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Author(s):  
Shalu Kumari Pathak ◽  
Arvind Sonwane ◽  
Subodh Kumar

Background: Programmable nucleases are very promising tools of genome editing (GE), but they suffer from limitations including potential risk of genotoxicity which led to the exploration of safer approach of GE based on RNA-guided recombinase (RGR) platform. RNA-guided recombinase (RGR) platform operates on a typical recognition or target site comprised of the minimal pseudo-core recombinase site, a 5 to 6-base pair spacer flanking it and whole this central region is flanked by two guide RNA-specified DNA sequences or Cas9 binding sites followed by protospacer adjacent motifs (PAMs). Methods: The current study focuses on analysis of entire cattle genome to prepare a detailed map of target sites for RNA-guided hyperactivated recombinase Gin with spacer length six. For this, chromosome wise whole genomic sequence data was retrieved from Ensembl. After that search pattern for recombinase Gin with spacer length six was designed. By using this search pattern, RGR target sites were located by using dreg program of Emboss package. Result: Total number of RGR target sites identified in bovine genome for recombinase Gin was 677 with spacer length six. It was also investigated that whether these RGR target sites are present with in any gene or not and it was found that RGR target sites lies in both genic and intergenic region. Besides this, description of genes in context with these target sites was identified.


2021 ◽  
Author(s):  
Vaibhav Purwar ◽  
Rajeev Gupta ◽  
Pramod Kumar Tiwari ◽  
Sarvesh Dubey

Abstract Dielectric Pocket Double-Gate-All-Around (DP-DGAA) MOSFETs are one of the preferred choices for ULSI applications because of significantly low off-current, reduced power dissipation, and high immunity to short channel effect. However, DP-DGAA MOSFETs suffer from self-heating owing to the unavailability of proper heat take-out paths. In this paper, the electrothermal (ET) simulations have been performed with hydrodynamic and thermodynamic transport models to analyze the self-heating effects (SHEs) in DP-DGAA MOSFETs. The electrothermal characteristics against various device parameters such as spacer length, device thickness, thermal contact resistance, and drain voltage have been investigated. The effect of SHE on the drive current has also been evaluated. Further, the impact of thermal contact resistance and ambient temperature variations of the device on SHE and thermal noise have been analyzed using Sentaurus TCAD simulator.


2021 ◽  
Author(s):  
Hiral Sanghavi ◽  
Richa Rashmi ◽  
Anirban Dasgupta ◽  
Sharmistha Majumdar

Abstract Guanine nucleotide binding proteins are characterized by a structurally and mechanistically conserved GTP-binding domain (G domain), indispensable for binding GTP. The G domain comprises five adjacent consensus motifs called G boxes, which are separated by amino acid spacers of different lengths. Several G proteins, discovered over time, are characterized by diverse function and sequence. This sequence diversity is also observed in the G box motifs (specifically the G5 box) as well as the inter-G box spacer length. The Spacers and Mismatch Algorithm (SMA) introduced in this study can predict G-domains in a given protein sequence, based on user-specified constraints for approximate G-box patterns and inter-box gaps in each G protein family. The SMA parameters can be customized as more G proteins are discovered and characterized structurally. Family-specific G box motifs including the less characterized G5 box were predicted with higher accuracy. Overall, our analysis suggests the possible classification of G protein families based on family-specific G box sequences and lengths of inter-G box spacers. SMA can be implemented via a web-based server at https://labs.iitgn.ac.in/datascience/gboxes/


2021 ◽  
pp. 100032
Author(s):  
Christina Barth ◽  
Hendrik Spreen ◽  
Dennis Mulac ◽  
Lucas Keuter ◽  
Matthias Behrens ◽  
...  

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Carlo A. Klein ◽  
Marc Teufel ◽  
Carl J. Weile ◽  
Patrick Sobetzko

AbstractTranscription, the first step to gene expression, is a central coordination process in all living matter. Besides a plethora of regulatory mechanisms, the promoter architecture sets the foundation of expression strength, timing and the potential for further regulatory modulation. In this study, we investigate the effects of promoter spacer length and sequence composition on strength and supercoiling sensitivity in bacteria. Combining transcriptomics data analysis and standardized synthetic promoter libraries, we exclude effects of specific promoter sequence contexts. Analysis of promoter activity shows a strong variance with spacer length and spacer sequence composition. A detailed study of the spacer sequence composition under selective conditions reveals an extension to the -10 region that enhances RNAP binding but damps promoter activity. Using physiological changes in DNA supercoiling levels, we link promoter supercoiling sensitivity to overall spacer GC-content. Time-resolved promoter activity screens, only possible with a novel mild treatment approach, reveal strong promoter timing potentials solely based on DNA supercoiling sensitivity in the absence of regulatory sites or alternative sigma factors.


Membranes ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 950
Author(s):  
Jasleen Kaur Daljit Singh ◽  
Minh Tri Luu ◽  
Jonathan F. Berengut ◽  
Ali Abbas ◽  
Shelley F. J. Wickham ◽  
...  

DNA nanotechnology provides methods for building custom membrane-interacting nanostructures with diverse functions, such as shaping membranes, tethering defined numbers of membrane proteins, and transmembrane nanopores. The modification of DNA nanostructures with hydrophobic groups, such as cholesterol, is required to facilitate membrane interactions. However, cholesterol-induced aggregation of DNA origami nanostructures remains a challenge. Aggregation can result in reduced assembly yield, defective structures, and the inhibition of membrane interaction. Here, we quantify the assembly yield of two cholesterol-modified DNA origami nanostructures: a 2D DNA origami tile (DOT) and a 3D DNA origami barrel (DOB), by gel electrophoresis. We found that the DOT assembly yield (relative to the no cholesterol control) could be maximised by reducing the number of cholesterols from 6 to 1 (2 ± 0.2% to 100 ± 2%), optimising the separation between adjacent cholesterols (64 ± 26% to 78 ± 30%), decreasing spacer length (38 ± 20% to 95 ± 5%), and using protective ssDNA 10T overhangs (38 ± 20% to 87 ± 6%). Two-step folding protocols for the DOB, where cholesterol strands are added in a second step, did not improve the yield. Detergent improved the yield of distal cholesterol configurations (26 ± 22% to 92 ± 12%), but samples re-aggregated after detergent removal (74 ± 3%). Finally, we confirmed functional membrane binding of the cholesterol-modified nanostructures. These findings provide fundamental guidelines to reducing the cholesterol-induced aggregation of membrane-interacting 2D and 3D DNA origami nanostructures, improving the yield of well-formed structures to facilitate future applications in nanomedicine and biophysics.


2021 ◽  
Author(s):  
V. Bharath Sreenivas ◽  
Vadthiya Narendar

Abstract The main aim of this work is to study the effect of symmetric and asymmetric spacer length variations towards source and drain on n-channel SOI JL vertically stacked (VS) nanowire (NW) FET at 10 nm gate length (LG). Spacer length is proved to be one of the stringent metrics in deciding device performance along with width, height and aspect ratio (AR). The physical variants in this work are symmetric spacer length (LSD), source side spacer length (LS) and drain side spacer length (LD). The simulation results give highest ION/IOFF ratio with LD variation compared to LS and LSD, whereas latter two variations have similar effect on ION/IOFF ratio. At 25 nm (2.5 × LG) of LD, the device gives appreciable ON current with the highest ION/IOFF ratio (2.19 × 108) with optimum subthreshold slope (SS) and ensures low power and high switching drivability. Moreover, it is noticed that among optimal values of LS and LD, the device ION/IOFF ratio has an improvement of 22.69% as compared to other variations. Moreover, the effect of various spacer dielectrics on optimized device is also investigated. Finally, the CMOS inverter circuit analysis is performed on the optimized symmetric and asymmetric spacer lengths.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3306-3306
Author(s):  
Mabel M. Jung ◽  
Koichi R. Katsumura ◽  
Peng Liu ◽  
Kirby D. Johnson ◽  
Joel P. Mackay ◽  
...  

Abstract Genomic analyses in clinical and experimental contexts have accelerated discoveries of human genetic variants. While elucidating the consequences of conspicuously loss-of-function variants is highly tractable, decoding the impact of missense or non-coding variants is considerably more challenging. Previously, we described a germline variant in GATA2 in a patient with GATA2-deficiency syndrome, which inserts nine amino acids between the two zinc fingers (9aa-Ins), one of which mediates sequence-specific DNA binding (Cavalcante de Andrade Silva M. et al., Leukemia, 2021). Unlike other GATA2 coding region and enhancer variants identified (Bresnick E.H. et al., Blood Adv., 2020), it was unclear whether the altered zinc finger spacing would be inhibitory, stimulatory or of no consequence. The 9aa-Ins variant was defective in activating several target genes (Hdc, Ear2 and Tpsb2) in a genetic complementation assay with Gata2 -77 enhancer-mutant (-77 -/-) primary hematopoietic progenitor cells. As only several target genes were tested, we used RNA-seq to conduct a genome-wide comparison of the capacity of GATA2 and 9aa-Ins to activate and repress transcription. To elucidate mechanisms, we considered the following models: 1) 9aa-Ins fails to regulate all genes normally controlled by GATA2; 2) 9aa-Ins fails to repress all genes normally controlled by GATA2; 3) 9aa-Ins fails to activate genes normally controlled by GATA2; 4) 9aa-Ins ectopically regulates genes not controlled by GATA2. Using a genetic complementation approach with -77 -/- cells that were immortalized by the Hoxb8 transcription factor (hi-77 -/-) (Wang G.G. et al., Nat. Methods, 2006; Johnson K.D. et al., JEM, 2020), we compared GATA2 and 9aa-Ins activities when expressed at a comparable level. This analysis revealed 2,138 GATA2-regulated, 525 GATA2 and 9aa-Ins-regulated, and 414 ectopically-regulated genes (at least two-fold change, adjusted P-value <0.05). A similar number of genes were GATA2-activated (1,061) and repressed (1,077). Only 144 out of the 1,061 (14%) were 9aa-Ins-activated and 381 out of 1,077 (35%) were 9aa-Ins-repressed, illustrating the severe consequences of this mutation and a greater impact on activation versus repression. Statistical analysis with a range of P-values constraints (0.01 to 0.1) verified that activation by 9aa-Ins was more significantly impaired than repression (86% were no longer activated, and 65% were no longer repressed, P = 5.4 x 10 -6). Gene ontology analysis revealed that the 9aa insertion impaired GATA2-mediated activation of genes related to GPCR signaling and GATA2-mediated repression of genes related to innate immune machinery. The ectopically-regulated genes did not conform to a particular mechanism or pathway. Since it was unclear whether the transcriptional defects of the 9aa-Ins mutant reflect a unique inhibitory activity imparted by the 9aa sequence, we systematically varied the inter-zinc finger spacer length to establish whether any alterations can be tolerated. Using the genetic complementation assay, 2, 4, 6, 8, and 9aa spacer variants were compared with GATA2 for their capacity to regulate GATA2-activated genes (Hdc, Il1rl1, Gata1 and S1pr1) and repressed genes (Irf8, Il6st, Il6ra and Tifab). GATA2-mediated activation was compromised by insertions of two amino acids or more, whereas repression tolerated two and four amino acid insertions; 6, 8 and 9 were more inhibitory. Quantitative analyses revealed that a 6aa insertion reduced activation of the GATA2-activated genes by >50% of the wild type value, whereas the GATA2-repressed genes were still repressed by at least 50% (18% retention of activation and 83% retention of repression, P = 0.001). Thus, zinc finger spacing alterations differentially impacted activation versus repression. These results provide a rigorous foundation for interpreting variants that alter zinc finger spacing without disrupting vital finger residues. In vitro and in vivo functional analyses and molecular modeling are ongoing to further dissect the underlying mechanisms and ascertain the importance of genetic networks and circuits that are sensitive or resistant to human disease variants. Disclosures No relevant conflicts of interest to declare.


Polymers ◽  
2021 ◽  
Vol 13 (20) ◽  
pp. 3462
Author(s):  
Nurul Asma Razali ◽  
Zuhair Jamain

Liquid crystal is an intermediate phase between the crystalline solid and an isotropic liquid, a very common substance in our daily lives. Two major classes of liquid crystal are lyotropic, where a liquid crystal is dissolved in a specific solvent under a particular concentration and thermotropic, which can be observed under temperature difference. This review aims to understand how a structure of a certain azo compound might influence the liquid crystal properties. A few factors influence the formation of different liquid crystals: the length of the alkyl terminal chain, inter/intra-molecular interaction, presence of spacer, spacer length, polarization effects, odd-even effects, and the presence of an electron-withdrawing group or an electron-donating group. As final observations, we show the compound’s different factors, the other liquid crystal is exhibited, and the structure–property relationship is explained. Liquid crystal technology is an ideal system to be applied to products to maximize their use, especially in the electronic and medical areas.


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