DNA analogues are currently being intensely investigated owing to their potential as gene-targeted drugs. Furthermore, their properties and interaction with DNA and RNA could provide a better ...understanding of the structural features of natural DNA that determine its unique chemical, biological and genetic properties. We recently designed a DNA analogue, PNA, in which the backbone is structurally homomorphous with the deoxyribose backbone and consists of N-(2-aminoethyl)glycine units to which the nucleobases are attached. We showed that PNA oligomers containing solely thymine and cytosine can hybridize to complementary oligonucleotides, presumably by forming Watson-Crick-Hoogsteen (PNA)2-DNA triplexes, which are much more stable than the corresponding DNA-DNA duplexes, and bind to double-stranded DNA by strand displacement. We report here that PNA containing all four natural nucleobases hybridizes to complementary oligonucleotides obeying the Watson-Crick base-pairing rules, and thus is a true DNA mimic in terms of base-pair recognition.
Although the importance of the nucleobases in the DNA double helix is well understood, the evolutionary significance of the deoxyribose phosphate backbone and the contribution of this chemical entity ...to the overall helical structure and stability of the double helix is not so clear. Peptide nucleic acid (PNA) is a DNA analogue with a backbone consisting of N-(2-aminoethyl)glycine units (Fig. 1) which has been shown to mimic DNA in forming Watson-Crick complementary duplexes with normal DNA. Using circular dichroism spectroscopy we show here that two complementary PNA strands can hybridize to one another to form a helical duplex. There is a seeding of preferred chirality which is induced by the presence of an L- (or D-) lysine residue attached at the carboxy terminus of the PNA strand. These results indicate that a (deoxy)ribose phosphate backbone is not an essential requirement for the formation of double helical DNA-like structures in solution.
Oligonucleotides that specifically recognize messenger RNA or double-stranded DNA present unique opportunities for inhibiting protein synthesis (the antisense approach) or for modulating gene ...expression, e.g., via triple helix formation. The deoxyribose phosphate backbone of DNA has been modified in a number of ways to increase nuclease stability and cell membrane permeability, assuming that major changes would be deleterious to the "DNA hybridization properties". Consequently, derivatives such as mono- or dithiophosphates, methyl phosphonates, borano phosphates, etc., as well as formacetal, carbamate, and siloxane, or dimethylenethio-, -sulfoxido-, and -sulfono-linked, species were prepared. The synthesis of peptides is more versatile than oligonucleotide synthesis, allowing the facile design of an achiral backbone and relatively large-scale production. We therefore designed peptide nucleic acids (PNA), i.e., molecules where the individual nucleobases were linked to an achiral peptide backbone.
The synthesis and DNA binding properties of bis-PNA (peptide nucleic acid) are reported. Two PNA segments each of seven nucleobases in length were connected in a continuous synthesis via a flexible ...linker composed of three 8-amino-3,6-dioxaoctanoic acid units. The sequence of the first strand was TCTCTTT (C- to N-terminal), while the second strand was TTTCTCT or TTTJTJT, where J is pseudoisocytosine. These bis-PNAs form triple-stranded complexes of somewhat higher thermal stability than monomeric PNA with complementary oligonucleotides and the thermal melting transition shows very little hysteresis. When the J base is placed in the strand parallel to the DNA complement (‘Hoogsteen strand’), the DNA binding was pH independent. The bis-PNAs were also superior to monomeric PNAs for targeting double-stranded DNA by strand invasion.
The effects of PNA (peptide nucleic acid) bound to double-stranded (ds) DNA targets positioned downstream from phage T3 or T7 promoters in pBluescriptKS+ derived plasmids on transcription by RNA ...polymerases T3 or T7 have been studied. The dsDNA targets A10, 5'-A5GA4 or 5'-A2GA2GA4, and the corresponding PNAs T10, T5CT4 and T2CT2CT4 were used and the target-PNA strand displacement complexes were performed in low-salt buffer, since PNA does not bind efficiently to ds DNA in higher salt than 50 mM. It is shown that transcription elongation is arrested at the target site with PNA bound to the template strand, whereas only a marginal effect is observed with PNA bound to the non-template strand. With PNA T10, transcription arrest occurs at the first base of the PNA-binding site, while the arrest with the PNA T5CT4 takes place 2-3 nt inside the PNA binding site. In the case of PNA T2CT2CT4 the arrest is less efficient and occurs at the last 1-3 nt of the binding site. Transcription arrest was also shown for PNAs T6 and T8, although with a much lower efficiency. These results show that efficient transcription elongation arrest can be obtained by PNA targeting of the template DNA strand.
Peptide nucleic acid (PNA) is an achiral nucleic acid mimic with a backbone consisting of partly flexible aminoethyl glycine units. By replacing the aminoethyl portion of the backbone by an amino ...cyclohexyl moiety, either in the (S, S) or the (R, R) configuration, we have synthesized conformationally constrained PNA residues. PNA oligomers containing (S, S)‐cyclohexyl residues were able to form hybrid complexes with DNA or RNA, with little effect on the thermal stability (Tm = 1°C per (S, S) unit, depending on their number and the sequence). In contrast, incorporation of the (R, R) isomer resulted in a drastic decrease in the stability of the PNA‐DNA (or RNA) complex (Tm = −8°C per (R, R) unit). In PNA‐PNA duplexes, however, the (R, R)‐ and (S, S)‐cyclohexyl residues only exerted a minor effect on the stability, and the complexes formed with the two isomers are of opposite handedness, as evidenced from circular dichroism spectroscopy. In some cases the introduction of a single (S, S) residue in a PNA 15‐mer improves its sequence specificity for DNA or RNA. From the thermal stabilities and molecular modeling based on the solution structure of a PNA‐DNA duplex determined by NMR techniques, we conclude that the right‐handed helix can accommodate the (S, S) isomer more easily than the (R, R) isomer. Thermodynamic measurements of H and S upon PNA‐DNA duplex formation show that the introduction of an (S, S)‐cyclohexyl unit in the PNA does indeed decrease the entropy loss, indicating a more conformationally constrained structure. However, the more favorable entropic contribution is balanced by a reduced enthalpic gain, indicating that the structure constrained by the cyclohexyl group is not so well suited for DNA hybridization.
Peptide nucleic acid (PNA) oligomers containing one or more (S,S)‐cyclo‐hexyl thymine units (right) hybridize as efficiently as regular PNA to complementary DNA or RNA oligomers. The binding occurs with decreased entropy loss but also with similarly decreased enthalpy gain compared to normal PNA, thereby emphasizing the difficulties in obtaining conformationally constrained ligands that adopt the most active binding conformation.
The photoreactions of 9-6-(4-azidobenzamido)hexylaminoacridine (AHA) and 9-6-(2-diazocyclopentadienylcarbonyloxy)hexylaminoacridine (DHA) with double stranded DNA result in formation of single strand ...nicks and alkali labile sites (adducts) with an efficiency of 6 x 10(-3) nicks per AHA and 3 x 10(-2) nicks per DHA molecule. The alkali dependent DNA cleavage by AHA shows a pronounced A+G preference whereas that by DHA is practically sequence independent. In the presence of diacridines, however, DHA exhibits a preference for cleavage at guanosines. These DNA photocleaving reagents could be useful for DNA photofootprinting and photosequencing.
Nitrobenzamido ligands linked to the DNA intercalator 9-aminoacridine via poly(methylene) chains induce single-strand nicks in DNA upon irradiation with long-wavelength ultraviolet light (lambda ...greater than or equal to 300 nm). Optimal photocleavage activity was found for the reagent 9-6-(4-nitrobenzamido)hexylamino-acridine. Removal of the acridinyl ligand or changing the position of the nitro group from the 4- to the 2-position caused a 10-fold decrease in photocleavage efficiency, whereas a change to the 3-position caused a 30-fold reduction. The DNA cleavage was 5-fold enhanced by subsequent piperidine treatment and showed some sequence dependency with predominant cleavage at G and T residues. Furthermore, significant differences in cleavage preference were observed when the poly(methylene) linker length was changed.
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