Direct Selection of an Allosteric RNA-Cleaving DNAzyme for a Protein Target: A Specific DNAzyme for Eosinophil Peroxidase to Monitor Airway Eosinopilia

ABSTRACT: In this project, in vitro selection was carried out to generate DNAzymes for Eosinophil peroxidase using a synthetic DNA library. Total 15 rounds of selections were carried out. The DNA molecules obtain in round 15, was applied in Illumina MiSeq deep sequencing which provided fastq files. Sequencing samples were prepared from each parallel SELEX experiment by PCR tagging with Illumina sequencing primers. Samples were size purified by agarose gel electrophoresis prior to being quantified by measuring absorbance at 260 nm. Tagged samples were pooled and paired-end sequenced on an Illumina MiSeq high-throughput DNA sequencer. Sequence data processing was performed on a Windows 10 computer running Ubuntu 20.04 under WSL2. Raw paired-end reads were trimmed of sequencing and library primers using cutadapt 3.4. Trimmed paired-end reads were then: 1) merged into a consensus sense read; 2) dereplicated; and, 3) clustered at 90% identity using USEARCH v11.0.667_i86linux32. Sequence frequencies and ranking lists were generated using custom Python scripts. Multiple sequence alignments were performed using MUSCLE v3.8.1551 and converted to sequence logos using WebLogo 3.7.8. Processed sequencing data and cluster linkage data were stored on a MySQL 8.0.22 database. Analysis of sequence copy number, frequency, cluster linkage and data plots were performed using the database and Microsoft Excel Top 20 sequences were tested for cleavage performance. The most active DNAzyme was characterized and optimized. At the end, fluorescence and lateral flow assays were developed and evaluated in real patients' sputums.

ORGANISM(S): synthetic construct

PROVIDER: GSE217946 | GEO | 2023/11/15

REPOSITORIES: GEO