Ligang Xing,Yanmei Zhao,Mingzhu Gong,Xia Liu,Yuhui Zhang,Dan Li,Zefeng He,Ping Yan,Jidong Yang
In this study, Graphene Oxide (GO) was used to screen the binding with the aptamers of L-carnitine chiral enantiomers . The ssDNA library was prepared by the method of Lambda exonuclease . In addition, a simple casing device was designed to improve the purification and recovery efficiency of the small ssDNA fragments in the process of screening. Finally, more than 160,000 aptamer sequences were obtained by high-throughput sequencing. We determined the strongest affinity aptamer sequence, CA04, by the Resonance Rayleigh scattering (RRS) technology. We also analyzed the key binding sites (in the 16th position case) of the truncated aptamer sequence CAD10. Interestingly, we found that aptamer CA10 and CA06 were both C-rich bases through sequence alignment and analysis, and the aptamer CA10 was confirmed that the CA10 and CA06 were formed under acidic conditions (pH 4.5) by CD spectrum and ESI-MS analysis. The interaction between gold nanoparticles (AuNPs) and functionalized aptamer CA10 was analyzed. We used Site-directed mutagenesis design and QGRS Mapper to optimize aptamer CA10, where an optimal aptamer CA10-03 were obtained after affinity analysis. It is also proved to be an effective method to obtain stronger affinity aptamer. Meanwhile, Native-PAGE and UV spectrum analysis were performed on the mutation sequences, and the interaction with ThT was analyzed. Finally, it is hoped that my study can provide help for later identification and detection of L-carnitine .
Human T-cell lymphophilic virus type 1 (HTLV-1), the known retrovirus causing cancer in humans, is closely associated with adult T-cell leukemia/lymphoma and HTLV-1 associated myelopathy/tropical spastic paraparesis. Due to its ability to evade the host's defense mechanisms, early tracking of HTLV-1 becomes crucial. In this study, we integrate λ-Exonuclease (λ-Exo)-assisted target recycling with a terminal deoxynucleotidyl transferase (TdT)-mediated template-free DNA extension process to develop an electrochemical analysis platform for the specific and sensitive detection of HTLV-1 DNA. During the λ-Exo-assisted target recycling, HTLV-1 DNA is recognized by hairpin DNA (Hp-DNA), forming double-stranded DNA (dsDNA) through DNA hybridization. The dsDNA, featuring blunt 5' terminal phosphorylation, is cleaved by λ-Exo, generating abundant short output sequence (sDNA). HTLV-1 DNA is released, initiating a cyclic hybridization-cleavage process. Subsequently, thiol-labelled capture DNA (CP-DNA) assembled on gold electrode surface captures a substantial amount of the generated sDNA, forming CP-DNA-sDNA nanostructures. When TdT and dNTPs are present on the electrode surface, the 3'-OH terminal of sDNA extends to generate long single-stranded DNA (ssDNA) structure. Methylene blue (MB) is selected as the electrochemical signal molecule. MB not only binds with ssDNA but also interacts specifically with dsDNA, resulting in a significantly enhanced electrochemical signal on modified electrode surface. The detection limit of HTLV-1 DNA is as low as 19 amol/L ( S / N = 3) when the two signal amplification strategies are combined. The analysis platform exhibits excellent analytical performance and holds promise as a novel tool for the early tracing and diagnosis of HTLV-1 DNA.
The employment of DNAzyme probes for visual biodetections has received increasing interest recently due to the simple nature of this type of assay. However, achieving high sensitivity and detecting targets beyond nucleic acids remain two major challenges in DNAzyme-based visual detections. In this work, based on a new quadratic amplification strategy, we developed a sensitive and visual detection method for cytokines by using hairpin aptamer DNAzyme probes. The target cytokine, interferon γ (IFN-γ), associates with the aptamer sequences and unfolds the hairpin structure of the probes, leading to simultaneous recycling of the target IFN-γ (assisted by Bst-polymerase) and the DNA sequences (aided by λ exonuclease) to achieve quadratic amplification. This quadratic amplification results in the generation of numerous peroxidase-mimicking DNAzymes, which cause significantly intensified color change of the probe solution for highly sensitive detection of IFN-γ by the naked eye down to 50 pM. The proposed visual sensing method shows also high selectivity toward the target IFN-γ and can be performed in homogeneous solutions with using completely unmodified, synthetic aptamer DNAzyme probes. These distinct advantages of our developed assay protocol make it a potential platform for detecting various types of biomolecules with careful probe designs.
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