Eric B Kmiec, University of Delaware, Newark, Delaware, USA
Published online: January 2006
DOI: 10.1038/npg.els.0005763
The information gained from the Human Genome Project must be formulated such that the function of newly discovered genes can be elucidated. A tool to aid in defining gene function is targeted gene repair, a technique that may also be valuable in human gene therapy.
Keywords: chimera; gene repair; chimeric RNA–DNA oligonucleotide; transfection; RAD51; yeast
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Figure 1. Generalized design features of the chimeric ribonucleic acid–deoxyribonucleic acid (RNA–DNA) oligonucleotide. The upper strand consists of RNA residues (1). Both ends of the molecule are configured into a hairpin structure (2). Each RNA residue has a 2¢-O-methyl modification (3). The lower strand, which is all DNA residues, directs the nucleotide exchange during gene repair (4). A single, unligated phosphodiester bond exists to allow intertwining of the chimera with the target site (5).
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Figure 2. Current working hypothesis of the mechanism of chimera-directed repair. The chimera (2) reaches the target site either through enzymatic or nonenzymatic activities. The double D-loop structure, reaction intermediate, may be matured and/or stabilized by Rad59p (3). Protein Rad52p acts at an early stage to retard the reaction intermediate. The double D-loop (4) containing a mismatched base pair G/T is recognized by a group of proteins, including those from the mismatch repair and nucleotide excision repair pathways (5). The excised base is replaced with a C residue, presumably by the action of a DNA polymerase (6). The chimera dissociates (between 7 and 8) and Msh2p and other mismatch repair activities correct the mismatch A/C left in the target sequence (8).
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Figure 3. Progress in targeted gene repair. References are shown for each seminal event in the development of chimera-directed gene repair.
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Liu H,
Agarwal S,
Kmiec E and
Davis BR
(2002)
Targeted beta-globin gene conversion in human hematopoietic CD34 | |
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| Further Reading | |
| Andersen MS, Sorensen CB, Bolund L and Jensen TG (2002) Mechanism underlying targeted gene correction using chimeric RNA/DNA and single-stranded DNA oligonucleotides. Journal of Molecular Medicine 80: 770–781. | |
| Liu C-M, Liu D-P and Liang C-C (2002) Oligonucleotide-mediated gene repair at DNA level: The potential applications for gene therapy. Journal of Molecular Medicine 80: 620–628. | |
| Rando TA (2002) Oligonucleotide-mediated gene therapy for muscular dystrophies. Neuromuscular Disorders 12: S55–S60. | |
| Thorpe PH, Stevenson BJ and Porteous DJ (2002) Functional correction of episomal mutations with short DNA fragments and RNA–DNA oligonucleotides. Journal of Gene Medicine 4: 195–204. | |
| Yanez RJ and Porter ACG (1999) Gene targeting is enhanced in human cells overexpressing hRAD51. Gene Therapy 6: 1282–1290. | |
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