Homopyrimidine peptide nucleic acids (PNAs) form loop structures when binding to complementary double-stranded DNA by strand displacement, and we now show that RNA polymerase recognizes these and ...initiates RNA transcription from PNA/double-stranded DNA strand displacement complexes at an efficiency comparable to that of the strong Escherichia coli lacUV5 promoter. Thus PNA targets can be considered as artificial promoters controlled positively by the corresponding PNA as a transcription factor. Our results have implications for the mechanism of action of RNA polymerase and suggest the use of PNA as specific gene activating reagents and drugs.
Polymers capable of recognizing adenine in aqueous medium were developed by the molecular imprinting technique using methacrylic acid and 4(5)-vinylimidazole as comonomers with ethylene glycol ...dimethacrylate as the cross-linking agent under different polymerization conditions. The affinity of these polymers for adenine and other nucleotide bases was compared. The association constant for the binding of adenine to the polymer is calculated to be 4.3 x 10(3)M(-1). Furthermore, binding of adenosine 5'-triphosphate (ATP) to the polymers was evaluated, and an enhanced binding compared to adenine was observed. The binding of ATP is pH dependent, with the maximum around pH 3. Results have been explained based on the hydrogen bonding and ionic interactions between ATP and the ligands on the polymer matrix. The catalytic effect of these polymers for the hydrolysis of ATP is briefly discussed.
A novel method for determination of psoralen photo-cross-linking sites in double-stranded DNA is described, which is based on a pronounced inhibition of Bal31 exonuclease activity by psoralen-DNA ...interstrand cross-links. The results using a 51 base pair fragment of plasmid pUC19 and a 346 base pair fragment of pBR322 show that 5'-TA sequences are preferred cross-linking sites compared to 3'-TA sequences. They also indicate that sequences flanking the 5'-TA site influence the cross-linking efficiency at the site. The DNA photo-cross-linking by 4,5',8-trimethylpsoralen and 8-methoxypsoralen was analyzed, and these two psoralens showed identical site specificity. The 5'-TA preference is rationalized on the basis of the local DNA structure in terms of the pi-pi electronic interaction between the thymines and the intercalated psoralens, as well as on the base tilt angles of the DNA.
It was recently found that polyamide nucleic acid (PNA) analogues consisting of thymines attached to an aminoethylglycine backbone bind strongly and sequence-selectively to adenine sequences of ...oligonucleotides and double-stranded DNA Nielsen, P. E., Egholm, M., Berg, R. H. \& Buchardt, O. (1991) Science 254, 1497-1500. It was concluded that the binding to double-stranded DNA was accomplished via strand displacement, in which the PNA bound to the Watson-Crick complementary adenine-containing strand, whereas the thymine-containing strand was extruded in a virtually single-stranded conformation. This model may provide a general way in which to obtain sequence-specific recognition of any sequence in double-stranded DNA by Watson-Crick hydrogen-bonding base-pair recognition, and it is thus paramount to rigorously establish this binding mode for synthetic DNA-binding ligands. We now report such results from electron microscopy. Furthermore, we show that binding of PNA to closed circular DNA results in unwinding of the double helix corresponding to approximately one turn of the double helix per 10 base pairs. The DNA·PNA complex, which is formed at low salt concentration (only a small portion of DNA molecules show complex formation at NaCl concentration higher than 40 mM), is exceptionally kinetically stable and cannot be dissociated by increasing salt concentration up to 500 mM.
Plasmlds containing double-stranded 10-mer PNA (peptide nucleic acid chimera) targets proximally flanked by two restriction enzyme sites were challenged with the complementary PNA or PNAs having one ...or two mismatches, and the effect on the restriction enzyme cleavage of the flanking sites was assayed. The following PNAs were used: T10-LysNH2, T5CT4-LysNH2 and T2CT2CT4-LysNH2 and the corresponding targets cloned Into pUC 19 were flanked by SamH1, Sa1 or Pst sites, respectively. In all cases It was found that complete inhibition of restriction enzyme cleavage was obtained with the complementary PNA, a significantly reduced effect was seen with a PNA having one mismatch, and no effect was seen with a PNA having two mismatches. These results show that PNA can be used as sequence specific blockers of DNA recognizing proteins.
A novel method for sequence specific double strand DNA cleavage using PNA (peptide nucleic acid) targeting Is described. Nuclease S1 digestion of double stranded DNA gives rise to double strand ...cleavage at an occupied PNA strand displacement binding site, and under optimized conditions complete cleavage can be obtalned. The efficiency of this cleavage is more than 10 fold enhanced when a tandem PNA site Is targeted, and additionally enhanced If this site Is In trans rather than in cis orientation. Thus In effect, the PNA targeting makes the single strand specific nuclease S1 behave like a pseudo restriction endonuclease.
4-Azido, 4-amino, 4-amido and 4-alkoxy compounds related to the lignans podophyllotoxin and 4'-demethylepipodophyllotoxin have been synthesized, and their structures elucidated. The Ritter reaction ...was shown to be useful in the preparation of the 4-amido compounds with the required stereochemistry. A preparative method for 4-chloro-4-deoxypicrophyllotoxin, for which all earlier synthetic attempts resulted in the two dehydrated compounds, alpha- and beta-apopicropodophyllotoxin, was developed. Supplementary preliminary studies of the biological activities of some of the compounds were performed. All compounds had pronounced inhibitory effect on the in vitro growth of human cervical cancer cells and TC-mouse cells with 4-amino-4-deoxypodophyllotoxin and 4-azido-4-deoxypodophyllotoxin showing the highest activity. Alkaline elution studies indicate that the toxicity of the 4'-demethoxy derivatives is due to protein-mediated DNA nicking. None of the compounds were found to have antiviral effect against herpes simplex type 2 (HSV-2), human immunodeficiency (HIV), and cytomegalovirus (CMV) in doses not toxic to the cells.
Peptide nucleic acids (PNAs) are novel DNA mimics in which the sugar-phosphate backbone has been replaced with a backbone based on amino acids. PNAs exhibit sequence-specific binding to DNA and RNA ...with higher affinities and specificities than unmodified DNA. They are resistant to nuclease and protease attack in serum and cellular extracts and, thus, appear very promising as diagnostic and biomolecular probes, and possibly as antisense and antigene drugs.
The Interactions of two representative mixed-sequence (one with an AT-stretch) PNA - DNA duplexes (10 or 15 base-pairs) and a PNA2/DNA triplex with the DNA binding reagents distamycln A, ...4',6-diamidlno-2-phenylindole (DAPI), ethldium bromide, 8-methoxy-psoralen and the ▵ and ▵ enantiomers of Ru(phen)2-dppz2− have been Investigated using optical spectro-scopic methods. The behaviour of these reagents versus two PNA- PNA duplexes has also been Investigated. With triple helical poly(dA)/(H-T10-Lys-NH2)2 no significant intercalate binding was detected for any of the DNA Intercalators, whereas DAPI, a DNA minor groove binder, was found to exhibit a circular dlchroism with a positive sign and amplitude consistent with minor groove binding. Similarly, a PNA-DNA duplex containing a central AATA motif, a typical minor groove binding site for the DNA minor groove binders dlsta-mycin A and DAPI, showed binding for both of these drugs, though with strongly reduced affinity. No important interactions were found for any of the ligands with a PNA - DNA duplex consisting of a ten base-pair mixed purine-pyrlmldlne sequence with only two AT base-pairs in the centre. Nor did any of the ligands show any detectable binding to the PNA-PNA duplexes (one containing an AATT motif). Various PNA derivatives with extentions of the backbone, believed to increase the flexibility of the duplex to opening of an intercalation slot, were tested for intercalation of ethidium bromide or 8-methoxypsoralen into the mixed sequence PNA-DNA duplex, however, without any observation of improved binding. The importance of the Ionic contribution of the deoxyribose phosphate backbone, versus interactions with the nucleobases, for drug binding to DNA is discussed in the light of these findings.
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