3'-minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures.

DNA probes with conjugated minor groove binder (MGB) groups form extremely stable duplexes with single-stranded DNA targets, allowing shorter probes to be used for hybridization based assays. In this paper, sequence specificity of 3'-MGB probes was explored. In comparison with unmodified DNA, MGB probes had higher melting temperature (T(m)) and increased specificity, especially when a mismatch was in the MGB region of the duplex. To exploit these properties, fluorogenic MGB probes were prepared and investigated in the 5'-nuclease PCR assay (real-time PCR assay, TaqMan assay). A 12mer MGB probe had the same T(m)(65 degrees C) as a no-MGB 27mer probe. The fluorogenic MGB probes were more specific for single base mismatches and fluorescence quenching was more efficient, giving increased sensitivity. A/T rich duplexes were stabilized more than G/C rich duplexes, thereby leveling probe T(m)and simplifying design. In summary, MGB probes were more sequence specific than standard DNA probes, especially for single base mismatches at elevated hybridization temperatures.

Linkers designed to intercalate the double helix greatly facilitate DNA alkylation by triplex-forming oligonucleotides carrying a cyclopropapyrroloindole reactive moiety.

Triplex-forming oligonucleotides (TFOs) bind sequence-specifically in the major groove of double-stranded DNA. Cyclopropapyrroloindole (CPI), the electrophilic moiety that comprises the reactive subunit of the antibiotic CC-1065, gives hybridization-triggered alkylation at the N-3 position of adenines when bound in the minor groove of double-stranded DNA. In order to attain TFO-directed targeting of CPI, we designed and tested linkers to 'thread' DNA from the major groove-bound TFO to the minor groove binding site of CPI. Placement of an aromatic ring in the linker significantly enhanced the site-directed reaction, possibly due to a 'threading' mechanism where the aromatic ring is intercalated. All of the linkers containing aromatic rings provided efficient alkylation of the duplex target. The linker containing an acridine ring system, the strongest intercalator in the series, gave a small but clearly detectable amount of non-TFO-specific alkylation. An equivalent-length linker without an aromatic ring was very inefficient in DNA target alkylation.

Antisense oligonucleotides containing modified bases inhibit in vitro translation of Leishmania amazonensis mRNAs by invading the mini-exon hairpin.

Complementary oligodeoxynucleotides (ODNs) that contain 2-aminoadenine and 2-thiothymine interact weakly with each other but form stable hybrids with unmodified complements. These selectively binding complementary (SBC) agents can invade duplex DNA and hybridize to each strand (Kutyavin, I. V., Rhinehart, R. L., Lukhtanov, E. A., Gorn, V. V., Meyer, R. B., and Gamper, H. B. (1996) Biochemistry 35, 11170-11176). Antisense ODNs with similar properties should be less encumbered by RNA secondary structure. Here we show that SBC ODNs strand invade a hairpin in the mini-exon RNA of Leishmania amazonensis and that the resulting heteroduplexes are substrates for Escherichia coli RNase H. SBC ODNs either with phosphodiester or phosphorothioate backbones form more stable hybrids with RNA than normal base (NB) ODNs. Optimal binding was observed when the entire hairpin sequence was targeted. Translation of L. amazonensis mRNA in a cell-free extract was more efficiently inhibited by SBC ODNs complementary to the mini-exon hairpin than by the corresponding NB ODNs. Nonspecific protein binding in the cell-free extract by phosphorothioate SBC ODNs rendered them ineffective as antisense agents in vitro. SBC phosphorothioate ODNs displayed a modest but significant improvement of leishmanicidal properties compared with NB phosphorothioate ODNs.

Triplex targeting of a native gene in permeabilized intact cells: covalent modification of the gene for the chemokine receptor CCR5.

A 12 nucleotide oligodeoxyribopurine tract in the gene for the chemokine receptor CCR5 has been targeted and covalently modified in intact cells by a 12mer triplex forming oligonucleotide (TFO) bearing a reactive group. A nitrogen mustard placed on the 5'-end of the purine motif TFO modified a guanine on the DNA target with high efficiency and selectivity. A new use of a guanine analog in these TFOs significantly enhanced triplex formation and efficiency of modification, as did the use of the triplex-stabilizing intercalator coralyne. This site-directed modification of a native chromosomal gene in intact human cells under conditions where many limitations of triplex formation have been partially addressed underscores the potential of this approach for gene control via site-directed mutagenesis.

Modulation of Cm/T, G/A, and G/T triplex stability by conjugate groups in the presence and absence of KCl.

Apparent equilibrium association constants were determined by gel mobility shift analysis for triple strand formation between a duplex target containing a 21 base long A-rich homopurine run and several end-modified C(m)/T (pyrimidine motif; C(m) = 5-methylcytosine), G/A (purine motif), and G/T (purine-pyrimidine motif) triplex-forming oligonucleotides (TFOs). Incubations were carried out for 24 h at 37 degrees C in 20 mM HEPES, pH 7.2, 10 mM MgCl2, and 1 mM spermine. The purine motif triplex was the most stable (Ka = 6.2 x 10(8) M-1) even though the TFO self-associated as a linear duplex. Conjugation of a terminal hexanol or cholesterol group to the G/A-containing TFO reduced triplex stability by 1.6- or 13-fold, whereas an aminohexyl group or intercalating agent (acridine or psoralen) increased triplex stability by 1.3- or 13-fold. These end groups produced similar effects in C(m)/T and G/T triplexes, although the magnitude of the effect sometimes differed. Addition of 140 mM KCl to mimic physiological conditions decreased stability of the G/A triplex by 1900-fold, making it less stable than the C(m)/T triplex. The inhibitory effect of KCl on G/A triplex formation could be partially compensated for by conjugating the TFO to an intercalating agent (30-350-fold stabilization) or by adding the triplex selective intercalator coralyne (1000-fold stabilization). Although the G/T triplex responded similarly to these agents, the stability of the C(m)/T triplex was unaffected by the presence of coralyne and was only enhanced 1.4-2.8-fold when the TFO was linked to an intercalating agent. In physiological buffer supplemented with 40 microM coralyne, the G/A triplex (Ka = 3.0 x 10(8) M-1) was more stable than the C(m)/T and G/T triplexes by factors of 300 and 12, respectively.

Factors influencing the extent and selectivity of alkylation within triplexes by reactive G/A motif oligonucleotides.

G/A motif triplex-forming oligonucleotides (TFOs) complementary to a 21 base pair homopurine/homopyrimidine run were conjugated at one or both ends to chlorambucil. These TFOs were incubated with several synthetic duplexes containing the targeted homopurine run flanked by different sequences. The extent of mono and interstrand cross-linking was compared with the level of binding at equilibrium. Covalent modification took place within a triple-stranded complex and usually occurred at guanine residues in the flanking double-stranded DNA. The efficiency of alkylation was dependent upon the sequence of the flanking duplex, the solution conditions, and the rate of triplex formation relative to the rate of chlorambucil reaction. Self-association of the TFOs as parallel duplexes was demonstrated and this did not interfere with triple strand formation. With an optimal target, cross-linking of the triplex was very efficient when incubation was carried in a physiological buffer supplemented with the triplex selective intercalator coralyne.

Oligonucleotides with conjugated dihydropyrroloindole tripeptides: base composition and backbone effects on hybridization.

The ability of conjugated minor groove binding (MGB) residues to stabilize nucleic acid duplexes was investigated by synthesis of oligonucleotides bearing a tethered dihydropyrroloindole tripeptide (CDPI3). Duplexes bearing one or more of these conjugated MGBs were varied by base composition (AT- or GC-rich oligonucleotides), backbone modifications (phosphodiester DNA, 2'-O-methyl phosphodiester RNA or phosphorothioate DNA) and site of attachment of the MGB moiety (5'- or 3'-end of either duplex strand). Melting temperatures of the duplexes were determined. The conjugated CDPI3 residue enhanced the stability of virtually all duplexes studied. The extent of stabilization was backbone and sequence dependent and reached a maximum value of 40-49 degrees C for d(pT)8. d(pA)8. Duplexes with a phosphorothioate DNA backbone responded similarly on CDPI3 conjugation, although they were less stable than analogous phosphodiesters. Modest stabilization was obtained for duplexes with a 2'-O-methyl RNA backbone. The conjugated CDPI3 residue stabilized GC-rich DNA duplexes, albeit to a lesser extent than for AT-rich duplexes of the same length.

Minor groove DNA alkylation directed by major groove triplex forming oligodeoxyribonucleotides.

We describe sequence-specific alkylation in the minor groove of double-stranded DNA by a hybridization-triggered reactive group conjugated to a triplex forming oligodeoxyribonucleotide (TFO) that binds in the major groove. The 24 nt TFOs (G/A motif) were designed to form triplexes with a homopurine tract within a 65 bp target duplex. They were conjugated to an N 5-methyl-cyclopropapyrroloindole (MCPI) residue, a structural analog of cyclopropapyrroloindole (CPI), the reactive subunit of the potent antibiotic CC-1065. These moieties react in the DNA minor groove, alkylating adenines at their N3 position. In order to optimize alkylation efficiency, linkers between the TFO and the MCPI were varied both in length and composition. Quantitative alkylation of target DNA was achieved when the dihydropyrroloindole (DPI) subunit of CC-1065 was incorporated between an octa(propylene phosphate) linker and MCPI. The required long linker traversed one strand of the target duplex from the major groove-bound TFO to deliver the reactive group to the minor groove. Alkylation was directed by relative positioning of the TFOs. Sites in the minor groove within 4-8 nt from the end of the TFO bearing the reactive group were selectively alkylated.

Direct, solid phase assembly of dihydropyrroloindole peptides with conjugated oligonucleotides.

A new controlled pore glass (CPG) support is described that allows for the direct synthesis of oligonucleotide derivatives carrying a minor groove binding (MGB) agent at the 3'-terminus. The MGB consisted of three repeating 1,2-dihydro-3H-pyrrolo[2,3-e]indole-7-carboxylate (DPI) subunits. The DPI trimer (DPI3) was prepared directly on the CPG support using repeated addition of the DPI subunit. The subunit was protected at the N-3-position with tert-butyloxycarbonyl residue and activated at the 7-carboxy residue by esterification with the 2,3,5,6-tetrafluorophenyl group. A linker, which provided the starting point for oligonucleotide synthesis, was introduced by reaction of the terminal N-3 with p-nitrophenyl 4-[bis(4-methoxyphenyl)phenylmethoxy]butyrate. When used as a support for oligonucleotide synthesis, this modified CPG gave the desired 3'-DPI3-octathymidylate [(dTp)8-DPI3] conjugate in good yield. This conjugate formed hyperstabilized complexes with complementary polyribo- (Tmax = 35 degrees C) and polydeoxyriboadenylic (Tmax = 69 degrees C) acids. In contrast to the N-carbamoyl derivative reported earlier by us, it demonstrated higher cooperativity of melting transitions.

Oligonucleotides containing 2-aminoadenine and 2-thiothymine act as selectively binding complementary agents.

A pair of complementary oligodeoxynucleotides (ODNs) uniformly substituted with 2-amino-adenine (A') in place of adenine and 2-thiothymine (T') in place of thymine did not hybridize to each other but did form very stable hybrids with unmodified complementary ODNs. These unusual properties were a consequence of the hydrogen-bonding properties of the two base analogs. Thermal denaturation studies of short duplexes which contained these bases demonstrated that the A'-T and A-T' doublets formed stable base pairs whereas the A'-T' doublet acted like a mismatch. Complementary ODNs substituted with these base analogs are referred to as SBC or selectively binding complementary ODNs. When used as a pair, these single-stranded ODNs invaded the ends of homologous duplexes and formed stable three-arm junctions under conditions where unmodified ODNs failed to give a product. SBC ODNs have a fundamental thermodynamic advantage in hybridizing to short segments of double-stranded nucleic acid and represent a new approach for the design of oligomeric probes and antisense agents. Many secondary structure features present in long single-stranded nucleic acids should be accessible to these reagents.

Rapid and efficient hybridization-triggered crosslinking within a DNA duplex by an oligodeoxyribonucleotide bearing a conjugated cyclopropapyrroloindole.

The antitumor antibiotic CC-1065 binds in the minor groove of double-stranded DNA, and the cyclopropapyrroloindole (CPI) subunit of the drug alkylates adjacent adenines at their N-3 position. We have attached racemic CPI to oligodeoxyribonucleotides (ODNs) via a terminal phosphorothioate at either the 3'- or 5'-end of the ODNs. These conjugates were remarkably stable in aqueous solution at neutral pH even in the presence of strong nucleophiles. When a 3'-CPI-ODN conjugate was hybridized to a complementary DNA strand at 37 degrees C, the CPI moiety alkylated nearby adenine bases of the complement efficiently and rapidly, with a half-life of a few minutes. The 4'-CPR- ODN conjugate showed very little reactivity within the duplex. CPI-ODN conjugates should be highly effective sequence-specific inhibitors of single-stranded viral DNA replication or gene selective inhibitors of transcription initiation.

Oligodeoxyribonucleotides with conjugated dihydropyrroloindole oligopeptides: preparation and hybridization properties.

Synthesis of a new class of conjugates between oligodeoxyribonucleotides (ODNs) and minor groove binders (MGBs) is described. The MGBs are analogs of the potent antibiotic CC-1065 and consist of repeating 1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylate (DPI) subunits with N-3 carbamoyl or tert-butyloxycarbonyl groups (CDPI or BocDPI subunits, respectively). The ODN-MGB conjugates were obtained by postsynthetic modification of 5'- or 3'-amino-tailed ODNs with the 2,3,5,6-tetrafluorophenyl (TFP) esters of CDPI1-3 or BocDPI1-2 or by ODN synthesis using a CDPI3-modified controlled pore glass (CPG) support. The hybridization properties of MGB-tailed octathymidylates were determined; they varied with respect to the site of conjugation (3' or 5'), the nature of the linker, the length of the DPI oligopeptide, and the type of N-3 substitution. Optical melting studies showed that the linkage of CDPI1-3 residues to (dTp)8 significantly increased the stability of hybrids formed by the latter with poly(dA). The extent of stabilization increased with the length of the peptide. When CDPI3 was conjugated to either end of (dTp)8, the melting temperature (Tm) of the hybrid formed with poly(dA) was increased by 43-44 degrees C. Free CDPI3 stabilized the (dTp)8-poly(dA) hybrid by only 2 degrees C, thus demonstrating the importance of conjugation. (dTp)8-CDPI1-3 conjugates also formed stabilized duplexes with poly(rA). The extent of stabilization was half that observed with poly(dA).

Synthesis and high stability of complementary complexes of N-(2-hydroxyethyl)phenazinium derivatives of oligonucleotides.

Two simple methods for the synthesis of oligonucleotides bearing a N-(2-hydroxyethyl)phenazinium (Phn) residue at the 5'- and/or 3'-terminal phosphate groups are proposed. By forming complexes between a dodecanucleotide d(pApApCpCpTpGpTpTpTpGpGpC), a heptanucleotide d(pCpCpApApApCpA), and Phn derivatives of the latter, it is shown that the introduction of a dye at the end of an oligonucleotide chain strongly stabilizes its complementary complexes. The Tmax and the thermodynamic parameters (delta H, delta S, delta G) of complex formation were determined. According to these data, coupling of a dye with the 5'-terminal phosphate group is the most advantageous: delta G(37 degrees C) is increased by 3.59 +/- 0.04 kcal/mol compared to 2.06 +/- 0.04 kcal/mol for 3'-Phn derivatives. The elongation of the linker, which connects the dye to the oligonucleotide, from a dimethylene up to a heptamethylene usually leads to destabilization of the oligonucleotide complex. The complementary complex formed by the 3',5'-di-Phn derivative of the heptanucleotide was found to be the most stable among all duplexes investigated. Relative to the unmodified complex the increase in free energy was 4.96 +/- 0.04 kcal/mol. The association constant of this modified complex at 37 degrees C is 9.5 x 10(6) M-1, whereas the analogous value for the unmodified complex is only 3 x 10(3) M-1.

N-(2-hydroxyethyl)phenazinium derivatives of oligonucleotides as effectors of the sequence-specific modification of nucleic acids with reactive oligonucleotide derivatives.

It has been found that mono- and especially diphenazinium derivatives of oligonucleotides complementary to the DNA sequence adjacent to the target sequence of the addressed alkylation of DNA, significantly enhance the extent and specificity of alkylation with p-(N-2-chloroethyl-N-methylamino)benzylamide derivatives of the addressing oligonucleotides, thus playing the role of effector of the sequence-specific (complementary addressed) modification.

Sequence-specific chemical modification of a hybrid bacteriophage M13 single-stranded DNA by alkylating oligonucleotide derivatives.

Alkylating oligonucleotide derivatives react with the complementary sequences in hybrid M13mp7 bacteriophage single-stranded DNA and destroy the infecting ability of the DNA. The reagents do not damage M13mp9 single-stranded DNA lacking the target nucleotide sequence.

Studies of the functional topography of Escherichia coli RNA polymerase. Affinity labelling of RNA polymerase in a promoter complex by phosphorylating derivatives of primer oligonucleotides.

Amidation of the 5'-phosphate group of the heptanucleotide pdApdApdApdTpdCpdGprC and of its derivatives of the general formula (pdN)npdGprC (n = 0-5) with imidazole, N-methylimidazole, and 4-dimethylaminopyridine afforded a series of phosphorylating affinity reagents. The parent oligonucleotides of this series complementary to promoter A2 of T7 phage over the region (-5 to +2) are known to be efficient primers of the synthesis of RNA by Escherichia coli RNA polymerase with promoter A2 as template. Treatment of the complex RNA-polymerase X promoter-A2 with affinity reagents followed by addition of [alpha-32P]UTP resulted in labelling of RNA polymerase by the residues -(pdN)npdGprCprU (p = radioactive phosphate). This affinity labelling was highly selective because elongation of the covalently bound residues (pdN)npdGprC by prU residues was catalyzed by the active center of RNA polymerase. The most efficient reagents were N-methylimidazolides. A dramatic change of the pattern of labelling of the subunits beta, beta', and sigma took place with changing n. Maximum labelling of the beta subunit occurred at n = 1 and of the sigma subunit at n = 5. The targets in both the subunits were His residues. The alpha subunit was not specifically labelled.

Oligonucleotides complementary to a promoter over the region -8...+2 as transcription primers for E. coli RNA polymerase.

Primer-dependent transcription by E. coli RNA polymerase on T7 promoter A2 has been studied. Synthetic deoxyribonucleotides complementary to the promoter over the region -8...+2 were taken as primers. A ribonucleoside residue was present at the 3'-end of some of these oligonucleotides. The octanucleotide complementary to the region -8...-1 appeared to be an active primer. Oligonucleotides having lengths from 3 to 6 nucleotide residues complementary to the promoter over the region -4...+2 also exhibited primer activity. The latter was some 5-10 times greater in the case of oligonucleotides having a ribonucleoside residue at the 3'-end. Oligonucleotides which on complementary binding do not reach the center of phosphodiester bond synthesis, as well as the decanucleotides (-8...+2) and octanucleotides (-6...+2) of both the ribo- and deoxyribo-series were inactive as primers.