The Validation and Amplification of Random DNA Libraries with Modified Nucleobases for Click-SELEX

Комарова, Н. В.; Panova, Olga; Titov, Aleksey

doi:10.3390/iecbm2022-13692

Cited by 1 publication

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“…We used a similar strategy to construct BDs, and the first step is to produce scaffold DNAs containing reactive groups for later attachment of side chain DNA strands (Figure B). 5-Modifications of cytosine and uracil are among the nucleobase modifications that are mostly used for DNA labeling, due to their good recognition by DNA polymerases. ,, To produce a large quantity of nucleobase-modified scaffold DNAs for the construction of BDs, we PCR amplified 150-bp-long DNA fragments with defined sequences from pre-constructed plasmid templates with dC Am TP, dATP, dTTP, and dGTP using Taq DNA polymerase, such that all the dC nucleotides in the newly synthesized DNA strands were 5-modified with a propargylamino group (Figure B).…”

Section: Resultsmentioning

confidence: 99%

Bottlebrush DNA-Primed Rolling Circle Amplification for Sensitive Detection of Biomolecules

Chen,

Zhang,

Peng

et al. 2023

ACS Appl. Nano Mater.

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Development of nucleic acids containing unnatural moieties has expanded the scope of genetic materials, and modifications of nucleobases have been extensively explored and found broad applications. However, previous efforts have mainly focused on modifying nucleobases with small molecules, while nucleobase modification with macromolecules has barely been explored. In this work, we first demonstrated the construction of highly programmable bottlebrush-like DNA structures by enzymatic production of DNA containing nucleobases modified with coupling handles and chemical attachment of single-stranded DNAs onto these handles. Employing these bottlebrush DNAs (BDs), we then developed a method named bottlebrush DNA-primed rolling circle amplification (BDP-RCA), which could rapidly produce branched DNA products with ultrahigh molecular weights in a controllable manner. BDP-RCA generated up to approximately 20-fold more products than ordinary RCA within 10 min. Moreover, the BDP-RCA product demonstrated a netlike structure in aqueous solution and a structure consisted of uniformly sized nanoflowers with a diameter of approximately 0.7 μm when lyophilized. These observations suggested the great potential of BDP-RCA in the development of materials and methods for biosensing, which were demonstrated with several examples. First, ultrasensitive detection of human α-thrombin with a limit of detection (LOD) of 1.1 pM was achieved by using BDP-RCA to amplify the detection signal in an ELISA-like assay. Second, magnetic beads immobilizing the BDP-RCA product were successfully applied for the efficient capture, enrichment, and preservation of a protein or bacterial cells to be detected. The LOD for the detection of human α-thrombin with this method was 68.5 pM.

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Section: Resultsmentioning

confidence: 99%