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Simple and efficient method to purify DNA-protein conjugates and its sensing applications.

ABSTRACT: DNA-protein conjugates are very useful in analytical chemistry for target recognition and signal amplification. While a number of methods for conjugating DNA with proteins are known, methods for purification of DNA-protein conjugates from reaction mixture containing unreacted proteins are much less investigated. In this work, a simple and efficient approach to purify DNA-invertase conjugates from reaction mixture via a biotin displacement strategy to release desthiobiotinylated DNA-invertase conjugates from streptavidin-coated magnetic beads was developed. The conjugates purified by this approach were utilized for quantitative detection of cocaine and DNA using a personal glucose meter through structure-switching DNA aptamer sensors and competitive DNA hybridization assays, respectively. In both cases, the purified DNA-invertase conjugates showed better performance compared to the same assays using unpurified conjugates. The approach demonstrated here can be further expanded to other DNA and proteins to generate purified DNA-protein conjugates for analytical and other applications.

SUBMITTER: Zhou Z

PROVIDER: S-EPMC4004194 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Simple and efficient method to purify DNA-protein conjugates and its sensing applications.

Zhou Zhaojuan Z Xiang Yu Y Tong Aijun A Lu Yi Y

Analytical chemistry 20140328 8

DNA-protein conjugates are very useful in analytical chemistry for target recognition and signal amplification. While a number of methods for conjugating DNA with proteins are known, methods for purification of DNA-protein conjugates from reaction mixture containing unreacted proteins are much less investigated. In this work, a simple and efficient approach to purify DNA-invertase conjugates from reaction mixture via a biotin displacement strategy to release desthiobiotinylated DNA-invertase con ...[more]

PMID: 24605905

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Project description:Background: CRISPR-Cas9-based technologies have revolutionized experimental manipulation of mammalian genomes. None-the-less, limitations of the delivery and efficacy of these technologies restrict their application in primary cells. Aims: To create an optimized protocol for penetrant, reproducible, and fast targeted genome editing in cell cultures derived from primary cells, using patient-derived glioblastoma (GBM) stem-like cells (GSCs) and human neural stem/progenitor cells (NSCs) for proof-of-concept experiments. Methods and results: We employed transient nucleofection of Cas9:sgRNA ribonucleoprotein complexes composed of chemically synthesized 2'-O-methyl 3’phosphorothioate-modified sgRNAs and purified Cas9 protein. Insertion-deletion mutation (indel) frequency and size distribution were measured via computational deconvolution of Sanger sequencing trace data. We found that this optimized technique routinely allows for >90% indel formation in only 3 days, without the need to create clonal lines for simple loss-of-function experiments. Using Western blotting, we observed near-total protein loss of target genes in cell pools. Additionally, we found that this approach allows for the creation of targeted genomic deletions. By using RNA-seq in edited NSCs to assess gene expression changes resulting from knockout of tumor suppressors commonly altered in GBM, we also demonstrated the utility of this method for quickly creating a series of gene knockouts that allow for the study of oncogenic activities. Conclusion: Our data suggest that this relatively simple method can be used for highly efficient and fast gene knockout, as well as for targeted genomic deletions, even in hyperdiploid cells (such as GSCs). This represents an extremely useful tool for the cancer research community when wishing to inactivate not only coding genes, but also non-coding RNAs, UTRs, enhancers, and promoters. This method can be readily applied to diverse cell types by varying the nucleofection conditions.

Dataset Information

Simple and efficient method to purify DNA-protein conjugates and its sensing applications.

Publications

Simple and efficient method to purify DNA-protein conjugates and its sensing applications.

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