Strand Displacement
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Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1739
Author(s):  
Chen-Yu Lo ◽  
Yang Gao

Bacteriophages have long been model systems to study the molecular mechanisms of DNA replication. During DNA replication, a DNA helicase and a DNA polymerase cooperatively unwind the parental DNA. By surveying recent data from three bacteriophage replication systems, we summarized the mechanistic basis of DNA replication by helicases and polymerases. Kinetic data have suggested that a polymerase or a helicase alone is a passive motor that is sensitive to the base-pairing energy of the DNA. When coupled together, the helicase–polymerase complex is able to unwind DNA actively. In bacteriophage T7, helicase and polymerase reside right at the replication fork where the parental DNA is separated into two daughter strands. The two motors pull the two daughter strands to opposite directions, while the polymerase provides a separation pin to split the fork. Although independently evolved and containing different replisome components, bacteriophage T4 replisome shares mechanistic features of Hel–Pol coupling that are similar to T7. Interestingly, in bacteriophages with a limited size of genome like Φ29, DNA polymerase itself can form a tunnel-like structure, which encircles the DNA template strand and facilitates strand displacement synthesis in the absence of a helicase. Studies on bacteriophage replication provide implications for the more complicated replication systems in bacteria, archaeal, and eukaryotic systems, as well as the RNA genome replication in RNA viruses.


2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Vijay Lakshmi Jamwal ◽  
Natish Kumar ◽  
Rahul Bhat ◽  
Piyush Singh Jamwal ◽  
Kaurab Singh ◽  
...  

Abstract Background Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19 pandemic, has infected more than 179 million people worldwide. Testing of infected individuals is crucial for identification and isolation, thereby preventing further spread of the disease. Presently, Taqman™ Reverse Transcription Real Time PCR is considered gold standard, and is the most common technique used for molecular testing of COVID-19, though it requires sophisticated equipments, expertise and is also relatively expensive. Objective Development and optimization of an alternate molecular testing method for the diagnosis of COVID-19, through a two step Reverse Transcription Loop-mediated isothermal AMPlification (RT-LAMP). Results Primers for LAMP were carefully designed for discrimination from other closely related human pathogenic coronaviruses. Care was also taken that primer binding sites are present in conserved regions of SARS-CoV2. Our analysis shows that the primer binding sites are well conserved in all the variants of concern (VOC) and variants of interest (VOI), notified by World Health Organization (WHO). These lineages include B.1.1.7, B.1.351, P.1, B.1.617.2, B.1.427/B.1.429, P.2, B.1.525, P.3, B.1.526 and B.1.617.1. Various DNA polymerases with strand displacement activity were evaluated and conditions were optimized for LAMP amplification and visualization. Different LAMP primer sets were also evaluated using synthetic templates as well as patient samples. Conclusion In a double blind study, the RT-LAMP assay was validated on more than 150 patient samples at two different sites. The RT-LAMP assay appeared to be 89.2% accurate when compared to the Taqman™ rt-RT-PCR assay.


2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Mohsen Mohammadniaei ◽  
Ming Zhang ◽  
Jon Ashley ◽  
Ulf Bech Christensen ◽  
Lennart Jan Friis-Hansen ◽  
...  

AbstractThe current nucleic acid signal amplification methods for SARS-CoV-2 RNA detection heavily rely on the functions of biological enzymes which imposes stringent transportation and storage conditions, high cost and global supply shortages. Here, a non-enzymatic whole genome detection method based on a simple isothermal signal amplification approach is developed for rapid detection of SARS-CoV-2 RNA and potentially any types of nucleic acids regardless of their size. The assay, termed non-enzymatic isothermal strand displacement and amplification (NISDA), is able to quantify 10 RNA copies.µL−1. In 164 clinical oropharyngeal RNA samples, NISDA assay is 100 % specific, and it is 96.77% and 100% sensitive when setting up in the laboratory and hospital, respectively. The NISDA assay does not require RNA reverse-transcription step and is fast (<30 min), affordable, highly robust at room temperature (>1 month), isothermal (42 °C) and user-friendly, making it an excellent assay for broad-based testing.


2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Hong Kang ◽  
Tong Lin ◽  
Xiaojin Xu ◽  
Qing-Shan Jia ◽  
Richard Lakerveld ◽  
...  

AbstractWe present a simple and effective scheme of a dynamic switch for DNA nanostructures. Under such a framework of toehold-free strand displacement, blocking strands at an excess amount are applied to displace the complementation of specific segments of paired duplexes. The functional mechanism of the scheme is illustrated by modelling the base pairing kinetics of competing strands on a target strand. Simulation reveals the unique properties of toehold-free strand displacement in equilibrium control, which can be leveraged for information processing. Based on the controllable dynamics in the binding of preformed DNA nanostructures, a multi-input-multi-output (MIMO) Boolean function is controlled by the presence of the blockers. In conclusion, we implement two MIMO Boolean functions (one with 4-bit input and 2-bit output, and the other with 16-bit input and 8-bit output) to showcase the controllable dynamics.


2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Qiu-Long Zhang ◽  
Liang-Liang Wang ◽  
Yan Liu ◽  
Jiao Lin ◽  
Liang Xu

AbstractLigand-oligonucleotide transduction provides the critical pathway to integrate non-nucleic acid molecules into nucleic acid circuits and nanomachines for a variety of strand-displacement related applications. Herein, a general platform is constructed to convert the signals of ligands into desired oligonucleotides through a precise kinetic control. In this design, the ligand-aptamer binding sequence with an engineered duplex stem is introduced between the toehold and displacement domains of the invading strand to regulate the strand-displacement reaction. Employing this platform, we achieve efficient transduction of both small molecules and proteins orthogonally, and more importantly, establish logical and cascading operations between different ligands for versatile transduction. Besides, this platform is capable of being directly coupled with the signal amplification systems to further enhance the transduction performance. This kinetically controlled platform presents unique features with designing simplicity and flexibility, expandable complexity and system compatibility, which may pave a broad road towards nucleic acid-based developments of sophisticated transduction networks.


2021 ◽  
pp. 338927
Author(s):  
Hanxiao Wang ◽  
Chi Zhang ◽  
Xinan An ◽  
Gaiping Li ◽  
Baoxian Ye ◽  
...  

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