Structure–Activity Studies on the Fluorescent Indicator in a Displacement Assay for the Screening of Small Molecules Binding to RNA

A series of xanthone and thioxanthone derivatives with aminoalkoxy substituents were synthesized as fluorescent indicators for a displacement assay in the study of small‐molecule–RNA interactions. The RNA‐binding properties of these molecules were investigated in terms of the improved binding selectivity to the loop region in the RNA secondary structure relative to 2,7‐bis(2‐aminoethoxy)xanthone (X2S) by fluorimetric titration and displacement assay. An 11‐mer double‐stranded RNA and a hairpin RNA mimicking the stem loop IIB of Rev response element (RRE) RNA of HIV‐1 mRNA were used. The X2S derivatives with longer aminoalkyl substituents showed a higher affinity to the double‐stranded RNA than the parent molecule. Introduction of a methyl group on the aminoethoxy moiety of X2S effectively modulated the selectivity to the RNA secondary structure. Methyl group substitution at the C1′ position suppressed the binding to the loop regions. Substitution with two methyl groups on the amino nitrogen atom resulted in reducing the affinity to the double‐stranded region by a factor of 40 %. The effect of methyl substitution on the amino nitrogen atom was also observed for a thioxanthone derivative. Titration experiments, however, suggested that thioxanthone derivatives showed a more prominent tendency of multiple binding to RNA than xanthone derivatives. The selectivity index calculated from the affinity to the double‐stranded and loop regions suggested that the N,N‐dimethyl derivative of X2S would be suitable for the screening of small molecules binding to RRE.     

Naphthyridine‐Benzoazaquinolone: Evaluation of a Tricyclic System for the Binding to (CAG)n Repeat DNA and RNA

The expansion of CAG repeats in the human genome causes the neurological disorder Huntington's disease. The small‐molecule naphthyridine‐azaquinolone NA we reported earlier bound to the CAG/CAG motif in the hairpin structure of the CAG repeat DNA. In order to investigate and improve NA ‐binding to the CAG repeat DNA and RNA, we conducted systematic structure‐binding studies of NA to CAG repeats. Among the five new NA derivatives we synthesized, surface plasmon resonance (SPR) assay showed that all of the derivatives modified from amide linkages in NA to a carbamate linkage failed to bind to CAG repeat DNA and RNA. One derivative, NBzA , modified by incorporating an additional ring to the azaquinolone was found to bind to both d(CAG)₉ and r(CAG)₉. NBzA binding to d(CAG)₉ was similar to NA binding in terms of large changes in the SPR assay and circular dichroism (CD) as well as pairwise binding, as assessed by electron spray ionization time‐of‐flight (ESI‐TOF) mass spectrometry. For the binding to r(CAG)₉, both NA and NBzA showed stepwise binding in ESI‐TOF MS, and NBzA ‐binding to r(CAG)₉ induced more extensive conformational change than NA ‐binding. The tricyclic system in NBzA did not show significant effects on the binding, selectivity, and translation, but provides a large chemical space for further modification to gain higher affinity and selectivity. These studies revealed that the linker structure in NA and NBzA was suitable for the binding to CAG DNA and RNA, and that the tricyclic benzoazaquinolone did not interfere with the binding.     

Formation of a Ligand‐Assisted Complex of Two RNA Hairpin Loops

The hairpin structure is one of the most common secondary structures in RNA and holds a central position in the stream of RNA folding from a non‐structured RNA to structurally complex and functional ribonucleoproteins. Since the RNA secondary structure is strongly correlated to the function and can be modulated by the binding of small molecules, we have investigated the modulation of RNA folding by a ligand‐assisted formation of loop–loop complexes of two RNA hairpin loops. With a ligand (NCT6), designed based on the ligand binding to the G–G mismatches in double‐stranded DNA, we successfully demonstrated the formation of both inter‐ and intra‐molecular NCT6‐assisted complex of two RNA hairpin loops. NCT6 selectively bound to the two hairpin loops containing (CGG)₃ in the loop region. Native polyacrylamide gel electrophoresis analysis of two doubly‐labeled RNA hairpin loops clearly showed the formation of intermolecular NCT6‐assisted loop–loop complex. Förster resonance energy‐transfer studies of RNA constructs containing two hairpin loops, in which each hairpin was labeled with Alexa488 and Cy3 fluorophores, showed the conformational change of the RNA constructs upon binding of NCT6. These experimental data showed that NCT6 simultaneously bound to two hairpin RNAs at the loop region, and can induce the conformational change of the RNA molecule. These data strongly support that NCT6 functions as molecular glue for two hairpin RNAs.     

Efficient Access to Peptidyl‐RNA Conjugates for Picomolar Inhibition of Non‐ribosomal FemXWv Aminoacyl Transferase

Peptidyl–RNA conjugates have various applications in studying the ribosome and enzymes participating in tRNA‐dependent pathways such as Fem transferases in peptidoglycan synthesis. Herein a convergent synthesis of peptidyl–RNAs based on Huisgen–Sharpless cycloaddition for the final ligation step is developed. Azides and alkynes are introduced into tRNA and UDP‐MurNAc‐pentapeptide, respectively. Synthesis of 2′‐azido RNA helix starts from 2′‐azido‐2′‐deoxyadenosine that is coupled to deoxycytidine by phosphoramidite chemistry. The resulting dinucleotide is deprotected and ligated to a 22‐nt RNA helix mimicking the acceptor arm of Ala‐tRNA^Ala by T4 RNA ligase. For alkyne UDP‐MurNAc‐pentapeptide, meso‐cystine is enzymatically incorporated into the peptidoglycan precursor and reduced, and L ‐Cys is converted to dehydroalanine with O‐(mesitylenesulfonyl)hydroxylamine. Reaction of but‐3‐yne‐1‐thiol with dehydroalanine affords the alkyne‐containing UDP‐MurNAc‐pentapeptide. The Cu^I‐catalyzed azide alkyne cycloaddition reaction in the presence of tris[(1‐hydroxypropyl‐1H‐1,2,3‐triazol‐4‐yl)methyl]amine provided the peptidyl‐RNA conjugate, which was tested as an inhibitor of non‐ribosomal FemX_Wv aminoacyl transferase. The bi‐substrate analogue was found to inhibit FemX_Wv with an IC₅₀ of (89±9) pM , as both moieties of the peptidyl–RNA conjugate contribute to high‐affinity binding.     

Triple‐Helix‐Stabilizing Effects in Collagen Model Peptides Containing PPII‐Helix‐Preorganized Diproline Modules

Collagen model peptides (CMPs) serve as tools for understanding stability and function of the collagen triple helix and have a potential for biomedical applications. In the past, interstrand cross‐linking or conformational preconditioning of proline units through stereoelectronic effects have been utilized in the design of stabilized CMPs. To further study the effects determining collagen triple helix stability we investigated a series of CMPs containing synthetic diproline‐mimicking modules (ProMs), which were preorganized in a PPII‐helix‐type conformation by a functionalizable intrastrand C₂ bridge. Results of CD‐based denaturation studies were correlated with calculated (DFT) conformational preferences of the ProM units, revealing that the relative helix stability is mainly governed by an interplay of main‐chain preorganization, ring‐flip preference, adaptability, and steric effects. Triple helix integrity was proven by crystal structure analysis and binding to HSP47.     

Synthesis of Naphthyridine Dimers with Conformational Restriction and Binding to DNA and RNA

One of the important determinants in the efficiency of a molecular interaction is the necessity for conformational changes in host and/or guest molecules upon binding. In small‐molecule interactions with nucleic acids, conformational changes on both molecules are often involved, especially in intercalating binding. Mismatch binding ligands (MBLs) we described here consist of two heterocycles that predominantly exist in one conformation, so it is of interest to determine if such molecules can bind to any DNA and RNA structures. One molecule, 1 ‐NHR, which predominantly exists as the unstacked conformation in aqueous solvent, has been successfully synthesized and characterized. Compound 1 ‐NHR did not efficiently bind to GX/Y DNA and RNA sequences, but the binding pattern is different from that of authentic MBL naphthyridine carbamate dimer. In vitro selection of RNA that specifically binds to 1 ‐NHR was performed from pre‐miR‐29a loop library RNA, and one RNA, to which 1 ‐NHR bound with high affinity, has been successfully identified. Although it was anticipated that 1 ‐NHR, with a predominantly unstacked conformation, would show entropy‐driven binding, isothermal titration calorimetry analysis suggested that the binding of 1 ‐NHR to RNA was enthalpy driven with an apparent K _d of about 100 nm .     

The crystal structure of the CeNA:RNA hybrid ce(GCGTAGCG):r(CGCUACGC)

Cyclohexenyl nucleic acids (CeNA) are characterised by the carbon–carbon double bond replacing the O4′‐oxygen atom of the natural D ‐2′‐deoxyribose sugar ring in DNA. CeNAs exhibit a high conformational flexibility, are stable against nuclease activity and their hybridisation is RNA selective. Additionally, CeNA has been shown to induce an enhanced biological activity when incorporated in siRNA. This makes CeNA a good candidate for siRNA and synthetic aptamer applications. The crystal structure of the synthetic CeNA:RNA hybrid ce(GCGTAGCG):r(CGCUACGC) has been solved with a resolution of 2.50 Å. The CeNA:RNA duplex adopts an anti‐parallel, right‐handed double helix with standard Watson–Crick base pairing. Analyses of the helical parameters revealed the octamer to form an A‐like double helix. The cyclohexenyl rings mainly adopt the ³H₂ conformation, which resembles the C3′‐endo conformation of RNA ribose ring. This C3′‐endo ring puckering was found in most of the RNA residues and is typical for A‐family helices. The crystal structure is stabilised by the presence of hexahydrated magnesium ions. The fact that the CeNA:RNA hybrid adopts an A‐type double helical conformation confirms the high potential of CeNAs for the construction of efficient siRNAs which can be used for therapeutical applications.     

Use of an Octapeptide–Guanidiniocarbonylpyrrole Conjugate for the Formation of a Supramolecular β‐Helix that Self‐Assembles into pH‐Responsive Fibers

Peptides that adopt β‐helix structures are predominantly found in transmembrane protein domains or in the lipid bilayer of vesicles. Constructing a β‐helix structure in pure water has been considered difficult without the addition of membrane mimics. Herein, we report such an example; peptide 1 self‐assembles into a supramolecular β‐helix in pure water based on charge interactions between the individual peptides. Peptide 1 further showed intriguing transitions from small particles to helical fibers in a time‐dependent process. The fibers can be switched to vesicles by changing the pH value.     

Sensing of Double‐Stranded DNA/RNA Secondary Structures by Water Soluble Homochiral Perylene Bisimide Dyes

A broad series of homochiral perylene bisimide (PBI) dyes were synthesized that are appended with amino acids and cationic side chains at the imide positions. Self‐assembly behavior of these ionic PBIs has been studied in aqueous media by UV/Vis spectroscopy, revealing formation of excitonically coupled H‐type aggregates. The interactions of these ionic PBIs with different ds‐DNA and ds‐RNA have been explored by thermal denaturation, fluorimetric titration and circular dichroism (CD) experiments. These PBIs strongly stabilized ds‐DNA/RNA against thermal denaturation as revealed by high melting temperatures of the formed PBI/polynucleotide complexes. Fluorimetric titrations showed that these PBIs bind to ds‐DNA/RNA with high binding constants depending on the number of the positive charges in the side chains. Thus, spermine‐containing PBIs with six positive charges each showed higher binding constants (logK_s=9.2–9.8) than their dioxa analogues (logK_s=6.5–7.9) having two positive charges each. Induced circular dichroism (ICD) of PBI assemblies created within DNA/RNA grooves was observed. These ICD profiles are strongly dependent on the steric demand of the chiral substituents of the amino acid units and the secondary structure of the DNA or RNA. The observed ICD effects can be explained by non‐covalent binding of excitonically coupled PBI dimer aggregates into the minor groove of DNA and major groove of RNA which is further supported by molecular modeling studies.     

Guanidiniocarbonylpyrrole–Aryl Derivatives: Structure Tuning for Spectrophotometric Recognition of Specific DNA and RNA Sequences and for Antiproliferative Activity

We present a systematic study of different guanidiniocarbonylpyrrole‐aryl derivatives designed to interact with DNA or RNA both through intercalation of an aromatic moiety into the base stack of the nucleotide and through groove binding of a guanidiniocarbonylpyrrole cation. We varied 1) the size of the aromatic ring (benzene, naphthalene, pyrene and acridine), 2) the length and flexibility of the linker connecting the two binding groups, and 3) the total number of positive charges present at different pH values. The compounds and their interactions with DNA and RNA were studied by UV/Vis, fluorescence and CD spectroscopy. Antiproliferative activities against human tumour cell lines were also determined. Our studies show that efficient interaction with, for example, DNA requires a significantly large aromatic ring (pyrene) connected through a flexible linker to the pyrrole moiety. However, a positive charge, as in 12 , is also needed. Compound 12 allows for base‐pair‐selective recognition of ds‐DNA at physiological pH values. The antiproliferative activities of these compounds correlate with their binding affinities towards DNA, suggesting that their biological effects are most probably due to DNA binding.     

A gold nanoparticle based approach for screening triplex DNA binders

Nanoparticle assemblies interconnected with DNA triple helixes can be used to colorimetrically screen for triplex DNA binding molecules and simultaneously determine their relative binding affinities based on melting temperatures. Nanoparticles assemble only when DNA triple helixes form between DNA from two different particles and a third strand of free DNA. In addition, the triple helix structure is unstable at room temperature and only forms in the presence of triplex DNA binding molecules which stabilize the triple helix. The resulting melting transition of the nanoparticle assembly is much sharper and at a significantly higher Tm than the analogous triplex structure without nanoparticles. Upon nanoparticle assembly, a concomitant red-to-blue color change occurs. The assembly process and color change do not occur in the presence of duplex DNA binders and therefore provide a significantly better screening process for triplex DNA binding molecules compared to standard methods.     

Native Top‐Down Mass Spectrometry of TAR RNA in Complexes with a Wild‐Type tat Peptide for Binding Site Mapping

Ribonucleic acids (RNA) frequently associate with proteins in many biological processes to form more or less stable complex structures. The characterization of RNA–protein complex structures and binding interfaces by nuclear magnetic resonance (NMR) spectroscopy, X‐ray crystallography, or strategies based on chemical crosslinking, however, can be quite challenging. Herein, we have explored the use of an alternative method, native top‐down mass spectrometry (MS), for probing of complex stoichiometry and protein binding sites at the single‐residue level of RNA. Our data show that the electrostatic interactions between HIV‐1 TAR RNA and a peptide comprising the arginine‐rich binding region of tat protein are sufficiently strong in the gas phase to survive phosphodiester backbone cleavage of RNA by collisionally activated dissociation (CAD), thus allowing its use for probing tat binding sites in TAR RNA by top‐down MS. Moreover, the MS data reveal time‐dependent 1:2 and 1:1 stoichiometries of the TAR–tat complexes and suggest structural rearrangements of TAR RNA induced by binding of tat peptide.     

Shape-Selective Targeting on RNA Bulges by Peptidomimetic Metallohelices

RNA bulges represent one of the most common motifs in the RNA secondary structure and serve in a variety of biological functions. Compounds stabilizing RNA bulges are important for probing RNA structure and function and for therapy of some diseases. Here, the ability of a series of enantiomeric pairs of optically pure bimetallic metallohelices with different flexible linkers to target various RNA bulges is investigated. The results show that binding affinities of the metallohelices to bulged RNA differ and strongly depend on the size of the bulge and the base composition of the bulge loop. Notably, the shorter, more compact, and less flexible metallohelices bind to RNA bulges most efficiently and selectively. Interestingly, the ability of the metallohelices to bind to RNA bulges correlates with their previously reported antimicrobial activity, which suggests that the selective recognition of bulged regions in RNA by the metallohelices might also contribute to their biological activity.     

Minimum sequence requirements for selective RNA-ligand binding: a molecular mechanics algorithm using molecular dynamics and free-energy techniques

In vitro evolution techniques allow RNA molecules with unique functions to be developed. However, these techniques do not necessarily identify the simplest RNA structures for performing their functions. Determining the simplest RNA that binds to a particular ligand is currently limited to experimental protocols. Here, we introduce a molecular-mechanics based algorithm employing molecular dynamics simulations and free-energy methods to predict the minimum sequence requirements for selective ligand binding to RNA. The algorithm involves iteratively deleting nucleotides from an experimentally determined structure of an RNA-ligand complex, performing energy minimizations and molecular dynamics on each truncated structure, and assessing which truncations do not prohibit RNA binding to the ligand. The algorithm allows prediction of the effects of sequence modifications on RNA structural stability and ligand-binding energy. We have implemented the algorithm in the AMBER suite of programs, but it could be implemented in any molecular mechanics force field parameterized for nucleic acids. Test cases are presented to show the utility and accuracy of the methodology.