Mechanistic investigation of bead-based padlock rolling circle amplification under molecular crowding conditions

Sasaki, Naoki; Kase, Chikako; Chou, Masaki; Nakazato, Genki; Sato, Kae

doi:10.1016/j.ab.2020.113596

Cited by 13 publications

(7 citation statements)

References 10 publications

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“…PEG and PVP are two common polymers, and they had a weak effect on LAMP acceleration in the absence of AuNPs (accelerated for ∼5 to 10 min, Figure A, C, and D). PEG was reported to increase the activity of polymerase, which could also be helpful for the isothermal nucleic acid amplification. , When the AuNPs were added to PEG and PVP, similarly boosted LAMP amplifications were obtained, and they showed ∼10 min acceleration compared to using the AuNPs alone (Figure D). Therefore, the effect of these polymers and AuNPs appeared to be additive.…”

Section: Resultsmentioning

confidence: 92%

Capping Gold Nanoparticles to Achieve a Protein-like Surface for Loop-Mediated Isothermal Amplification Acceleration and Ultrasensitive DNA Detection

Jiang

Yang

Liu

2022

ACS Appl. Mater. Interfaces

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Loop-mediated isothermal amplification (LAMP) is a popular DNA amplification method. Gold nanoparticles (AuNPs) were reported to enhance the efficiency of LAMP, although the underlying mechanism remained elusive. Since AuNPs strongly adsorb a range of ligands, preadsorbed ligands cannot be easily displaced. In this work, we systematically investigated the effect of surface-modified AuNPs on LAMP by varying the order of mixing of AuNPs with each reagent in the LAMP system (Mg 2+ , template DNA, dNTPs, primers, and polymerase). Mixing the AuNPs with the primers delayed the LAMP based on SYBR green I fluorescence. While other orders of mixing had little effect, all accelerated the reaction. We then tested other common ligands including polymers (polyethylene glycol and polyvinylpyrrolidone), inorganic ions (Br − ), proteins, glutathione (GSH), and DNA (A 15 ) on AuNP-LAMP. The boosted AuNP performance on LAMP was most obvious when the AuNPs formed a protein-like surface. Finally, using GSH-capped AuNPs, a detection limit of around 100 copies/μL −1 of target DNA was achieved. This work has identified a ligand-capped AuNP strategy to boost LAMP and yielded a higher sensitivity in DNA sensing, which also deepens our understanding of AuNP-assisted LAMP.

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Section: Resultsmentioning

confidence: 92%

Capping Gold Nanoparticles to Achieve a Protein-like Surface for Loop-Mediated Isothermal Amplification Acceleration and Ultrasensitive DNA Detection

Jiang

Yang

Liu

2022

ACS Appl. Mater. Interfaces

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“…In a control experiment with streptavidin-modified sepharose beads, primer DNA was immobilized on beads prior to the bead-based padlock/RCA, as previously described, 11 with some modifications. Briefly, suspensions of 1 × 10 4 streptavidinmodified sepharose beads were centrifuged at 200g for 1 min to remove the solution.…”

Section: Optimization Of Primer Dna Immobilizationmentioning

confidence: 99%

“…10 In addition, using PEG as a crowder, we have investigated the mechanism of the increase in the number of products and evaluated the effect of PEG molecular weight and concentration on the number of products. 11 However, we believe that further studies are necessary to understand the mechanism of this method and to improve its performance. The first consideration is the timing and amount of the primer DNA to be immobilized to the beads.…”

Section: Introductionmentioning

confidence: 99%

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Bead-based Padlock Rolling Circle Amplification under Molecular Crowding Conditions: The Effects of Crowder Charge and Size

2021

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Bead-based padlock rolling circle amplification under molecular crowding conditions, which we have developed for ultrasensitive detection of DNA, is examined to improve the detection efficiency and sensitivity of the method as well as to gain insight into the mechanism of the method. Both non-magnetic and magnetic sepharose microbeads were employed. Biotinylated DNA had to be pre-immobilized onto the microbeads in order to obtain products on the magnetic beads. The optimal concentration of biotinylated DNA was found to be about 5 μM, above which the number of products decreased. The effect of the crowder charge was examined, and neutral polymers were found to be effective on ligation and the hybridization step, while charged polymers were only effective on the hybridization step and inhibited the ligation and primer extension. The effect of the molecular weight of neutral dextran on the number of products was investigated, and the number of products was found to be increased with an increase in the molecular weight of dextran.

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“…Although, nontemplate ligation gives a low yield of the target product, this method is indispensable in studies of NA samples of unknown composition and in the analysis of short-stranded NAs, e.g., fragmented DNA or small RNAs. In addition, the methods are known in which the yield of the CT during ligation is increased by using molecular crowding agents, such as polyethylene glycol [ 14 , 15 ].…”

Section: Molecular Bases Of Rolling Circle Amplificationmentioning

confidence: 99%

Rolling Circle Amplification as a Universal Method for the Analysis of a Wide Range of Biological Targets

et al. 2021

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— Detection and quantification of biotargets are important analytical tasks, which are solved using a wide range of various methods. In recent years, methods based on the isothermal amplification of nucleic acids (NAs) have been extensively developed. Among them, a special place is occupied by rolling circle amplification (RCA), which is used not only for the detection of a specific NA but also for the analysis of other biomolecules, and is also a versatile platform for the development of highly sensitive methods and convenient diagnostic devices. The present review reveals a number of methodical aspects of RCA-mediated analysis; in particular, the data on its key molecular participants are presented, the methods for increasing the efficiency and productivity of RCA are described, and different variants of reporter systems are briefly characterized. Differences in the techniques of RCA-mediated analysis of biotargets of various types are shown. Some examples of using different RCA variants for the solution of specific diagnostic problems are given.

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Mechanistic investigation of bead-based padlock rolling circle amplification under molecular crowding conditions

Cited by 13 publications

References 10 publications

Capping Gold Nanoparticles to Achieve a Protein-like Surface for Loop-Mediated Isothermal Amplification Acceleration and Ultrasensitive DNA Detection

Capping Gold Nanoparticles to Achieve a Protein-like Surface for Loop-Mediated Isothermal Amplification Acceleration and Ultrasensitive DNA Detection

Bead-based Padlock Rolling Circle Amplification under Molecular Crowding Conditions: The Effects of Crowder Charge and Size

Rolling Circle Amplification as a Universal Method for the Analysis of a Wide Range of Biological Targets

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