Stabilization of DNA Triple Helices Using Conjugates of Oligonucleotides and Synthetic Ligands

The possibility is discussed of stabilizing a DNA triple helix by covalent conjugation to the third strand (through its terminal phosphate) of ligands that have affinity to double and triple helices. Two types of stabilizers are considered: minor groove binders based on oligopyrroles, and triplex-specific intercalators. As a target, a synthetic 29-mer duplex containing a natural polypurine sequence of the human immunodeficiency provirus was employed. The stabilization with minor groove binders requires several conditions to be respected: a sufficiently long linker capable of reaching the minor groove from the major groove, a specific double-stranded structure of the oligopyrrole fragment, and its in-phase fitness to the target sequence. The best stabilizers of a triplex were novel conjugates in which two parallel molecules containing six pyrrole units each are linked to the same 5"-phosphate of a 16-mer triplex-forming oligonucleotide. The stabilizing properties of these derivatives were comparable to those of benzoindoloquinoline (BIQ) intercalators attached to the terminal phosphate of triple-helix forming oligonucleotides.     

Conjugates of Oligo(2′-O-Methylribonucleotides) with Minor Groove Binders as New Sequence-Specific Agents Recognizing Both Grooves of Double-Stranded DNA

Abstract

Design, synthesis and physico-chemical studies of new pyrimidine oligo(2′-O-methylribonucleotide) conjugates with one or two oligo(pyrrolecarboxamide) minor groove binders (MGB) are described.     

Enhancement and Inhibition by 2′-O-Hydroxyethyl Residues of Gene Targeting Mediated by Triple Helix Forming Oligonucleotides

Abstract

Reagents that recognize and bind specific genomic sequences in living mammalian cells would have great potential for genetic manipulation, including gene knockout, strain construction, and gene therapy. Triple helix forming oligonucleotides (TFOs) bind specific sequences via the major groove, but pyrimidine motif TFOs are limited by their poor activity under physiological conditions. Base and sugar analogues that overcome many of these limitations have been described. In particular, 2′-O-modifications influence sugar pucker and third strand conformation, and have been important to the development of bioactive TFOs. Here we have analyzed the impact of 2′-O-hydroxyethyl (2′-HE) substitutions, in combination with other 2′ modifications. We prepared modified TFOs conjugated to psoralen and measured targeting activity in a gene knockout assay in cultured hamster cells. We find that 2′-HE residues enhance the bioactivity of TFOs containing 2′-O-methyl (2′-OMe) modifications, but reduce the bioactivity of TFOs containing, in addition, 2′-O-aminoethyl (2′-AE) residues.     

Properties of Triple Helices Formed by Oligonucleotides Containing 8-Aminopurines

Abstract

The synthesis of parallel hairpins carrying 8-aminopurines is described. These hairpins have a high affinity for specific polypyrimidine sequences resulting in the formation of very stable triplexes.     

Oligonucleotide–Minor Groove Binder Conjugates and Their Complexes with Complementary DNA: Effect of Conjugate Structural Factors on the Thermal Stability of Duplexes

Synthetic polycarboxamide minor groove binders (MGB) consisting of N‐methylpyrrole (Py), N‐methylimidazole (Im), N‐methyl‐3‐hydroxypyrrole (Hp) and β‐alanine (β) show strong and sequence‐specific interaction with the DNA minor groove in side‐by‐side antiparallel or parallel orientation. Two MGB moieties covalently linked to the same terminal phosphate of one DNA strand stabilize DNA duplexes formed by this strand with a complementary one in a sequence‐specific manner, similarly to the corresponding mono‐conjugated hairpin structures. The series of conjugates with the general formula Oligo‐(L‐MGB‐R)_m was synthesized, where m = 1 or 2, L = linker, R = terminal charged or neutral group, MGB = –(Py)_n–, –(Im)_n– or –[(Py/Im)_n–(CH₂)₃CONH–(Py/Im)_n–] and 1 < n < 5. Using thermal denaturation, we studied effects of structural factors such as m and n, linker L length, nature and orientation of the MGB monomers, the group R and the backbone (DNA or RNA), etc. on the stability of the duplexes. Structural factors are more important for linear and hairpin monophosphoroamidates than for parallel bis‐phosphoroamidates. No more than two oligocarboxamide strands can be inserted into the duplex minor groove. Attachment of the second sequence‐specific parallel ligand [–L(Py)₄R] to monophosphoroamidate conjugate CGTTTATT–L(Py)₄R leads to the increase of the duplex Tm, whereas attachment of [–L(Im)₄R] leads to its decrease. The mode of interaction between oligonucleotide duplex and attached ligands could be different (stacking with the terminal A:T pair of the duplex or its insertion into the minor groove) depending on the length and structure of the MGB.     

Oligonucleotide—Minor Groove Binder 1:2 Conjugates: Side by Side Parallel Minor Groove Binder Motif in Stabilization of DNA Duplex

Synthetic polycarboxamides consisting of N‐methylpyrrole (Py), N‐methylimidazole (Im), N‐methyl‐3‐hydroxypyrrole (Hp) and β‐alanine (β) show strong and sequence‐specific interaction with the DNA minor groove when they form hairpin structures with side‐by‐side antiparallel motifs. In the present paper, new conjugates containing two ligands linked to the same terminal phosphate of DNA strand were constructed. The paper describes optimized synthesis and properties of oligonucleotide‐linked polyamide strands that insert into the minor groove of a duplex in a parallel or antiparallel orientation. Strong stabilization of DNA duplexes by two attached minor groove ligands is demonstrated by the thermal denaturation method. The unmodified duplex 5′‐CGTTTATTp‐3′/5′‐AATAAACG‐3′ melts at 20°C. When one tetra(Py) residue was attached to the first strand of this duplex, denaturation temperature was increased to 46°C; attachment of the second tetra(Py) in a parallel orientation resulted in denaturation temperature of 60°C. It is even higher than in case of “classic” octapyrrole hairpin ligand (Tm = 58°C). Sequence‐specific character of stabilization by two conjugated ligands was demonstrated for G:C‐containing oligonucleotides attached to tetracarboxamide and octacarboxamide ligands constructed from Py, Im and β units according to established recognition rules (ΔTm = 20°C). The two‐strand parallel minor groove binder constructions attached to addressing oligonucleotides could be considered as site‐specific ligands recognizing single‐ and double‐stranded DNA similarly to already described hairpin MGB structures with antiparallel orientation of carboxamide units.     

Chemical Incorporation of 1-Methyladenosine,Minor tRNA Component,into Oligonucleotides

Abstract

The synthesis of suitably protected 1-methyladenosine derivatives has been developed and its successful chemical incorporation into oligonucleotides was achieved.     

A database of 32 DNA triplets to study triple helices by molecular mechanics and dynamics

We present here a database of 32 deoxyribonucleotide triplets, that can be used as building blocks of triple helix forming deoxyribonucleotides on a computer. This database is made of all the pairing schemes of the triplets ATT, GCC⁺, ATA and GCG where the third base forms two hydrogen bonds with the purine of the first two Watson-Crick strands. The essential features of the known triple helices were preserved in the resulting structures. A triple helix can be easily built from any combination of these basic triplets. Four homogeneous and alternate triple helices thus obtained were studied by molecular mechanics and dynamics in vacuo. The results are in agreement with known experimental observations for ATT and suggest a possible structure for the GCG triple helix. In order to characterize the geometry of the structures obtained, the definitions of nucleic acid structure parameters (R.E. Dickerson et al., EMBO J. 8 (1989) 1–4) have been extended to triple helical polynucleotides.     

Stabilization of RNA Bulges by Oligonucleotides Containing 2′-Naphthylmethyl-2′-deoxytubercidine

Abstract

A novel nucleoside analogue, 2′-naphthylmethyl-2′-deoxytubercidine, is synthesized and incorporated in oligonucleotides that stabilize bulges in partially complementary RNA.     

Nuclear condensation and free radical scavenging: a dual mechanism of bisbenzimidazoles to modulate radiation damage to DNA

The complexing of histones with DNA and the resulting condensation of chromatin protects mammalian cell, from radiation-induced strand breakage. In the present study, benzimidazoles DMA and TBZ showed marked radioprotection through drug-induced compaction of chromatin and direct quenching of free radicals generated by radiation. The mammalian cells were incubated with 100 μM concentration of DMA and TBZ and irradiated at 5 Gy; both the ligands showed nuclei condensation suggesting a probable mechanism to protect DNA from radiation damage. The bisubstituted analogs of Hoechst 33342 are found to be better free radical scavengers and protect DNA against radiation-induced damage at a lower concentration than the parent molecule. Both the ligands also quenched free radicals in isolated free radical system suggesting their dual mode of action against radiation-induced damage to DNA. Molecules binding to the chromatin alter gene expression, whereas in this study both the ligands have not shown any profound effect on the nucleosome assembly and gene expression in vitro and in vivo. Both ligands afford a 2-fold protection by altering DNA structure as well as through direct free radical quenching in bulk solution in comparison to the parent ligand, which acts only through quenching of free radicals. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Stabilization of DNA duplex by 2-substituted adenine as a minor groove modifier

2-(1-Naphthalenylethynyl)-2'-deoxyadenosine ((N)A) was synthesized and incorporated into oligodeoxynucleotides. DNA duplexes containing newly designed 5'-(N)AT-3'/3'-T(N)A-5' base pairs are considerably stabilized than unmodified duplexes by stacking interaction of naphthalene rings in the narrow minor groove as characterized by a new emission at longer wavelength and exciton coupled CD signals.     

Synthesis and Studies of Modified Oligonucleotides- Directed Triple Helix Formation at the Purine-Pyrimidine Interrupted Site

Abstract

Triple helix formation is still restricted to oligopurine-oligopyrimidine double stranded DNA target. Herein we focus on our progress achieved in nucleobase and oligonucleotide modifications area to address the chemical challenge to circumvent the recognition of a purine-pyrimidine base pair interruption in an oligopyrimidine-oligopurine DNA sequence.     

Electron microscopic studies of sequence-specific recognition of duplex DNA by different ligands

The following ligands were used to study sequence specific recognition of duplex DNA by electron microscopic techniques: methyltransferases BspR1 and EcoR124 (recognition sequences GGCC and GAAN7RTCG, respectively), a biotinylated deoxyoligonucleotide 5′-CTCTCTCTCTCTCT-3′ capable of forming triplex DNA, and PNA oligomer H-T₁₀-LysNH₂. For each ligand the best conditions for electron microscopic (EM)detection of stable specific complex formation were determined. It was demonstrated that EM allowed us to determine the position of the individual target site with an error of 15–20 bp, the relative affinities for individual target sites and kinetic parameters of the binding. These results open new possibilities for EM investigations of sequence-specific interactions with a wide range of other ligands of a similar nature. They also imply that a wide range of different sequences can be unambiguously and precisely mapped by EM and greatly extend the scope of EM applications for physical mapping of genomic DNA.     

Analysis of GAA/TTC DNA triplexes using nuclear magnetic resonance and electrospray ionization mass spectrometry

The formation of a GAA/TTC DNA triplex has been implicated in Friedreich's ataxia. The destabilization of GAA/TTC DNA triplexes either by pH or by binding to appropriate ligands was analyzed by nuclear magnetic resonance (NMR) and positive-ion electrospray mass spectrometry. The triplexes and duplexes were identified by changes in the NMR chemical shifts of H8, H1, H4, 15N7, and 15N4. The lowest pH at which the duplex is detectable depends upon the overall stability and the relative number of Hoogsteen C composite function G to T composite function A basepairs. A melting pH (pHm) of 7.6 was observed for the destabilization of the (GAA)2T4(TTC)2T4(CTT)2 triplex to the corresponding Watson-Crick duplex and the T4(CTT)2 overhang. The mass spectrometric analyses of (TTC)6.(GAA)6 composite function(TTC)6 triplex detected ions due to both triplex and single-stranded oligonucleotides under acidic conditions. The triplex ions disappeared completely at alkaline pH. Duplex and single strands were detectable only at neutral and alkaline pH values. Mass spectrometric analyses also showed that minor groove-binding ligands berenil, netropsin, and distamycin and the intercalating ligand acridine orange destabilize the (TTC)6.(GAA)6 composite function (TTC)6 triplex. These NMR and mass spectrometric methods may function as screening assays for the discovery of agents that destabilize GAA/TTC triplexes and as general methods for the characterization of structure, dynamics, and stability of DNA and DNA-ligand complexes.     

Conjugation of d-glucosamine to bovine trypsin increases thermal stability and alters functional properties

d-glucosamine was conjugated to bovine trypsin by carbodiimide chemistry, involving a water-soluble carbodiimide and a succinimide ester, with the latter being to increase the yield of the conjugation. Mass spectrometric data suggested that several glycoforms were formed, with around 12 d-glucosamine moieties coupled to each trypsin molecule on average. The moieties were probably coupled to eight carboxyl groups (of glutamyl and aspartyl residues) and to four tyrosyl residues on the surface of the enzyme. The glycated trypsin possessed increased thermal stability. When compared with its unmodified counterpart, T_50% was increased by 7 °C, thermal inactivation of the first step was increased 34%, and long-term stability assay revealed 71-times higher residual activity at 25 °C (without stabilizing Ca²⁺ ions in aqueous buffer) after 67 days. Furthermore, resistance against autolysis was increased almost two-fold. Altered functional properties of the glycated trypsin were also observed. The glycated trypsin was found to become increasingly basophilic, and was found to be slightly structurally altered. This was indicated by 1.2 times higher catalytic efficiency (k_cat/K_m) than unmodified trypsin against the substrate N-α-benzoyl-l-arginine-p-nitroanilide. Circular dichroism spectropolarimetry suggested a minor change in spatial arrangement of α-helix/helices, resulting in an increased affinity of the glycated trypsin for this small synthetic substrate.     

Selectively 13C-enriched DNA: 13C and 1H assignments of a triple helix by two-dimensional relayed HMQC experiments

Summary We present NMR studies of an intramolecular triple helix, the three strands of which have been linked by a hexaethylene glycol chain. To overcome the generally encountered difficulties of assignment in the homopyrimidine strands, the carbon C1 of the pyrimidines were selectively ¹³C-enriched. Assignments of the aromatic and sugar protons were obtained from NOESY-HMQC and TOCSY-HMQC spectra. We show that the recognition of a DNA duplex by a third strand via triplex formation is easily carried out in solution by observing the changes of the ¹H1–¹³C1 connectivities as a function of pH. Furthermore, the conformation of the sugars has been found to be C2-endo, on the basis of the coupling constant values directly measured in an HSQC spectrum.