1.50 [0.43], p = 4.7 × 10-3), respectively. Consistent results were also observed when using the US AIS summary statistic in three MR methods; however, no significant causality was observed when evaluating the effect of AIS on BMI.ConclusionsOur Mendelian randomization analysis using large studies of AIS and GWAS for BMI summary statistics revealed that genetic variants contributing to low BMI have a causal effect on the onset of AIS. This result was consistent with those of epidemiological studies and would contribute to the early detection of AIS

Multi-Arm Junctions for Dynamic DNA Nanotechnology

Nonenzymatic catalytic substrates have been engineered using toehold-mediated DNA strand displacement, and their programmable applications range from medical diagnosis to molecular computation. However, the complexity, stability, scalability, and sensitivity of those systems are plagued by network leakage. A novel way to suppress leakage is to increase its energy barrier through four-way branch migration. Presented here, we designed multi-arm junction substrates that simultaneously exploit four-way branch migration, with a high-energy barrier to minimize leakage, and three-way branch migration, with a low-energy barrier to maximize catalysis. Original feed forward, autocatalytic, and cross-catalytic systems have been designed with polynomial and exponential amplification that exhibit the modularity of linear substrates and the stability of hairpin substrates, creating a new phase space for synthetic biologist, biotechnologist, and DNA nanotechnologists to explore. A key insight is that high-performing circuits can be engineered in the absence of intensive purification and/or extensive rounds of design optimization. Without adopting established leakage suppression techniques, the ratio of the catalytic rate constant to the leakage rate constant is more than 2 orders of magnitude greater than state-of-the-art linear and hairpin substrates. Our results demonstrate that multi-arm junctions have great potential to become central building blocks in dynamic DNA nanotechnology