Site-specific binding of chelerythrine and sanguinarine to single pyrimidine bulges in hairpin DNA

Spectrofluorometric titration, electrospray ionization time-of-flight mass spectrometric and UV melting methods were employed to study the binding of chelerythrine and sanguinarine to bulged DNA. The results showed that both alkaloids bind specifically to single pyrimidine (C, T) bulge sites. The ability of sanguinarine to bind to both regular and bulged hairpins was found to be stronger than that of chelerythrine, but the binding selectivity of chelerythrine toward single-base bulges was much larger than that of sanguinarine. Figure Association constants for chelerythrine and sanguinarine toward regular and single-base bulged hairpins obtained from fluorometric analysis     

Crystal structures of an A-form duplex with single-adenosine bulges and a conformational basis for site-specific RNA self-cleavage

Background: Bulged nucleotides are common secondary structural motifs in RNA molecules and are often involved in RNA-RNA and RNA-protein interactions. RNA is selectively cleaved at bulge sites (when compared to other sites within stems) in the presence of divalent metal cations. The effects of bulge nucleotides on duplex stability and topology have been extensively investigated, but no detailed X-ray structures of bulge-containing RNA fragments have been available.Results: We have crystallized a self-complementary RNA-DNA chimeric 11-nucleotide sequence containing single-adenosine bulges under two different conditions, giving two distinct crystal forms. In both lattices the adenosines are looped out, leaving the stacking interactions in the duplex virtually unaffected. The bulges cause the duplex to kink in both cases. In one of the structures, the conformation of the bulged nucleotide places its modeled 2′-oxygen in line with the adjacent phosphate on the 3′ side, where it is poised for nucleophilic attack.Conclusions: Single adenosine bulges cause a marked opening of the normally narrow RNA major groove in both crystal structures, rendering the bases more accessible to interacting molecules compared with an intact stem. The geometries around the looped-out adenosines are different in the two crystal forms, indicating that bulges can confer considerable local plasticity on the usually rigid RNA double helix. The results provide a conformational basis for the preferential, metal-assisted self-cleavage of RNA at bulged sites.     

Battle for the bulge: directing small molecules to DNA and RNA defects

Small molecules were tailored to specifically bind bulged DNA by complementing the geometry and nucleotide size of the bulge site. The prospect of generating small molecules that influence the secondary structure of DNA and RNA holds great promise for clinical applications.     

Structural basis for bifunctional zinc(II) macrocyclic complex recognition of thymine bulges in DNA

The zinc(II) complex of 1-(4-quinoylyl)methyl-1,4,7,10-tetraazacyclododecane (cy4q) binds selectively to thymine bulges in DNA and to a uracil bulge in RNA. Binding constants are in the low-micromolar range for thymine bulges in the stems of hairpins, for a thymine bulge in a DNA duplex, and for a uracil bulge in an RNA hairpin. Binding studies of Zn(cy4q) to a series of hairpins containing thymine bulges with different flanking bases showed that the complex had a moderate selectivity for thymine bulges with neighboring purines. The dissociation constants of the most strongly bound Zn(cy4q)-DNA thymine bulge adducts were 100-fold tighter than similar sequences with fully complementary stems or than bulges containing cytosine, guanine, or adenine. In order to probe the role of the pendent group, three additional zinc(II) complexes containing 1,4,7,10-tetraazacyclododecane (cyclen) with aromatic pendent groups were studied for binding to DNA including 1-(2-quinolyl)methyl-1,4,7,10-tetraazacyclododecane (cy2q), 1-(4-biphenyl)methyl-1,4,7,10-tetraazacyclododecane (cybp), and 5-(1,4,7,10-tetraazacyclododecan-1-ylsulfonyl)-N,N-dimethylnaphthalen-1-amine (dsc). The Zn(cybp) complex binds with moderate affinity but little selectivity to DNA hairpins with thymine bulges and to DNA lacking bulges. Similarly, Zn(dsc) binds weakly both to thymine bulges and hairpins with fully complementary stems. The zinc(II) complex of cy2q has the 2-quinolyl moiety bound to the Zn(II) center, as shown by (1)H NMR spectroscopy and pH-potentiometric titrations. As a consequence, only weak (500 μM) binding is observed to DNA with no appreciable selectivity. An NMR structure of a thymine-bulge-containing hairpin shows that the thymine is extrahelical but rotated toward the major groove. NMR data for Zn(cy4q) bound to DNA containing a thymine bulge is consistent with binding of the zinc(II) complex to the thymine N3(-) and stacking of the quinoline on top of the thymine. The thymine-bulge bound zinc(II) complex is pointed into the major groove, and there are interactions with the guanine positioned 5' to the thymine bulge.     

Recognition of thymine in DNA bulges by a Zn(II) macrocyclic complex

A Zn(II) macrocyclic complex with appended quinoline is a bifunctional recognition agent that uses both the Zn(II) center and the pendent aromatic group to bind to thymine in bulges with good selectivity over DNA containing G, C or A bulges. Spectroscopic studies show that the stem containing the bulge stays largely intact in a DNA hairpin with the Zn(II) complex bound to the thymine bulge.     

Convenient synthesis of NCS-chromophore metabolite isosteres: binding agents for bulged DNA microenvironments

A designed molecule with capacity to bind DNA bulges (20) has been prepared from readily available starting materials. The spirocyclic template was modeled on a metabolite of neocarzinostatin chromophore (NCSi-gb) and is equipped with functionality to enable convenient bioassay. Preliminary studies confirm binding at specific bulged sequences and induction of polymerase-mediated slippage events. The target compound offers a convenient means to study affinity for unique bulged motifs and for use as a molecular biology reagent.     

Structure-based computational database screening, in vitro assay, and NMR assessment of compounds that target TAR RNA

There has been little prior effort to discover new drugs on the basis of a unique RNA structure. Binding of the viral transactivator Tat to the 5' bulge of the transactivation response (TAR) element is necessary for HIV-1 replication, so TAR RNA is a superb target. A computational approach was developed to screen a large chemical library for binding to a three-dimensional RNA structure. Scoring function development, flexible ligand docking, and limited target flexibility were essential. From the ranked list of compounds predicted to bind TAR, 43 were assayed for inhibition of the Tat-TAR interaction via electrophoretic mobility shift assays. Eleven compounds (between 0.1 and 1 microM) inhibited the Tat-TAR interaction, and some inhibited Tat transactivation in cells. NMR spectra verified specific binding to the 5' bulge and no interaction with other regions of TAR.     

Bulged Guanine is Uniquely Sensitive to Damage Caused by Visible‐Light Irradiation of Ethidium Bound to DNA: A Possible Role in Mutagenesis

The interaction of ethidium bromide (=3,8‐diamino‐5‐ethyl‐6‐phenylphenanthridinium bromide; EB) with a series of duplex DNA oligomers having single‐base bulges and single‐base mis‐pairs was investigated (Fig. 1). Physical and spectroscopic analysis reveals no definitive evidence for selective binding of EB at the bulge or mis‐pair. However, irradiation of the bound EB with VIS light leads to lesions in the DNA selectively in the sequence having a bulged guanine. This reaction is attributed to the formation of an exciplex between the lowest excited singlet state of the EB and the bulged guanine. The exciplex is trapped by H₂O, which initiates a sequence of reactions that lead to piperidine‐requiring strand cleavage at this site. Significantly, the damaged bulged guanine is not recognized by E. coli formamidopyrimidine glycosylase (Fpg), which is part of a base‐excision repair system for oxidative damage to DNA. Thus, DNA containing a bulged guanine and having a bound intercalator may be irreparably damaged by exposure to VIS light, even though normal duplex DNA is relatively inert under these conditions.     

Identification of ligands for the Tau exon 10 splicing regulatory element RNA by using dynamic combinatorial chemistry

We describe the use of dynamic combinatorial chemistry (DCC) to identify ligands for the stem-loop structure located at the exon 10-5'-intron junction of Tau pre-mRNA, which is involved in the onset of several tauopathies including frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17). A series of ligands that combine the small aminoglycoside neamine and heteroaromatic moieties (azaquinolone and two acridines) have been identified by using DCC. These compounds effectively bind the stem-loop RNA target (the concentration required for 50% RNA response (EC(50)): 2-58 μM), as determined by fluorescence titration experiments. Importantly, most of them are able to stabilize both the wild-type and the +3 and +14 mutated sequences associated with the development of FTDP-17 without producing a significant change in the overall structure of the RNA (as analyzed by circular dichroism (CD) spectroscopy), which is a key factor for recognition by the splicing regulatory machinery. A good correlation has been found between the affinity of the ligands for the target and their ability to stabilize the RNA secondary structure.     

RNA binding and thiolytic stability of a quinoline-containing helix-threading peptide

Helix-threading peptides (HTPs) bind selectively to sites predisposed to intercalation in folded RNA molecules placing peptide functional groups into the dissimilar grooves of the duplex. Here we report the design and synthesis of new HTPs with quinoline as the intercalation domain. A quinoline-containing HTP is shown to bind selectively to duplex RNA binding sites. Furthermore, the affinity cleavage pattern generated using an EDTA.Fe modified derivative is consistent with minor groove localization of its N-terminus. This compound binds base-pair steps flanked by single nucleotide bulges on the 3' side on both strands, whereas bulges on the 5' side of the intercalation site do not support binding. Furthermore, unlike acridine HTPs, the quinoline compound is resistant to thiolytic degradation that leads to loss of RNA-binding activity. The RNA-binding selectivity and stability observed for quinoline-containing HTPs make them excellent candidates for further development as regulators of intracellular RNA function.     

Targeting DNA bulged microenvironments with synthetic agents: lessons from a natural product

Bulged regions of nucleic acids are important structural motifs whose function has been linked to a number of key nuclear processes. Additionally, bulged intermediates have been implicated in the etiology of several genetic diseases and as targets for viral regulation. Despite these obvious ramifications, few molecules are capable of selective binding to bulged sequences. Prompted by the remarkable affinity of a natural product metabolite, we have designed and prepared a series of readily accessible synthetic agents with selective bulge binding activity. Furthermore, by screening a library of bulge-containing oligodeoxynucelotides, correlations between structure and affinity of the agents can be drawn. In addition to potential applications in molecular biology, the availability of these spirocyclic agents now opens the door for rational drug design.     

Biomechanical analysis of cancerous and normal cells based on bulge generation in a microfluidic device

This paper presents a new biomechanical analysis method for discrimination between cancerous and normal cells through compression by poly(dimethylsiloxane) (PDMS) membrane deflection in a microfluidic device. When a cell is compressed, cellular membrane will expand and then small bulges will appear on the peripheral cell membrane beyond the allowable strain. It is well known that the amount of F-actin in cancer cells is less than that of normal cells and bulges occur at the sites where cytoskeleton becomes detached from the membrane bilayer. Accordingly, we have demonstrated the difference of the bulge generation between breast cancer cells (MCF7) and normal cells (MCF10A). After excessive deformation, the bulges generated in MCF7 cells were not evenly distributed on the cell periphery. Contrary to this, the bulges of MCF10A cells showed an even distribution. In addition, the morphologies of bulges of MCF7 and MCF10A cells looked swollen protrusion and tubular protrusion, respectively. Peripheral strains at the moment of the bulge generation were also 72% in MCF7 and 46% in MCF10A. The results show that the bulge generation can be correlated with the cytoskeleton quantity inside the cell, providing the first step of a new biomechanical approach.     

Spirocyclic helical compounds as binding agents for bulged RNA, including HIV-2 TAR

Based on fluorescence binding studies and 1D 1H NMR studies, designed synthetic analogues of NCSi-gb bind specifically with two-base bulged RNA, including HIV-2 TAR RNA, making them potential lead compounds for antiviral drug development.     

A route for preparing new neamine derivatives targeting HIV-1 TAR RNA

In the search for molecules possessing antibiotic or antiviral properties and ribonuclease like activity, that is, able to induce the cleavage of bacterial or viral RNA targets, we report a new route for preparing selectively neamine derivatives modified at their 4′- and/or 5-hydroxyl functions. Using trityl protective groups for the amino functions and 4-methoxybenzyl groups for the hydroxyl functions, new neamine derivatives, such as histidine, phenanthroline, flavin, adenine conjugates were efficiently obtained after a single deprotection step under acid conditions. For the first time, 4′-modified neamine derivatives were prepared. Most of the 4′-derivatives showed affinity and selectivity for TAR RNA close to those of the corresponding 5-derivatives. The most potent compound is the 4′-histidine derivative 31 which binds more tightly to TAR RNA compared to its 5-isomer and neamine and recognizes selectively TAR oligonucleotides having a bulge.     

Targeting abasic sites and single base bulges in DNA with metalloinsertors

The site-specific recognition of abasic sites and single base bulges in duplex DNA by sterically expansive rhodium metalloinsertors has been investigated. Through DNA photocleavage experiments, Rh(bpy)2(chrysi)3+ is shown to bind both abasic sites and single base bulges site-specifically and, upon irradiation, to cleave the backbone of the defect-containing DNA. Photocleavage titrations reveal that the metal complex binds DNA containing an abasic site with high affinity (2.6(5) x 106 M-1), comparably to the metalloinsertor and a CC mismatch. The complex binds single base bulge sites with lower affinity (approximately 105 M-1). Analysis of cleavage products and the correlation of affinities with helix destabilization suggest that Rh(bpy)2(chrysi)3+ binds both lesions via metalloinsertion, as observed for Rh binding at mismatched sites, a binding mode in which the mismatched or unpaired bases are extruded from the helix and replaced in the base stack by the sterically expansive ligand of the metalloinsertor.     

Cleavage of RNA bulge loops by artificial RNases

Sensitivity of phosphodiester bonds in RNA bulge loops to cleavage by short cationic peptides and compounds based on 1,4-diazabicyclo[2.2.2]octane and its conjugates with imidazole was studied. Bulge loops containing from one to seven nucleotides were formed in RNA upon its hybridization with partially complementary oligodeoxyribonucleotides. The efficiency of RNA cleavage depends on the length of a bulge loop, the position of the cleaved phosphodiester bond in the loop, and the nature of the RNA-binding fragment of chemical ribonuclease (1,4-diazabicyclo[2.2.2]octane or a cationic peptide). In the absence of Mg²⁺ ions, the phosphodiester bond in the CA motif located in the apical position in 4-, 6-, or 7-membered loops is cleaved with the highest efficiency. In the presence of magnesium ions, the selectivity of RNA cleavage within bulge loops is substantially enhanced. In the case of 1,4-diazabicyclo[2.2.2]octane-based compounds, RNA is subjected to cleavage predominantly at the bonds in 4-, 6-, and 7-membered loops, whereas cleavage of other bonds is greatly suppressed. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 7, pp. 1236–1246, July, 2006.     

Synthesis and NMR binding study of a chiral spirocyclic helical analogue of a natural DNA bulge binder

[reaction: see text] Synthesis of chiral spirocyclic helical compounds which mimic the molecular architecture of the potent DNA bulge binder obtained from the antitumor agent NCS-chrom has been accomplished. Structural analysis of the compounds by CD and NMR is presented. NMR titration study indicates binding of P,alpha-helimer (1d) at a two-base bulge site in a DNA oligomer, providing insight to the design of agents as specific probes of a bulged structure in nucleic acids.     

Differentiate RNA Single‐Stranded Region of the Branched Structures and Hairpin Loops by an Octahedral Cobalt(II) Complex

Single‐stranded RNA molecules usually include secondary structural elements such as bulges, internal loops, and hairpin loops. These RNA secondary structural elements are often essential for the biological activity and functions of the RNA molecule. Chemical probe Co^II(HAPP)(TFA)₂ in the presence of H₂O₂ is found to differentiate single‐stranded RNA from branched structures and hairpin loops. This study uses Co^II(HAPP)(TFA)2 to analyze the structures of two RNA molecules: a fragment of HIV TAR RNA (TAR‐27) and the catalytic domain 5 of group II intron (D5‐29). The electrophoretic mobility of TAR‐27 does not shift in the presence of Co^II(HAPP)(TFA)₂, suggesting that the reagent does not change the conformation of RNA substrate. Cleavage of the RNA substrates by Co^II(HAPP)(TFA)₂ unambiguously differentiated RNA internal looped structures from hairpin loops. The results show that Co^II(HAPP)(TFA)2 is a sensitive, informative and convenient tool for analysis of RNA secondary structures.     

Synergistic DNA- and Protein-Based Recognition Promote an RNA-Templated Bio-orthogonal Reaction

Biomolecular assemblies composed of proteins and oligonucleotides play a central role in biological processes. While in nature, oligonucleotides and proteins usually assemble via non-covalent interactions, synthetic conjugates have been developed which covalently link both modalities. The resulting peptide-oligonucleotide conjugates have facilitated novel biological applications as well as the design of functional supramolecular systems and materials. However, despite the importance of concerted protein/oligonucleotide recognition in nature, conjugation approaches have barely utilized the synergistic recognition abilities of such complexes. Herein, the structure-based design of peptide-DNA conjugates that bind RNA through Watson-Crick base pairing combined with peptide-mediated major groove recognition is reported. Two distinct conjugate families with tunable binding characteristics have been designed to adjacently bind a particular RNA sequence. In the resulting ternary complex, their peptide elements are located in proximity, a feature that was used to enable an RNA-templated click reaction. The introduced structure-based design approach opens the door to novel functional biomolecular assemblies.