Exclusion of RNA strands from a purine motif triple helix.

Research concerning oligonucleotide-directed triple helix formation has mainly focused on the binding of DNA oligonucleotides to duplex DNA. The participation of RNA strands in triple helices is also of interest. For the pyrimidine motif (pyrimidine.purine.pyrimidine triplets), systematic substitution of RNA for DNA in one, two, or all three triplex strands has previously been reported. For the purine motif (purine.purine.pyrimidine triplets), studies have shown only that RNA cannot bind to duplex DNA. To extend this result, we created a DNA triple helix in the purine motif and systematically replaced one, two, or all three strands with RNA. In dramatic contrast to the general accommodation of RNA strands in the pyrimidine triple helix motif, a stable triplex forms in the purine motif only when all three of the substituent strands are DNA. The lack of triplex formation among any of the other seven possible strand combinations involving RNA suggests that: (i) duplex structures containing RNA cannot be targeted by DNA oligonucleotides in the purine motif; (ii) RNA strands cannot be employed to recognize duplex DNA in the purine motif; and (iii) RNA tertiary structures are likely to contain only isolated base triplets in the purine motif.     

Single-strand DNA triple-helix formation

Chemical modification studies provide evidence that single-stranded oligodeoxyribonucleotides can form stable intrastrand triple helices. Two oligonucleotides of opposite polarity were synthesized, each composed of a homopurine-homopyrimidine hairpin stem linked to a pyrimidine sequence which is capable of folding back on the hairpin stem and forming specific Hoogsteen hydrogen bonds. Using potassium permanganate as a chemical modification reagent, we have found that two oligodeoxyribonucleotides of sequence composition type 5'-(purine)8(N)4(pyrimidine)8(N)6(pyrimidine)8-3' and 5'-(pyrimidine)8N6(pyrimidine)8N4(purine)8-3' undergo dramatic structural changes consistent with intrastrand DNA triple-helix formation induced by lowering the pH or raising the Mg2+ concentration. The intrastrand DNA triple helix is sensitive to base mismatches.     

Investigation of the formation and intracellular stability of purine.(purine/pyrimidine) triplexes.

In a previous work we showed that a short triple helix-forming oligonucleotide (TFO) targeted to the murine c-pim-1 proto-oncogene promoter gives a very stable triple helix under physiological conditions in vitro . Moreover, this triplex was stable inside cells when preformed in vitro . However, we failed to detect triplex formation for this sequence inside cells in DMS footprinting studies. In the present work, in order to determine whether our previous in vivo results are limited to this particular short triplex or can be generalized to other purine.(purine/pyrimidine) triplexes, we have tested three other DNA targets already described in the literature. All these purine.(purine/pyrimidine) triplexes are specific and stable at high temperature in vitro . In vivo studies have shown that the preformed triplexes are stable inside cells for at least 3 days. This clearly demonstrates that intracellular conditions are favourable for the existence of purine. (purine/pyrimidine) triplexes. The triplexes can also be formed in nuclei. However, for all the sequences tested, we were unable to detect any triple helix formation in vivo in intact cells by DMS footprinting. Our results show that neither (i) chromatinization of the DNA target, (ii) intracellular K+concentration nor (iii) cytoplasmic versus nuclear separation of the TFO and DNA target are responsible for the intracellular arrest of triplex formation. We suggest the existence of a cellular mechanism, based on a compartmentalization of TFOs and/or TFO trapping, which separates oligonucleotides from the DNA target. Further work is needed to find oligonucleotide derivatives and means for their delivery to overcome the problem of triplex formation inside cells.     

Energetics of strand-displacement reactions in triple helices: a spectroscopic study.

DNA triple helices offer exciting new perspectives toward oligonucleotide-directed inhibition of gene expression. Purine and GT triplexes appear to be the most promising motifs for stable binding under physiological conditions compared to the pyrimidine motif, which forms at relatively low pH. There are, however, very little data available for comparison of the relative stabilities of the different classes of triplexes under identical conditions. We, therefore, designed a model system which allowed us to set up a competition between the oligonucleotides of the purine and pyrimidine motifs targeting the same Watson-Crick duplex. Several conclusions may be drawn: (i) a weak hypochromism at 260 nm is associated with purine triplex formation; (ii) delta H degree of GA, GT and TC triplex formation (at pH 7.0) was calculated as -0.1, -2.5 and -6.1 kcal/mol per base triplet, respectively. This unexpectedly low delta H degree for the purine triple helix formation implies that its delta G degree is nearly temperature-independent and it explains why these triplexes may still be observed at high temperatures. In contrast, the pyrimidine triplex is strongly favoured at lower temperatures; (iii) as a consequence, in a system where two third-strands compete for triplex formation, displacement of the GA or GT strand by a pyrimidine strand may be observed at neutral pH upon lowering the temperature. This original purine-to-pyrimidine triplex conversion shows a significant hypochromism at 260 nm and a hyperchromism at 295 nm which is similar to the duplex-to-triplex conversion in the pyrimidine motif. Further evidence for this triplex-to-triplex conversion is provided by mung bean-nuclease foot-printing assay.     

Effect of a 5'-phosphate on the stability of triple helix.

An effect of 5'-phosphorylation on the stability of triple helical DNA containing pyrimidine:purine:pyrimidine strands has been demonstrated by both gel electrophoresis and UV melting. A 5'-phosphate on the purine-rich middle strand of a triple helix lowers the stability of triple helix formation by approximately 1 kcal/mol at 25 degrees C. The middle strand is involved in both Watson-Crick and Hoogsteen base pairing. In contrast, a 5'-phosphate on the pyrimidine-rich strands, which are involved in either Watson-Crick or Hoogsteen base pairing, has a smaller effect on the stability of triple helix. The order of stability is: no phosphate on either strand > phosphate on both pyrimidine strands > phosphate on purine strand > phosphate on all three strands. Differential stability of triple helix species is postulated to stem from an increase in rigidity due to steric hindrance from the 5'-phosphate. This result indicates that labelling with 32P affect equilibrium in triplex formation.     

Modified Oligonucleotides with Triple-Helix Stabilization Properties

Abstract

Triple helix binding properties of several purine and pyrimidine derivatives are described. Introduction of an amino group at position 8 of adenine and guanine stabilize triple helix.     

Circular dichroism of helices formed by purine monomers with pyrimidine polynucleotides

The CD spectra of a number of helical complexes formed by purine monomers and complementary pyrimidine polyribonucleotides have been observed over the range 200–400 nm. Each of these spectra is quite similar to that of the corresponding polymer–polymer helix. The spectra are evidently determined by the geometry of the asymmetric array of bases, largely unperturbed by the ribose–phosphate backbone. The helix structure (A-form), on the other hand, is determined by the backbone of the pyrimidine homopolymer. Data on the monomer–polymer complexes support the conclusion that the CD spectra of ribohomopolymer helices depend primarily on interastrand interactions of the same transition within a given base and are relatively unaffected by transitions of the complementary base.     

Unusual duplex formation in purine rich oligodeoxyribonucleotides

The purine rich oligodeoxyribonucleotides 1C, d(ATGACGGAATA) and 2C, d(ATGAGCGAATA) alone exhibit highly cooperative melting transitions. Analysis of the concentration dependence of melting, and electrophoretic studies indicate that these oligomers can form an unusual purine rich offset double helix. The unusual duplex is predicted to contain four A.T, two G.C, and four G.A mismatch base pairs as well as a single A base stacked on the 3' end of each chain of the helix. Other possible models for the duplex are unlikely because they are predicted to contain many base pairs of low stability. Changing the central sequence to CGG or GGG should destabilize the duplex and this is observed. The unusual duplex of 2C is more stable than the duplex of 1C indicating that the stability of G.A base pairs is quite sensitive to the surrounding sequence. Addition of 1C and 2C to their complementary pyrimidine strands results in normal duplexes of similar stability. We feel that the unusual duplexes are significantly stabilized by the intrinsic stacking tendency of purine bases.     

Origin of ultraviolet damage in DNA

A novel ultraviolet (u.v.) footprinting technique has been used to analyze the formation of u.v. photoproducts at 250 bases of a 5 S rRNA gene under conditions where the gene is either double or single-stranded. Because many more types of u.v. damage can be detected by the u.v. footprinting technique than has been previously possible, we have been able to examine in detail why certain bases in DNA are damaged by u.v. light while others are not. Our measurements demonstrate that the ability of u.v. light to damage a given base in DNA is determined by two factors, the sequence of the DNA in the immediate vicinity of the photoproduct, and the flexibility of the DNA at the site of the photoproduct. For pyrimidines, the predominant photoreaction in double-stranded DNA involves covalent dimerization between adjacent pyrimidine residues. Dimerization is much easier in melted DNA because the geometrical changes required for adjacent pyrimidine residues to dimerize are easier in single-stranded DNA. The absorption of a u.v. photon cannot simultaneously induce the geometrical changes required for adjacent pyrimidines or other bases to dimerize with one another. Rather, upon the absorption of a u.v. photon, only those thermally excited bases that are in a geometry capable of easily forming a photodimer during excitation, can photoreact. In contrast to adjacent pyrimidines, non-adjacent pyrimidines (pyrimidines flanked on either side by a purine) do not readily form u.v. photoproducts in double-stranded DNA. Because photoreactions at non-adjacent pyrimidine residues are greatly enhanced in single-stranded DNA, their failure to form in double-helical DNA is attributed to torsional constraints imposed by the double helix which make it difficult for non-adjacent pyrimidines to adopt a geometry necessary for photoreaction. Although purines are believed to be resistant to u.v. damage, our measurements demonstrate that at moderate u.v. dosages purines which are flanked on their 5' side by two or more contiguous pyrimidines readily form u.v. photoproducts in double-stranded DNA. Flanking pyrimidines appear to activate purine photoreactions by transferring triplet excitation energy to the purine. Melting of the DNA helix greatly inhibits the ability of flanking pyrimidines to activate purine photoreactions, presumably by disrupting intimate orbital overlap required for triplet transfer.     

Uranyl photofootprinting of triple helical DNA.

Two triple helix structures (15-mers containing only T.A-T triplets or containing mixed T.A-T and C.G-C triplets) have been studied by uranyl mediated DNA photocleavage to probe the accessibility of the phosphates of the DNA backbone. Whereas the phosphates of the pyrimidine strand are at least as accessible as in double stranded DNA, in the phosphates of the purine strand are partly shielded and more so at the 5'-end of the strand. With the homo A/T target increased cleavage is observed towards the 3'-end on the pyrimidine strand. These results show that the third strand is asymmetrically positioned along the groove with the tightest triple strand double strand interactions at the 5'-end of the third strand. The results also indicate that homo-A versus mixed A/G 'Hoogsteen-triple helices' have different structures.     

Left-Handed Intercalated DNA Double Helix: Rendezvous of Ethidium and Actinomycin D in the Z-Helical Conformation Space

Abstract

It is now very well recognized that the DNA double helix is conformationally pluralistic and that this flexibility is derived from internal motions due to backbone torsions. But what is less apparent is that such internal motions can occur in a correlated fashion and express themselves in a wide variety of structural motifs and phenomena. For example, flexibility inherent in the DNA molecule can lead to a family of Z-DNA, LZ1 and LZ2 being the two extremes and correlated internal motion can cause LZ1?LZ2 transition. More interestingly, such motions manifest themselves as breathing modes on the DNA lattice resulting in the sequence specific intercalation sites. Following a detailed stereochemical analyses we observed that the intercalation site for ethidium is located at the dCpdG sequence of the intercalated LZ1 helix (LZ1*) while that for actinomycin D is located at the dGpdC sequence of the intercalated LZ2 helix (LZ2*). From the stereochemistry of the drug binding we make experimentally testable predictions which are in fact supported by a few recent experimental studies. These studies also show that a left-handed intercalated B-DNA model is a viable intermediate in the Z to B transition which can hold the drug with binding energy comparable to that of the intercalated right-handed B-DNA.     

Hucleotide sequence of the Bos indicus α‐lactalbumin 5’ flanking region: Comparison with the 605 taurus sequence

Abstract

The 5’ flanking region of the α‐lactalbumin gene from Bos indicus (Brahman) and Bos taurus (Holstein) have been sequenced. The sequences were compared to detect potential sequence variations. The 538 bp of 5’ flanking region contained nine sequence variations between the two breeds. Seven of the variations occur in the 5’ flanking region of the α‐lactalbumin gene and two occur in the region encoding the 5’ untranslated region of the mRNA. Seven of the variations are conservative purine to purine or pyrimidine to pyrimidine variations, while two are purine to pyrimidine variations.     

Trans-N-deoxyribosylase: purification by affinity chromatography and characterization.

trans-N-Deoxyribosylase (EC 2.4.2.6) is usually considered as a single protein catalyzing indifferently the transfer of the deoxyribosyl moiety to and from a purine or a pyrimidine base. Affinity chromatography of an extract from Lactobacillus helveticus with two types of ligands allowed the separation and purification of two distinct trans-N-deoxyribosylases. One catalyzes specifically the deoxyribosyl transfer to and from purine bases exclusively: trans-N-deoxyribosylase-I, the other catalyzes the transfer to and from pyrimidine and purine bases: trans-N-deoxyribosylase-II. A Tris inhibition study showed a markedly different susceptibility of the two enzymes. Preliminary results indicate that the purine-specific enzyme is a polymeric enzyme of molecular weight 86 000 (+/- 4000).     

Theoretical studies on alpha-helix--DNA interactions

The interaction energies between (Ala)10 and alpha-helix fragment and different nucleotide sequences in right-handed B-form have been optimized using semi-empirical potential energy functions. The energies are calculated for two different orientations of the alpha-helix, viz., when the alpha-helix axis taken in the N----C direction is (i) parallel and (ii) antiparallel to the 5'-3' ascending strand of DNA, proximal to it. When both the DNA molecule as well as the alpha-helix are treated as rigid molecules it is found that a polyalanine alpha-helix has slightly more favourable contacts when it is in the proximity of a four nucleotide sequence of 5'-(N-A-T-N)-3' type, where N is either a purine or a pyrimidine. However, when the two interacting molecules are allowed to undergo local structural variations then the interaction energy appears to be independent of the base sequence confirming the non-specific nature of these interactions.     

Physico-chemical studies on DNA triplexes containing an alternate third strand with a non-nucleotide linker

Differential scanning calorimetric (DSC), circular dichroism (CD) and molecular mechanics studies have been performed on two triple helices of DNA. The target duplex consists of 16 base pairs in alternate sequence of the type 5′-(purine)_m(pyrimidine)_m-3′. In both the triplexes, the third oligopyrimidine strand crosses the major groove at the purine–pyrimidine junction, with a simultaneous binding of the adjacent purine tracts on alternate strands of the Watson–Crick duplex. The switch is ensured by a non-nucleotide linker, the 1,2,3 propanetriol residue, that joins two 3′–3′ phosphodiester ends. The third strands differ from each other for a nucleotide in the junction region. The resulting triple helices were termed 14-mer-PXP and 15-mer-PXP (where P=phosphate and X=1,2,3-propanetriol residue) according to the number of nucleotides that compose the third strand. DSC data show two independent processes: the first corresponding to the dissociation of the third strand from the target duplex, the second to the dissociation of the double helix in two single strands. The two triple helices show the same stability at pH 6.6. At pH 6.0, the 15-mer-PXP triplex is thermodynamically more stable than the 14-mer-PXP triplex. Thermodynamic data are discussed in relation to structural models. The results are useful when considering the design of oligonucleotides that can bind in an antigene approach to the DNA for therapeutic purposes.     

Extension of the range of DNA sequences available for triple helix formation: stabilization of mismatched triplexes by acridine-containing oligonucleotides.

Triple helix formation usually requires an oligopyrimidine*oligopurine sequence in the target DNA. A triple helix is destabilized when the oligopyrimidine*oligopurine target contains one (or two) purine*pyrimidine base pair inversion(s). Such an imperfect target sequence can be recognized by a third strand oligonucleotide containing an internally incorporated acridine intercalator facing the inverted purine*pyrimidine base pair(s). The loss of triplex stability due to the mismatch is partially overcome. The stability of triplexes formed at perfect and imperfect target sequences was investigated by UV thermal denaturation experiments. The stabilization provided by an internally incorporated acridine third strand oligonucleotide depends on the sequences flanking the inverted base pair. For triplexes containing a single mismatch the highest stabilization is observed for an acridine or a propanediol tethered to an acridine on its 3'-side facing an inverted A*T base pair and for a cytosine with an acridine incorporated to its 3'-side or a guanine with an acridine at its 5'-side facing an inverted G*C base pair. Fluorescence studies provided evidence that the acridine was intercalated into the triplex. The target sequences containing a double base pair inversion which form very unstable triplexes can still be recognized by oligonucleotides provided they contain an appropriately incorporated acridine facing the double mismatch sites. Selectivity for an A*T base pair inversion was observed with an oligonucleotide containing an acridine incorporated at the mismatched site when this site is flanked by two T*A*T base triplets. These results show that the range of DNA base sequences available for triplex formation can be extended by using oligonucleotide intercalator conjugates.     

Nuclear magnetic resonance structural studies of intramolecular purine.purine.pyrimidine DNA triplexes in solution. Base triple pairing alignments and strand direction

Recently, P.A. Beal and P.B. Dervan, expanding on earlier observations by others, have established the formation of purine.purine.pyrimidine triple helices stabilized by G.GC, A.AT and T.AT base triples where the purine-rich third strand was positioned in the major groove of the Watson-Crick duplex and anti-parallel to its purine strand. The present nuclear magnetic resonance (n.m.r.) study characterizes the base triple pairing alignments and strand direction in a 31-mer deoxyoligonucleotide that intramolecularly folds to generate a 7-mer (R/Y-)n.(R+)n(Y-)n triplex with the strands linked by two T5 loops and stabilized by potential T.AT and G.GC base triples. (R and Y stand for purine and pyrimidine, respectively, while the signs establish the strand direction.) This intramolecular triplex gives well-resolved exchangeable and non-exchangeable proton spectra with Li+ as counterion in aqueous solution. These studies establish that the T1 to C7 pyrimidine and the G8 to A14 purine strands are anti-parallel to each other and align through Watson-Crick A.T and G.C pair formation. The T15 to G21 purine-rich third strand is positioned in the major groove of this duplex and pairs through Hoogsteen alignment with the purine strand to generate T.AT and G.GC triples. Several lines of evidence establish that the thymidine and guanosine bases in the T15 to G21 purine-rich third strand adopt anti glycosidic torsion angles under conditions where this strand is aligned anti-parallel to the G8 to A14 purine strand. We have also recorded imino proton n.m.r. spectra for an (R-)n.(R+)n(Y-)n triplex stabilized by G.GC and A.AT triples through intramolecular folding of a related 31-mer deoxyoligonucleotide with Li+ as counterion. The intramolecular purine.purine.pyrimidine triplexes containing unprotonated G.GC, A.AT and T.AT triples are stable at basic pH in contrast to pyrimidine.purine.pyrimidine triplexes containing protonated C+.GC and T.AT triples, which are only stable at acidic pH.     

Evidence for separate carriers for purine nucleosides and for pyrimidine nucleosides in the renal brush border membrane.

The aim of the present study was to test if the transport of all nucleosides in rat renal brush border membranes occurs via a common carrier or if specific carriers exist for various groups of nucleosides. We measured the inward transport of radiolabeled nucleosides into brush border vesicles. The effect of unlabeled nucleosides present inside of the vesicles (trans-stimulation) or outside of the vesicles (cis-inhibition) was studied. Uphill influx of a nucleoside into the vesicles could be driven by the efflux of another nucleoside (trans-stimulation) if they were both purines or both pyrimidines but not if one nucleoside was a purine and the other one a pyrimidine. Thus, there exist a carrier that transports various purine nucleosides, and a carrier that transports various pyrimidine nucleosides, but the tested purine nucleosides and the tested pyrimidine nucleosides do not appear to be transported by the same carrier. Uridine and thymidine were similarly potent for the inhibition of cytidine transport whereas uridine was much more potent than thymidine for the inhibition of adenosine transport. This suggests that cytidine and adenosine can use different carriers. Preincubation of the vesicles with N-ethylmaleimide resulted in a marked decrease of the rate of transport of purine nucleosides but it had little effect on the transport of pyrimidine nucleosides. These data are best explained by the presence in the renal brush border membrane of two carriers, one for purine nucleosides, the other one for pyrimidine nucleosides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Allotypes of the constant region of the rabbit T cell receptor beta-2 chain

Our laboratory previously reported that there was restriction fragment length polymorphism of TCR C beta genes in rabbits. EcoRI digests of DNA from different rabbits gave fragments of 9 and 6 kb (C beta a) or 14 and 6 kb (C beta b) that hybridized to a C beta cDNA probe. We also reported that the 9- and 14-kb types segregated as Mendelian traits and that there were allotypic differences in the first exon of the C beta 1 genes of C beta a and C beta b animals. Here we report the DNA sequence of the C beta 2 gene present in the 6-kb EcoRI fragment from a C beta b animal and compare the exon sequences with that of a cDNA from a C beta a animal. We find replacement changes in the first and third exons that probably represent allotypic forms of the rabbit C beta 2 gene. The genomic DNA 5' of exon 1 of both beta 1 and beta 2 contain alternating purine/pyrimidine repeat sequences. The genomic C beta 2 has an open reading frame of 69 amino acids in frame with exon 1 similar to a longer one previously found 5' of exon 1 of C beta 1. Further 5' of this region, rabbit C beta 1 and C beta 2 DNA sequences are only about 66% similar. Both the C beta 1 and C beta 2 sequences have two chi sequences; one in exon 1 with a perfect match and one in the intron downstream of exon 1 with one mismatch. Alternating purine/pyrimidine repeats and chi sequences found in rabbit C beta 1 and C beta 2 genes may have contributed to process(es) of gene duplication and/or conversion.