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. (c) 2005 Elsevier Inc. All rights reserved.