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Abstract: Synthetic oligonucleotides had been employed in DNA repair and promised great potentials in gene therapy. To test the ability of single-stranded oligonucleotide (SSO)-mediated gene repair within a chromosomal site in human cells, a HeLa cell line stably integrated with mutant enhanced green fluorescence protein gene (mEGFP) in the genome was established. Transfection with specific SSOs successfully repaired the mEGFP gene and resulted in the expression of functional fluorescence proteins, which could be detected by fluorescence microscopy and FACS assay. Western blot showed that EGFP was only present in the cells transfected with correction SSOs rather than the control SSOs. Furthermore, DNA sequencing confirmed that phenotype change resulted from the designated nucleotide correction at the target site. Using this reporter system, we determined the optimal structure of SSO by investigating the effect of length, modifications, and polarities of SSOs as well as the positions of the mismatch-forming nucleotide on the efficiency of SSO-mediated gene repair. Interestingly, we found that SSOs with mismatch-forming nucleotide positioned at different positions have varying potencies that homology at the 5′-end of SSOs was more crucial for the SSO’s activity. These results provided guidance for designing effective SSOs as tools for treating monogenic inherited diseases. [Copyright &y& Elsevier]

Abstract: Targeted gene repair mediated by single-stranded DNA oligonucleotides (SSOs) is a promising method to correct the mutant gene precisely in prokaryotic and eukaryotic systems. We used a HeLa cell line, which was stably integrated with mutant enhanced green fluorescence protein gene (mEGFP) in the genome, to test the efficiency of SSO-mediated gene repair. We found that the mEGFP gene was successfully repaired by specific SSOs, but the efficiency was only ∼0.1%. Then we synthesized a series of nonspecific oligonucleotides, which were single-stranded DNA with different lengths and no significant similarity with the SSOs. We found the efficiency of SSO-mediated gene repair was increased by 6-fold in nonspecific oligonucleotides-treated cells. And this improvement in repair frequency correlated with the doses of the nonspecific oligonucleotides, instead of the lengths. Our evidence suggested that this increased repair efficiency was achieved by the transient alterations of the cellular proteome. We also found the obvious strand bias that antisense SSOs were much more effective than sense SSOs in the repair experiments with nonspecific oligonucleotides. These results provide a fresh clue into the mechanism of SSO-mediated targeted gene repair in mammalian cells. [Copyright &y& Elsevier]

Cell-free DNA (cfDNA), as a non-invasive approach, has been introduced in a wide range of applications, including cancer diagnosis/ monitoring, prenatal testing, and transplantation monitoring. Yet, studies of cfDNA fragmentomics in physiological conditions are lacking. In this study, we aim to explore the correlation of fragmentation patterns of cfDNA with blood biochemical and hematological parameters in healthy individuals. We addressed the impact of physiological variables and abnormal blood biochemical and hematological parameters on cfDNA fragment size distribution. We also figured and validated that hematological inflammation markers, including leukocyte, lymphocyte, neutrophil, and platelet distribution width as well as aspartate transaminase levels were significantly correlated with the genome-wide cfDNA fragmentation pattern. Our findings suggest that cfDNA fragmentation profiles were associated with physiological parameters related to cardiovascular risk factors, inflammatory response and hepatocyte injury, which may provide insights for further research on the potential role of cfDNA fragmentation in diagnosis and monitor of several disease. • Blood triglyceride and glucose level correlated with short cfDNA fragment ratio. • cfDNA fragmentation correlated with inflammation-related hematological parameters. • AST was significantly correlated with cfDNA fragmentation patterns. [ABSTRACT FROM AUTHOR]