A 'retro-inverso' PNA: structural implications for DNA and RNA binding.

'Retro-inverso' peptide nucleic acid (PNA) monomers of thymine (T*: N-(amidomethyl)-N-(N1-thyminyl-acetyl)-beta-alanyl) (and adenine) have been prepared and introduced in PNA oligomers. A homo 'retro-inverso' T*8 PNA was found not to hybridize to a complementary DNA or RNA oligonucleotide, whereas introduction of one retro-inverso thymine unit into the middle of a normal PNA 15-mer resulted in a c.a. 8 degrees C destabilization of the complex of this oligomer with a complementary DNA or RNA oligomer. In an effort to compensate for the structural nucleobase 'phase-shift' caused by the T* monomer by also introducing a beta-alanine monomer it is concluded that the effect of the T* backbone is -7 degrees C when hybridizing to DNA and -4.5 degrees C when hybridizing to RNA. Nonetheless, the T* unit shows good sequence discrimination comparable to that of normal PNA. Molecular dynamics simulations indicate an unfavourable conformation of the backbone amide carbonyl group resulting in reduced interaction with the aqueous medium and an 'electrostatic clash' with the carbonyl of the nucleobase linker. These results show that a simple inversion of an amide bond in the PNA backbone has a dramatic, and hardly predictable, effect on the DNA mimicking properties of the oligomer.

Specificity of antibodies raised against a specific phosphoromonothioate oligonucleotide sequence.

The phosphoromonothioate oligonucleotide HPV (human papilloma virus) sequence (monothioate HPV) 5'-TTG,CTT,CCA,TCT,TCC,TCG,TC-3' was photocoupled via three different sites (the 5'-end, the 3'-end and the midpoint) to PPD (purified protein derivative) and OA (ovalbumin), and the three types of conjugates (5'-HPV/carrier, 3'-HPV/carrier and midpoint-HPV/carrier) were used for the immunization of mice. Furthermore, a group of mice were immunized with the HPV sequence alone. No detectable antibody response against the monothioate HPV oligonucleotide was seen in mice receiving only the unconjugated monothioate HPV sequence. The OA-coupled monothioate HPV sequence also failed to elicit a detectable antibody response against the monothioate HPV oligonucleotide. However the PPD-conjugated monothioate HPV sequences induced a significant anti-monothioate HPV antibody response in BCG (bacille Calmette Guérin)-primed mice, a result that must be ascribed to the effect of using PPD as a carrier in BCG-primed mice. The antisera from all groups were tested on plates coated with the corresponding OA conjugates. By far the strongest response was obtained in mice receiving the HPV sequence coupled at the midpoint position. Further, all three groups of antisera obtained by immunizing with the different PPD conjugates were tested on microtiter plates coated with one of the three different OA conjugates. The antisera differed in their response depending on which OA conjugate was used for coating of the plate. Again, the midpoint-HPV/PPD antiserum showed the highest response, and this conjugate apparently represents the most efficient immunogen. Results from inhibition experiments with various relevant analogs of the monothiate HPV sequence showed that the three antiserum pools contained antibodies predominantly directed against the conformation of the monothioate backbone structure, but that at least a subpopulation of the antibodies recognized structures, which depended on the specific HPV base sequence.

Molecular imprinting approach for the recognition of adenine in aqueous medium and hydrolysis of adenosine 5'-triphosphate.

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.

Solid-phase synthesis of peptide nucleic acids.

Peptide nucleic acids (PNA) were synthesized by a modified Merrifield method using several improvements. Activation by O-[benzotriazol-1-yl]-1,1,3,3-tetramethyluronium hexafluorophosphate in combination with in situ neutralization of the resin allowed efficient coupling of all four Boc-protected PNA monomers within 30 min. HPLC analysis of the crude product obtained from a fully automated synthesis of the model PNA oligomer H-CGGACTAAGTCCATTGC-Gly-NH2, indicated an average yield per synthetic cycle of 97.1%. N1-benzyloxycarbonyl-N6(3)-methylimidazole triflate substantially outperformed acetic anhydride as a capping reagent. The resin-bound PNAs were successfully cleaved by the 'low-high' trifluoromethanesulphonic acid procedure.

Efficient pH-independent sequence-specific DNA binding by pseudoisocytosine-containing bis-PNA.

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.

Interactions of DNA binding ligands with PNA-DNA hybrids.

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 distamycin A, 4',6-diamidino-2-phenylindole (DAPI), ethidium bromide, 8-methoxy-psoralen and the delta and lambda enantiomers of Ru(phen)2-dppz2+ have been investigated using optical spectroscopic 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 intercalative binding was detected for any of the DNA intercalators, whereas DAPI, a DNA minor groove binder, was found to exhibit a circular dichroism 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 distamycin 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-pyrimidine 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.

Sequence-specific transcription arrest by peptide nucleic acid bound to the DNA template strand.

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.

Evidence for (PNA)2/DNA triplex structure upon binding of PNA to dsDNA by strand displacement.

The binding of PNA (peptide nucleic acid) T2CT2CT4-LysNH2 to the double-stranded DNA target 5'-A2GA2GA4 was studied by KMnO4 and dimethylsulfate (DMS) probing. It is found that upon sequence-specific strand displacement binding of the PNA to the dsDNA target concomitant protection of the N-7 of guanines within the target takes place. It is furthermore shown that the binding of this PNA is more efficient at pH 5.5 than at pH 6.5 and very inefficient at pH 7.5. These results clearly indicate that C+G Hoogsteen base pairing is present and important for binding and that the strand displacement complex therefore involves a PNA.DNA-PNA triplex.

Structural characterization of PNA-DNA duplexes by NMR. Evidence for DNA in a B-like conformation.

The nucleic acid analogues PNA (peptide nucleic acids) hybridize with DNA of complementary sequence. The solution structures of two PNA-DNA duplexes, H-(GCTATGTC)-NH2.d(GACATAGC) and H-(GTAGATCACT)-NH2.d(AGTGATCTAC), have been studied by 1H NMR. It was found that the PNA-DNA hybrids are base paired by hydrogen bonds, most likely of the Watson-Crick type. From two-dimensional NOESY and COSY results it is concluded that the DNA strand in the PNA-DNA complex adopts a B-like structure with the deoxyribose sugars in the C2'-endo conformation.

Monoclonal antibodies to thioguanine: influence of coupling position on fine specificity.

Thioguanine derivatives with reactive ester groups at positions 6, 7, or 9 of the purine ring were synthesized and coupled to a protein carrier. The purified protein derivative of tuberculin was used as the carrier for immunizing bacillus Calmette-Guerin primed mice. This led to high antibody titers against the homologously coupled hapten, and spleen cells from the immunized mice were used to produce monoclonal antibodies against thioguanine. All monoclonal antibodies were selected for their ability to recognize free thioguanine and were analyzed for their fine specificity by inhibition experiments with a panel of thiopurine derivates. The specificity of the monoclonal antibodies showed a strong dependence on the coupling position of the thioguanine. Within each group of monoclonal antibodies, raised against one of the three different conjugates, there was a high degree of heterogeneity, with antibodies differing in their binding according to the substitution on the thioguanine analogues used in the inhibition experiments. This panel of antibodies may be used for quantitative assays of thiopurines and their metabolites in patients undergoing treatment with thioguanine, 6-mercaptopurine, and azathioprine.

Differential blocking of coagulation-activating pathways of Limulus amebocyte lysate.

The coagulation of Limulus amebocyte lysate (LAL) can be activated through two pathways, one initiated by endotoxin and the other by beta-glucans. The two pathways join at the step of activation of the proclotting enzyme. We report here that the endotoxin-activated pathway can be differentially inhibited by two methods in a Limulus enzyme-linked immunosorbent assay (ELISA), either by the combined use of dimethyl sulfoxide and polymyxin B or by a monoclonal antibody against Limulus factor C. LAL reactivities to 10 different endotoxin preparations could be inhibited by the former method by a factor of 10(4) to 10(6) and could be blocked almost totally by the latter method, irrespective of the source of endotoxin. The sensitivity of the assay was approximately 50 pg/ml both for curdlan from Alcaligenes faecalis and for laminarin from Laminaria digitata. We also found that the beta-glucan-activated pathway could be totally blocked by laminarin (> 1 microgram/ml) without affecting the endotoxin-activated pathway, allowing endotoxin to be quantitated specifically by the Limulus ELISA with a detection limit of 0.005 endotoxin unit per ml. The use of uninhibited and differentially inhibited ELISAs demonstrated that different LAL preparations showed much greater variation in assaying beta-glucans than in assaying endotoxins. The LAL reactivity of normal human plasma was found to be due to the activation of the beta-glucan pathway, but not the endotoxin pathway, of LAL.

Pitfalls in characterization of protein interactions using radioiodinated crosslinking reagents. Preparation and testing of a novel photochemical 125I-label transfer reagent.

Much attention has been focused on the study of protein interactions with radioiodinated photo-crosslinking reagents, and pitfalls in using this methodology are discussed. A new photochemical and cleavable heterobifunctional crosslinking reagent, succinimidyl N-14-(2-hydroxybenzoyl)-N-11-(4-azidobenzoyl)-9-oxo-8,11,14-triaza -4,5- dithiatetradecanoate (SHAD) was prepared, and its potential as a label transfer reagent was tested in model systems. SHAD was radioiodinated, and the labeled reagent (125I-SHAD) was converted to an amide (125I-HADM, as a mimicry of conjugation to protein 1) and photolyzed. When compared to the widely used SASD reagent (sulfosuccinimidyl 2-[[(4-azidosalicyl)-amino]ethyl]-1,3- dithiopropionate, Pierce), SHAD has a number of decisive advantages. The amide of 125I-SASD (125I-ASDM) was generated and photolyzed, and it was found that at least 50% of the radioactivity is released from 125I-ASDM after 3 min of irradiation, whereas only approximately 10% is liberated from 125I-HADM under similar conditions. Furthermore, 125I-HADM was photolyzed in the presence of excess amine (mimicry of crosslinking to protein 2), and the product was cleaved by reduction (mimicry of label transfer). The transformations in the course of photolysis were monitored by UV spectroscopy and TLC analysis, and a high degree of reagent cleavage upon reduction was demonstrated. 125I-SHAD was used to crosslink Lys78-plasminogen and fibrin. 125I-SHAD was conjugated to Lys78-plasminogen in the dark. Fibrinogen and thrombin were added, and Lys78-plasminogen was crosslinked to the fibrin clot by exposure to light.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA-like double helix formed by peptide nucleic acid.

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.

Peptide nucleic acid.DNA strand displacement loops as artificial transcription promoters.

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.

Sensitive quantitation of endotoxin by enzyme-linked immunosorbent assay with monoclonal antibody against Limulus peptide C.

Limulus peptide C, a 28-amino-acid fragment of coagulogen formed by the reaction of endotoxin with Limulus amebocyte lysate, was synthesized, and a monoclonal antibody against it was raised. A new microassay for endotoxin was developed, using this antibody in an enzyme-linked immunosorbent assay for generated peptide C-like immunoreactivity. A linear relationship between absorbance and endotoxin concentration was obtained. Control standard endotoxin in water could be detected to a level of 0.001 endotoxin unit per ml. The endotoxin levels in plasma samples from normal humans, rabbit, mice, and guinea pigs were generally found to be below the detection limit of 0.01 endotoxin unit per ml of plasma. The color and turbidity of specimens did not interfere with the assay. The consumption of Limulus amebocyte lysate in the assay was less than 5% of that in the gel-clot and chromogenic assays. With raw lysate, which was much more stable in solution than chloroform-treated lysate, the assay was still highly sensitive to endotoxin but was totally unresponsive to natural glucans. The monoclonal antibody cross-reacted with peptide C-like immunoreactivity generated in Tachypleus amebocyte lysate, which gave equal sensitivity in the endotoxin assay.

New compounds related to podophyllotoxin and congeners: synthesis, structure elucidation and biological testing.

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.

Single base pair mutation analysis by PNA directed PCR clamping.

A novel method that allows direct analysis of single base mutation by the polymerase chain reaction (PCR) is described. The method utilizes the finding that PNAs (peptide nucleic acids) recognize and bind to their complementary nucleic acid sequences with higher thermal stability and specificity than the corresponding deoxyribooligonucleotides and that they cannot function as primers for DNA polymerases. We show that a PNA/DNA complex can effectively block the formation of a PCR product when the PNA is targeted against one of the PCR primer sites. Furthermore, we demonstrate that this blockage allows selective amplification/suppression of target sequences that differ by only one base pair. Finally we show that PNAs can be designed in such a way that blockage can be accomplished when the PNA target sequence is located between the PCR primers.

PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen-bonding rules.

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.

Peptide nucleic acids and their potential applications in biotechnology.

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.