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What is Chimeraplasty?

Chimeraplasty, also known as targeted gene correction, is a technique in which a synthetically created molecule consisting of both RNA and DNA is used to repair single base pair mutations, deletions, or insertions in DNA.


Background Information

The technique of chimeraplasty was developed in the lab of Drs. Eric B. Kmiec, Kyonggeun Yoon, and Allyson Cole-Strauss, at Thomas Jefferson University in Philadelphia, Pennsylvania (Gura 1999 ). Kmiec studies homologous recombination, and he realized that the rate of recombination is increased for active genes being transcribed into mRNAs (Gura 1999). This led him to question whether synthetic RNA could be used in gene repair by tricking the cell to incorporate good DNA into mutated sites (Gura 1999). However, the problem with this idea was that RNA degrades much faster than DNA, and might not last long enough to repair the targeted DNA (Gura 1999 ). Finally, Kmiec realized that he could create a hybrid DNA-RNA molecule (a chimeraplast) that would be stable for long enough to allow gene repair (Gura 1999).


A chimeraplast consists of a paper-clip shaped, double-stranded stretch of DNA interspersed with short strands of RNA (Fig. 1) (Stephenson 1999). The design of the chimeraplast is a result of the discovery that hybrids of RNA and DNA are very active in homologous pairing reactions (Cole-Strauss and others 1996; Yoon, Cole-Strauss, and Kmiec 1996). Furthermore, researchers found that the hairpin caps at the ends of the molecules do not impede base pairing with target genes (Cole-Strauss and others 1996; Yoon, Cole-Strauss, and Kmiec 1996). The purpose of the short RNA strands is to activate the oligonucleotide ("oligo") for recombination, and the hairpin caps protect the molecule from destabilization or destruction by exonucleases or cellular helicases (Cole-Strauss and others 1996; Yoon, Cole-Strauss, and Kmiec 1996). Finally, the ribose of the RNA is 2'-O-methyl modified to add protection against cleavage by RNase H activities (Cole-Strauss and others1996; Yoon, Cole-Strauss, and Kmiec 1996).


When researchers design a chimeric oligonucleotide, they replicate a short portion of the base sequence of the target gene surrounding the base pair mutation so that it aligns perfectly with the gene, except for the one base pair where the mutation occurs (Yoon, Cole-Strauss, and Kmiec 1996 ;Cole-Strauss and others 1999). In this location, the correct base is substituted into the oligo (Yoon, Cole-Strauss, and Kmiec 1996 ;Cole-Strauss and others 1999). Therefore, when the chimera inserts between the strands of target DNA (Fig. 2), the mismatched base pair is recognized by the endogenous repair system, and the sequence is changed in either the chimeraplast (using the target DNA as a template), or in the target DNA (using the chimera as a template) (Yoon, Cole-Strauss, and Kmiec 1996 ;Cole-Strauss and others 1999).

Mechanism of Repair

In cell-free extracts, chimeric oligos probably work by pairing with a plasmid target based on homology (Fig. 2), using DNA pairing enzymes and complexes (Cole-Strauss and others 1999). After pairing, endogenous repair machinery recognizes the mismatch between the gene and the chimera, and uses mismatch repair to correct the "spelling mistake" by using the chimera as the template sequence (Fig. 2) (Cole-Strauss and others 1999). Then, the chimera decays, leaving only the corrected target DNA (Fig. 3) (Coghlan 1999).

For in vivo repair, the oligos can be attached to organ-specific ligands, as has been performed by Li-Wen Lai at the University of Arizona in Tucson (Smaglik 2000). Liposomes and synthetic polymers are also used to deliver chimeric molecules to the appropriate cells or tissue (Stephenson 1999). In plants, microscopic gold particles are coated with the chimeric molecules and fired into cells (Coghlan 1999). In all cases, the oligos that enter the nucleus can repair point mutations within the cell after pairing with their sequence-specific target DNA (Fig. 2) by causing the cell's repair machinery to perform mismatch repair on the point mutation (Cole-Strauss and others 1999). After correction, the chimera decays, leaving the corrected gene (Fig. 3) (Coghlan 1999).

Fig. 1. The chimeraplast is a paper-clip shaped, double stranded stretch of DNA interspersed with short strands of RNA (Stephenson 1999). The base sequence of the chimeraplast perfectly matches the sequence of the target gene, except at the location marked with a box ("Mismatched Bases") (Cole-Strauss and others 1996; Yoon, Cole-Strauss, and Kmiec 1996).

Fig. 2. The chimera integrates itself between the two strands of target DNA, and the hairpin binds to the target gene (Yoon, Cole-Strauss, and Kmiec 1996). Because of the mismatched bases, the endogenous repair mechanisms change the sequence of the target gene, using the chimera as a template (Cole-Strauss and others 1999; Coghlan 1999). This corrects the point mutation in the target gene.

Fig. 3. After correcting the target gene, the hairpin decays, leaving only the corrected target gene (Coghlan 1999).


Uses of Chimeraplasty



Coghlan A. 1999. Look, no new genes: A subtle technique tricks plant cells in doing their own genetic engineering. New Scientist 163 (2197): 4.

Cole-Strauss A, Gamper H, Holloman WK, Munoz M, Cheng N, Kmiec EB. 1999. Targeted gene repair directed by the chimeric RNA/DNA oligonucleotide in a mammalian cell-free extract. Nucleic Acids Research 27 (5): 1323-1330.

Cole-Strauss A, Yoon K, Xiang Y, Byrne BC, Rice MC, Gryn J, Holloman WK, Kmiec EB. 1996. Correction of the Mutation Responsible for Sickle Cell Anemia by an RNA-DNA Oligonucleotide. Science 273: 1386-1389.

Gura T. 1999. Repairing the Genome's Spelling Mistakes. Science 285: 316-318.

Robrish D. 1999 July 26. New technique permits faster, more accurate gene tailoring. CNEWS Science. <> Accessed 2000 Feb 20.

Smaglik P. 2000 Jan 10. Chimeraplasty Potential: As research advances, hopes rise, but efficiency and safety are still concerns. The Scientist. <> Accessed 2000 Feb 20.

Stephenson J. 1999. New Method to Repair Faulty Genes Stirs Interest in Chimeraplasty Technique. JAMA 281 (2): 119-121.

Yoon K, Cole-Strauss A, Kmiec EB. 1996. Targeted gene correction of episomal DNA in mammalian cells mediated by chimeric RNA-DNA oligonucleotide. Proceedings of the National Academy of Science 93: 2071-2076.

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