Skip to main content
NCBI home page
Biophysical Journal logoLink to Biophysical Journal
. 2001 Nov;81(5):2876–2885. doi: 10.1016/S0006-3495(01)75928-0

A simple model for gene targeting.

T Ratilainen 1, P Lincoln 1, B Nordén 1
PMCID: PMC1301752  PMID: 11606298

Abstract

Sequence-specific binding to genomic-size DNA sequences by artificial agents is of major interest for the development of gene-targeting strategies, gene-diagnostic applications, and biotechnical tools. The binding of one such agent, peptide nucleic acid (PNA), to a randomized human genome has been modeled with statistical mass action calculations. With the length of the PNA probe, the average per-base binding constant k(0), and the binding affinity loss of a mismatched base pair as main parameters, the specificity was gauged as a "therapeutic ratio" G = maximum safe [PNA](tot)/minimal efficient [PNA](tot). This general, though simple, model suggests that, above a certain threshold length of the PNA, the microscopic binding constant k(0) is the primary determinant for optimal discrimination, and that only a narrow range of rather low k(0) values gives a high therapeutic ratio G. For diagnostic purposes, the value of k(0) could readily be modulated by changing the temperature, due to the substantial Delta H degrees associated with the binding equilibrium. Applied to gene therapy, our results stress the need for appropriate control of the binding constant and added amount of the gene-targeting agent, to meet the varying conditions (ionic strength, presence of competing DNA-binding molecules) found in the cell.

Full Text

The Full Text of this article is available as a PDF (188.3 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Boffa L. C., Morris P. L., Carpaneto E. M., Louissaint M., Allfrey V. G. Invasion of the CAG triplet repeats by a complementary peptide nucleic acid inhibits transcription of the androgen receptor and TATA-binding protein genes and correlates with refolding of an active nucleosome containing a unique AR gene sequence. J Biol Chem. 1996 May 31;271(22):13228–13233. doi: 10.1074/jbc.271.22.13228. [DOI] [PubMed] [Google Scholar]
  2. Bonham M. A., Brown S., Boyd A. L., Brown P. H., Bruckenstein D. A., Hanvey J. C., Thomson S. A., Pipe A., Hassman F., Bisi J. E. An assessment of the antisense properties of RNase H-competent and steric-blocking oligomers. Nucleic Acids Res. 1995 Apr 11;23(7):1197–1203. doi: 10.1093/nar/23.7.1197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Carlsson C., Jonsson M., Nordén B., Dulay M. T., Zare R. N., Noolandi J., Nielsen P. E., Tsui L. C., Zielenski J. Screening for genetic mutations. Nature. 1996 Mar 21;380(6571):207–207. doi: 10.1038/380207a0. [DOI] [PubMed] [Google Scholar]
  4. Chan P. P., Lin M., Faruqi A. F., Powell J., Seidman M. M., Glazer P. M. Targeted correction of an episomal gene in mammalian cells by a short DNA fragment tethered to a triplex-forming oligonucleotide. J Biol Chem. 1999 Apr 23;274(17):11541–11548. doi: 10.1074/jbc.274.17.11541. [DOI] [PubMed] [Google Scholar]
  5. Daban J. R. Physical constraints in the condensation of eukaryotic chromosomes. Local concentration of DNA versus linear packing ratio in higher order chromatin structures. Biochemistry. 2000 Apr 11;39(14):3861–3866. doi: 10.1021/bi992628w. [DOI] [PubMed] [Google Scholar]
  6. Egholm M., Buchardt O., Christensen L., Behrens C., Freier S. M., Driver D. A., Berg R. H., Kim S. K., Norden B., Nielsen P. E. PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen-bonding rules. Nature. 1993 Oct 7;365(6446):566–568. doi: 10.1038/365566a0. [DOI] [PubMed] [Google Scholar]
  7. Gambacorti-Passerini C., Mologni L., Bertazzoli C., le Coutre P., Marchesi E., Grignani F., Nielsen P. E. In vitro transcription and translation inhibition by anti-promyelocytic leukemia (PML)/retinoic acid receptor alpha and anti-PML peptide nucleic acid. Blood. 1996 Aug 15;88(4):1411–1417. [PubMed] [Google Scholar]
  8. Giesen U., Kleider W., Berding C., Geiger A., Orum H., Nielsen P. E. A formula for thermal stability (Tm) prediction of PNA/DNA duplexes. Nucleic Acids Res. 1998 Nov 1;26(21):5004–5006. doi: 10.1093/nar/26.21.5004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Holbrook J. A., Capp M. W., Saecker R. M., Record M. T., Jr Enthalpy and heat capacity changes for formation of an oligomeric DNA duplex: interpretation in terms of coupled processes of formation and association of single-stranded helices. Biochemistry. 1999 Jun 29;38(26):8409–8422. doi: 10.1021/bi990043w. [DOI] [PubMed] [Google Scholar]
  10. Jensen K. K., Orum H., Nielsen P. E., Nordén B. Kinetics for hybridization of peptide nucleic acids (PNA) with DNA and RNA studied with the BIAcore technique. Biochemistry. 1997 Apr 22;36(16):5072–5077. doi: 10.1021/bi9627525. [DOI] [PubMed] [Google Scholar]
  11. Nielsen P. E., Egholm M., Berg R. H., Buchardt O. Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science. 1991 Dec 6;254(5037):1497–1500. doi: 10.1126/science.1962210. [DOI] [PubMed] [Google Scholar]
  12. Nielsen P. E. Peptide nucleic acids as therapeutic agents. Curr Opin Struct Biol. 1999 Jun;9(3):353–357. doi: 10.1016/S0959-440X(99)80047-5. [DOI] [PubMed] [Google Scholar]
  13. Nielsen P. E. Sequence-selective DNA recognition by synthetic ligands. Bioconjug Chem. 1991 Jan-Feb;2(1):1–12. doi: 10.1021/bc00007a001. [DOI] [PubMed] [Google Scholar]
  14. Peyret N., Seneviratne P. A., Allawi H. T., SantaLucia J., Jr Nearest-neighbor thermodynamics and NMR of DNA sequences with internal A.A, C.C, G.G, and T.T mismatches. Biochemistry. 1999 Mar 23;38(12):3468–3477. doi: 10.1021/bi9825091. [DOI] [PubMed] [Google Scholar]
  15. Ratilainen T., Holmén A., Tuite E., Nielsen P. E., Nordén B. Thermodynamics of sequence-specific binding of PNA to DNA. Biochemistry. 2000 Jul 4;39(26):7781–7791. doi: 10.1021/bi000039g. [DOI] [PubMed] [Google Scholar]
  16. Ren J., Chaires J. B. Sequence and structural selectivity of nucleic acid binding ligands. Biochemistry. 1999 Dec 7;38(49):16067–16075. doi: 10.1021/bi992070s. [DOI] [PubMed] [Google Scholar]
  17. Rufer N., Dragowska W., Thornbury G., Roosnek E., Lansdorp P. M. Telomere length dynamics in human lymphocyte subpopulations measured by flow cytometry. Nat Biotechnol. 1998 Aug;16(8):743–747. doi: 10.1038/nbt0898-743. [DOI] [PubMed] [Google Scholar]
  18. Weiler J., Gausepohl H., Hauser N., Jensen O. N., Hoheisel J. D. Hybridisation based DNA screening on peptide nucleic acid (PNA) oligomer arrays. Nucleic Acids Res. 1997 Jul 15;25(14):2792–2799. doi: 10.1093/nar/25.14.2792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Wittung P., Nielsen P., Nordén B. Extended DNA-recognition repertoire of peptide nucleic acid (PNA): PNA-dsDNA triplex formed with cytosine-rich homopyrimidine PNA. Biochemistry. 1997 Jul 1;36(26):7973–7979. doi: 10.1021/bi963136b. [DOI] [PubMed] [Google Scholar]
  20. Zhu J., Wartell R. M. The relative stabilities of base pair stacking interactions and single mismatches in long RNA measured by temperature gradient gel electrophoresis. Biochemistry. 1997 Dec 9;36(49):15326–15335. doi: 10.1021/bi9716783. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES