Aptamer-guided gene targeting in yeast and human cells.

Gene targeting is a genetic technique to modify an endogenous DNA sequence in its genomic location via homologous recombination (HR) and is useful both for functional analysis and gene therapy applications. HR is inefficient in most organisms and cell types, including mammalian cells, often limiting the effectiveness of gene targeting. Therefore, increasing HR efficiency remains a major challenge to DNA editing. Here, we present a new concept for gene correction based on the development of DNA aptamers capable of binding to a site-specific DNA binding protein to facilitate the exchange of homologous genetic information between a donor molecule and the desired target locus (aptamer-guided gene targeting). We selected DNA aptamers to the I-SceI endonuclease. Bifunctional oligonucleotides containing an I-SceI aptamer sequence were designed as part of a longer single-stranded DNA molecule that contained a region with homology to repair an I-SceI generated double-strand break and correct a disrupted gene. The I-SceI aptamer-containing oligonucleotides stimulated gene targeting up to 32-fold in yeast Saccharomyces cerevisiae and up to 16-fold in human cells. This work provides a novel concept and research direction to increase gene targeting efficiency and lays the groundwork for future studies using aptamers for gene targeting.

RPA Stabilization of Single-Stranded DNA Is Critical for Break-Induced Replication.

DNA double-strand breaks (DSBs) are cytotoxic lesions that must be accurately repaired to maintain genome stability. Replication protein A (RPA) plays an important role in homology-dependent repair of DSBs by protecting the single-stranded DNA (ssDNA) intermediates formed by end resection and by facilitating Rad51 loading. We found that hypomorphic mutants of RFA1 that support intra-chromosomal homologous recombination are profoundly defective for repair processes involving long tracts of DNA synthesis, in particular break-induced replication (BIR). The BIR defects of the rfa1 mutants could be partially suppressed by eliminating the Sgs1-Dna2 resection pathway, suggesting that Dna2 nuclease attacks the ssDNA formed during end resection when not fully protected by RPA. Overexpression of Rad51 was also found to suppress the rfa1 BIR defects. We suggest that Rad51 binding to the ssDNA formed by excessive end resection and during D-loop migration can partially compensate for dysfunctional RPA.

Real-Time PCR-Coupled CE-SELEX for DNA Aptamer Selection.

Aptamers are short nucleic acid or peptide sequences capable of binding to a target molecule with high specificity and affinity. Also known as "artificial antibodies," aptamers provide many advantages over antibodies. One of the major hurdles to aptamer isolation is the initial time and effort needed for selection. The systematic evolution of ligands by exponential enrichment (SELEX) is the traditional procedure for generating aptamers, but this process is lengthy and requires a large quantity of target and starting aptamer library. A relatively new procedure for generating aptamers using capillary electrophoresis (CE), known as CE-SELEX, is faster and more efficient than SELEX but requires laser-induced fluorescence (LIF) to detect the aptamer-target complexes. Here, we implemented an alternative system without LIF using real-time- (RT-) PCR to indirectly measure aptamer-target complexes. In three rounds of selection, as opposed to ten or more rounds common in SELEX protocols, a specific aptamer for bovine serum albumin (BSA) was obtained. The specificity of the aptamer to BSA was confirmed by electrophoretic mobility shift assays (EMSAs), an unlabeled competitor assay, and by a supershift assay. The system used here provides a cost effective and a highly efficient means of generating aptamers.