Screening substrate-binding positions by rolling circle amplification suggesting a binding model of Nt.BstNBI

2019 ◽  
Vol 476 (10) ◽  
pp. 1483-1496
Author(s):  
Hua Wei ◽  
Suming Tang ◽  
Xuying Duan ◽  
Yifu Guan ◽  
Guojie Zhao
Keyword(s):  
Catalytic Mechanism ◽  
Rolling Circle Amplification ◽  
Locked Nucleic Acid ◽  
Dna Duplex ◽  
Recognition Sequence ◽  
Acid Modification ◽  
Binding Model ◽  
Rolling Circle ◽  
Structure Information ◽  
Potential Binding

Abstract Nicking endonucleases (NEs) become increasingly attractive for their promising applications in isothermal amplification. Unfortunately, in comparison with their applications, their catalytic mechanism studies have relatively lagged behind due to a paucity of crystal structure information. Nt.BstNBI is one of those widely used NEs. However, many aspects of its catalytic mechanism still remained to be explored. Herein, we employed only rolling circle amplification (RCA) assay as a major analytic tool and succeeded in identifying the potential binding positions and regions of the DNA substrate based on locked nucleic acid modification, DNA duplex length of substrate, and substrate mismatch designs. Based on these data, we, for the first time, revealed that Nt.BstNBI was likely to recognize six adjacent positions of the recognition sequence (G1rt, A2rt, G3rt, A2rb, C3rb, and T4rb) in the major groove and hold three positions of the cleavage sequence (N3ct, N4ct, and N7cb) in the minor groove of DNA duplex for nicking. Moreover, this work also demonstrated the unexpected efficiency of RCA to study the macromolecular interaction for certain kind of nucleases in an easy and high-throughput way.

An electrochemiluminescence biosensor for Kras mutations based on locked nucleic acid functionalized DNA walkers and hyperbranched rolling circle amplification

2017 ◽  
Vol 53 (20) ◽  
pp. 2910-2913 ◽  
Author(s):  
Ying Zhang ◽  
Lixu Wang ◽  
Fang Luo ◽  
Bin Qiu ◽  
Longhua Guo ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Rolling Circle Amplification ◽  
Locked Nucleic Acid ◽  
Specific Detection ◽  
Kras Mutations ◽  
Rolling Circle ◽  
Dna Walkers ◽  
Mutant Genes

Herein, an electrochemiluminescence (ECL) biosensor for ultrasensitive and specific detection of Kras mutant genes has been developed.

Ferromagnetic Resonance Biosensor for Homogeneous and Volumetric Detection of DNA

2017 ◽  
Author(s):  
Bo Tian ◽  
Peter Svedlindh ◽  
Mattias Strömberg ◽  
Erik Wetterskog
Keyword(s):  
Magnetic Nanoparticles ◽  
Ferromagnetic Resonance ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Rolling Circle Amplification ◽  
Resonance Field ◽  
Isothermal Amplification ◽  
Detection Sensitivity ◽  
Serum Samples ◽  
Rolling Circle

In this work, we demonstrate for the first time, a ferromagnetic resonance (FMR) based homogeneous and volumetric biosensor for magnetic label detection. Two different isothermal amplification methods, <i>i.e.</i>, rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP) are adopted and combined with a standard electron paramagnetic resonance (EPR) spectrometer for FMR biosensing. For RCA-based FMR biosensor, binding of RCA products of a synthetic Vibrio cholerae target DNA sequence gives rise to the formation of aggregates of magnetic nanoparticles. Immobilization of nanoparticles within the aggregates leads to a decrease of the net anisotropy of the system and a concomitant increase of the resonance field. A limit of detection of 1 pM is obtained with an average coefficient of variation of 0.16%, which is superior to the performance of other reported RCA-based magnetic biosensors. For LAMP-based sensing, a synthetic Zika virus target oligonucleotide is amplified and detected in 20% serum samples. Immobilization of magnetic nanoparticles is induced by their co-precipitation with Mg<sub>2</sub>P<sub>2</sub>O<sub>7</sub> (a by-product of LAMP) and provides a detection sensitivity of 100 aM. The fast measurement, high sensitivity and miniaturization potential of the proposed FMR biosensing technology makes it a promising candidate for designing future point-of-care devices.<br>

scholarly journals Addressing widespread misidentifications of traditional medicinal mushrooms in Sanghuangporus (Basidiomycota) through ITS barcoding and designation of reference sequences

IMA Fungus ◽  
2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Shan Shen ◽  
Shi-Liang Liu ◽  
Ji-Hang Jiang ◽  
Li-Wei Zhou
Keyword(s):  
Species Identification ◽  
Rolling Circle Amplification ◽  
Practical Method ◽  
Morphological Characters ◽  
Its Sequences ◽  
Commercial Development ◽  
Medicinal Mushrooms ◽  
Rolling Circle ◽  
Reference Sequences ◽  
Its Barcoding

Abstract“Sanghuang” refers to a group of important traditionally-used medicinal mushrooms belonging to the genus Sanghuangporus. In practice, species of Sanghuangporus referred to in medicinal studies and industry are now differentiated mainly by a BLAST search of GenBank with the ITS barcoding region as a query. However, inappropriately labeled ITS sequences of “Sanghuang” in GenBank restrict accurate species identification and, to some extent, the utilization of these species as medicinal resources. We examined all available 271 ITS sequences related to “Sanghuang” in GenBank including 31 newly submitted sequences from this study. Of these sequences, more than half were mislabeled so we have now corrected the corresponding species names. The mislabeled sequences mainly came from strains utilized by non-taxonomists. Based on the analyses of ITS sequences submitted by taxonomists as well as morphological characters, we separate the newly described Sanghuangporus subbaumii from S. baumii and treat S. toxicodendri as a later synonym of S. quercicola. Fourteen species of Sanghuangporus are accepted, with intraspecific distances up to 1.30% (except in S. vaninii, S. weirianus and S. zonatus) and interspecific distances above 1.30% (except between S. alpinus and S. lonicerinus, and S. baumii and S. subbaumii). To stabilize the concept of these 14 species of Sanghuangporus, their taxonomic information and reliable ITS reference sequences are provided. Moreover, ten potential diagnostic sequences are provided for Hyperbranched Rolling Circle Amplification to rapidly confirm three common commercial species, viz. S. baumii, S. sanghuang, and S. vaninii. Our results provide a practical method for ITS barcoding-based species identification of Sanghuangporus and will promote medicinal studies and commercial development from taxonomically correct material.

scholarly journals MiRNA Detection Using a Rolling Circle Amplification and RNA-Cutting Allosteric Deoxyribozyme Dual Signal Amplification Strategy

Biosensors ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 222
Author(s):  
Chenxin Fang ◽  
Ping Ouyang ◽  
Yuxing Yang ◽  
Yang Qing ◽  
Jialun Han ◽  
...  
Keyword(s):  
Signal Amplification ◽  
Rolling Circle Amplification ◽  
Padlock Probe ◽  
Rolling Circle ◽  
Sensing Platform ◽  
Mirna Detection ◽  
Substrate Cleavage ◽  
Amplification Strategy ◽  
Circular Template ◽  
Dual Signal Amplification

A microRNA (miRNA) detection platform composed of a rolling circle amplification (RCA) system and an allosteric deoxyribozyme system is proposed, which can detect miRNA-21 rapidly and efficiently. Padlock probe hybridization with the target miRNA is achieved through complementary base pairing and the padlock probe forms a closed circular template under the action of ligase; this circular template results in RCA. In the presence of DNA polymerase, RCA proceeds and a long chain with numerous repeating units is formed. In the presence of single-stranded DNA (H1 and H2), multi-component nucleic acid enzymes (MNAzymes) are formed that have the ability to cleave substrates. Finally, substrates containing fluorescent and quenching groups and magnesium ions are added to the system to activate the MNAzyme and the substrate cleavage reaction, thus achieving fluorescence intensity amplification. The RCA–MNAzyme system has dual signal amplification and presents a sensing platform that demonstrates broad prospects in the analysis and detection of nucleic acids.

scholarly journals Evaluation of Immuno-Rolling Circle Amplification for Multiplex Detection and Profiling of Antigen-Specific Antibody Isotypes

2021 ◽  
Vol 93 (15) ◽  
pp. 6169-6177
Author(s):  
Sara Horta ◽  
Felix Neumann ◽  
Shu-hao Yeh ◽  
Christoffer Mattsson Langseth ◽  
Kadri Kangro ◽  
...  
Keyword(s):  
Specific Antibody ◽  
Rolling Circle Amplification ◽  
Multiplex Detection ◽  
Rolling Circle ◽  
Antibody Isotypes
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