Solid‐Phase synthesis of peptide nucleic acids

Christensen, Leif; Fitzpatrick, Richard; Gildea, Brian; Petersen, Kenneth H.; Hansen, Henrik F.; Koch, Troels; Egholm, Michael; Buchardt, Ole; Nielsen, Peter E.; Coull, James; Berg, Rolf H.

doi:10.1002/psc.310010304

Cited by 368 publications

(330 citation statements)

References 12 publications

Supporting

Mentioning

318

Contrasting

Unclassified

Order By: Relevance

“…PNA oligomers were synthesized from N-Boc protected (2-aminoethyl)glycine monomers (25). The AQ-containing monomers are shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Peptide nucleic acid–DNA duplexes: Long range hole migration from an internally linked anthraquinone

Armitage

Ly²,

Koch³

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

The discovery that peptide nucleic acids (PNA) mimic DNA and RNA by forming complementary duplex structures following Watson-Crick base pairing rules opens fields in biochemistry, diagnostics, and medicine for exploration. Progress requires the development of modified PNA duplexes having unique and well defined properties. We find that anthraquinone groups bound to internal positions of a PNA oligomer intercalate in the PNA-DNA hybrid. Their irradiation with near-UV light leads to electron transfer and oxidative damage at remote GG doublets on the complementary DNA strand. This behavior mimics that observed in related DNA duplexes and provides the first evidence for long range electron (hole) transport in PNA-DNA hybrid. Analysis of the mechanism for electron transport supports hole hopping.Peptide nucleic acid (PNA) oligomers are DNA͞RNA analogs (see Fig. 1) in which the natural sugar-phosphate backbone is replaced by a synthetic peptide backbone (1). PNA oligomers that contain purine and pyrimidine nucleobases hybridize with complementary DNA and RNA strands to form right-handed, double-helical complexes according to the Watson-Crick rules of hydrogen bond-mediated base pair formation (2). Although much has been learned about the structural (3) and thermodynamic (4) factors involved in hybridization, little is known about the chemical reactivity of PNA͞DNA hybrids. It is crucial to understand how PNA͞DNA hybrids mimic the reactions and functions of duplex DNA. Of immediate importance for their application as clinical diagnostic agents is investigation of the conductivity of DNA and its PNA analogs (5, 6).DNA must balance the dual requirements of chemical stability and ease of transcription and replication (7). It is clear that DNA is far from inert toward a variety of different reactive species, particularly oxidizing agents. Oxidative damage to DNA produced by normal metabolism, deep-UV laser irradiation (8), gamma rays (9), or pulse radiolysis (10) accumulates at guanine residues, an effect attributed to one-dimensional migration of a radical cation (''hole'') along the DNA helix (11). Both the low oxidation potential and reactivity of the guanine radical cation contribute to the effectiveness of guanine as a trap for the migrating hole. Because guanine lesions may be the major cause of mutations (12), intense attention is focused on understanding the conductivity properties of DNA to elucidate the mechanisms by which migration of oxidative damage occurs (13). In this regard, the recent reports by Barton and coworkers are particularly important (14, 15). They describe a system consisting of a rhodium complex that is covalently linked to one end of a DNA duplex. Irradiation caused damage to the DNA more than 30 Å from where the complex was presumed to intercalate. This observation opens up exciting opportunities to study the factors that control hole migration in nucleic acids.Photosensitizers often react with nucleic acids by single electron transfer to oxidize a base. Recent findings reveal that the...

show abstract

“…PNA oligomers were synthesized from N-Boc protected (2-aminoethyl)glycine monomers (25). The AQ-containing monomers are shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Peptide nucleic acid–DNA duplexes: Long range hole migration from an internally linked anthraquinone

Armitage

Ly²,

Koch³

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

show abstract

“…The bis-PNAs PNA1021 [Biotin-(eg1) 3 -TTJ TTJ TTTTLys-aha-Lys-aha-Lys-TTTT CTT CTT-Lys-NH 2 ] (23) and PNA3593 [Dig-(eg1) 3 -(DMLys) 3 -TJT JTT TJT T-(eg1) 3 -TTC TTT CTC T-Gly-NH 2 (DMLys = e-N,N-dimethyl lysine; aha = 6-aminohaxanoic acid, eg1 = 8-amino-3,6-oxaoctanoic acid)] were synthesized using solid phase Boc chemistry according to published procedures (23,33,34). Digoxigenin was conjugated to the bis-PNA in solution using Digoxigenin NHS-ester.…”

Section: Methodsmentioning

confidence: 99%

DNA supercoiling enhances cooperativity and efficiency of an epigenetic switch

Nørregaard

Andersson

Sneppen

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Bacteriophage λ stably maintains its dormant prophage state but efficiently enters lytic development in response to DNA damage. The mediator of these processes is the λ repressor protein, CI, and its interactions with λ operator DNA. This λ switch is a model on the basis of which epigenetic switch regulation is understood. Using single molecule analysis, we directly examined the stability of the CI-operator structure in its natural, supercoiled state. We marked positions adjacent to the λ operators with peptide nucleic acids and monitored their movement by tethered particle tracking. Compared with relaxed DNA, the presence of supercoils greatly enhances juxtaposition probability. Also, the efficiency and cooperativity of the λ switch is significantly increased in the supercoiled system compared with a linear assay, increasing the Hill coefficient. The decision between the dormant state (the lysogenic state) and the vegetative state (the lytic state) made by bacteriophage λ upon infection of an Escherichia coli was the first epigenetic switch to be deciphered (1) and continues to provide insights into biological processes (2, 3). Once the lysogenic state is established, it is exceptionally stable and departure in the absence of the DNA damage sensing system is nearly always caused by mutation (4-6). Maintenance of lysogeny is mediated by the λ repressor protein, CI, and its formation of a complex with phage DNA. The CI protein binds at two regulatory regions called operator right (OR) and operator left (OL), located about 2.3 kbp apart on the phage genome. Each operator is a constellation of three adjacent subsites that each bind dimers of CI in a hierarchical manner (1, 7). In a lysogenic cell, CI prevents lytic growth by directly repressing the lytic promoters pR and pL. This repression is enhanced by longrange cooperative interactions between CI dimers bound to the OL1-OL2 and OR1-OR2 regions, thus looping the DNA that lies between them (8) (Fig. 1 A and B). CI-mediated DNA looping brings OR3 in proximity to OL3. This juxtaposition allows a CI dimer bound to the strong OL3 to facilitate another CI dimer to bind to the intrinsically weak OR3, thus reducing the concentration of CI necessary to occupy OR3. In this manner CI can autoregulate its own expression by binding to OR2 (activation) or by binding to OR3 (repression). This autoregulation is crucial for the phage to maintain repression while preventing excessive accumulation of CI. When DNA in the lysogen is damaged, the bacterial DNA-damage-sensing system is triggered, leading to CI inactivation by self-cleavage (9) and to an efficient switch to the lytic state, eventually leading to production of progeny phage and cell death (1).The switch from the lysogenic to the lytic state must be decisive. Partial entry into vegetative growth may kill the host without producing a full burst of progeny phage. Because the in vivo state of DNA is supercoiled (10), we sought to examine protein-mediated DNA looping on native, supercoiled DNA. In vitro single molecule exper...

show abstract

“…The PNAs were synthesized by solid-phase synthesis as described elsewhere (10) and the carboxylic acid derivatives of the dyes were coupled to the terminal residue as follows. Ten to 20 mg t-Boc protected resinbound PNA (Ϸ0.15 mmol/g) was swelled overnight in dichloromethane.…”

Section: Probe Synthesismentioning

confidence: 99%

“…with an amino linker or by coupling a carboxylic acid derivative of the dye directly to the terminal PNA residue during solid-phase synthesis (10).…”

mentioning

confidence: 99%

Light-Up Probes: Thiazole Orange-Conjugated Peptide Nucleic Acid for Detection of Target Nucleic Acid in Homogeneous Solution

Svanvik

Westman

Wang³

et al. 2000

Analytical Biochemistry

237

147

View full text Add to dashboard Cite

We have constructed light-up probes for nucleic acid detection. The light-up probe is a peptide nucleic acid (PNA) oligonucleotide to which the asymmetric cyanine dye thiazole orange (TO) is tethered. It combines the excellent hybridization properties of PNA and the large fluorescence enhancement of TO upon binding to DNA. When the PNA hybridizes to target DNA, the dye binds and becomes fluorescent. Free probes have low fluorescence, which may increase almost 50-fold upon hybridization to complementary nucleic acid. This makes the light-up probes particularly suitable for homogeneous hybridization assays, where separation of the bound and free probe is not necessary. We find that the fluorescence enhancement upon hybridization varies among different probes, which is mainly due to variations in free probe fluorescence. For eight probes studied the fluorescence quantum yield at 25°C in the unbound state ranged from 0.0015 to 0.08 and seemed to depend mainly on the PNA sequence. The binding of the light-up probes to target DNA is highly sequence specific and a single mismatch in a 10-mer target sequence was readily identified.

show abstract

Solid‐Phase synthesis of peptide nucleic acids

Cited by 368 publications

References 12 publications

Peptide nucleic acid–DNA duplexes: Long range hole migration from an internally linked anthraquinone

Peptide nucleic acid–DNA duplexes: Long range hole migration from an internally linked anthraquinone

DNA supercoiling enhances cooperativity and efficiency of an epigenetic switch

Light-Up Probes: Thiazole Orange-Conjugated Peptide Nucleic Acid for Detection of Target Nucleic Acid in Homogeneous Solution

Contact Info

Product

Resources

About