A novel RNA motif based on the structure of unusually stable 2',5'-linked r(UUCG) loops

We have recently shown that hairpins containing 2',5'-linked RNA loops exhibit superior thermodynamic stability compared to native hairpins comprised of 3',5'-RNA loops [Hannoush, R. N.; Damha, M. J. J. Am. Chem. Soc. 2001, 123, 12368-12374]. A remarkable feature of the 2',5'-r(UUCG) tetraloop is that, unlike the corresponding 3',5'-linked tetraloop, its stability is virtually independent of the hairpin stem composition. Here, we determine the solution structure of unusually stable hairpins of the sequence 5'-G(1)G(2)A(3)C(4)-(U(5)U(6)C(7)G(8))-G(9)(U/T(10))C(11)C(12)-3' containing a 2',5'-linked RNA (UUCG) loop and either an RNA or a DNA stem. The 2',5'-linked RNA loop adopts a new fold that is completely different from that previously observed for the native 3',5'-linked RNA loop. The 2',5'-RNA loop is stabilized by (a). U5.G8 wobble base pairing, with both nucleotide residues in the anti-conformation, (b). extensive base stacking, and (c). sugar-base and sugar-sugar contacts, all of which contribute to the extra stability of this hairpin structure. The U5:G8 base pair stacks on top of the C4:G9 loop-closing base pair and thus appears as a continuation of the stem. The loop uracil U6 base stacks above U5 base, while the cytosine C7 base protrudes out into the solvent and does not participate in any of the stabilizing interactions. The different sugar pucker and intrinsic bonding interactions within the 2',5'-linked ribonucleotides help explain the unusual stability and conformational properties displayed by 2',5'-RNA tetraloops. These findings are relevant for the design of more effective RNA-based aptamers, ribozymes, and antisense agents and identify the 2',5'-RNA loop as a novel structural motif.     

Expanding functionality of RNA: synthesis and properties of RNA containing imidazole modified tandem G-U wobble base pairs

Imidazole modification at C-5 of uridine that is part of tandem G-U wobble base pairs causes slight reduction of thermal stability (DeltaDeltaG(0)(310) < 0.4 kcal mol(-1)) and relatively small change in hydration of short RNA helices.     

Probing RNA hairpins with cobalt(III)hexammine and electrospray ionization mass spectrometry

In this work, electrospray ionization mass spectrometry (ESI MS) was employed to study the interactions of cobalt(III) hexammine, Co(NH3)6(3+), with five RNA hairpins representing the 790 loop of 16S ribosomal RNA and 1920 loop of 23S ribosomal RNA. The RNAs varied in mismatch identity (G.U versus A.C) and level of base modification (pseudouridine versus uridine). Co(NH3)6(3+) binding was observed with the four RNA hairpins that contained a G.U wobble pair in the stem region. ESI MS revealed 1:1 and 1:2 complex formation with all RNAs. Weaker binding was observed with the fifth RNA hairpin that contained an A.C wobble pair in the stem region. The effects of pH on Co(NH3)6(3+) binding were also examined.     

Kinetic preference for interaction of cisplatin with the G-C-rich wobble basepair region in both tRNAAla and MhAla

The anticancer active complex cisplatin interacts preferentially with the common, G-C rich, wobble base pair region of both tRNA(Ala) and Mh(Ala) in a reaction that at pH 6.3 is rate limited by the acid hydrolysis of the metal complex.     

Platinum interference with siRNA non-seed regions fine-tunes silencing capacity

Knowledge concerning the molecular mechanisms governing the influence of non-coding RNAs on protein production has emerged rapidly during the past decade. Today, two main research areas can be identified, one oriented toward the use of artificially introduced siRNAs for manipulation of gene expression, and the other one focused on the function of endogenous miRNAs. In both cases, the active molecule consists of a ～20-nucleotide-long RNA duplex. In the siRNA case, improved systemic stability is of central interest for its further development toward clinical applications. With respect to miRNA processing and function, understanding its influence on mRNA targeting and the silencing ability of individual miRNAs, e.g., under pathological conditions, remains a scientific challenge. In the present study, a model system is presented where the influence of the two clinically used anticancer drugs, cisplatin and oxaliplatin, on siRNA's silencing capacity has been evaluated. More specifically, siRNAs targeting the 3' UTR region of Wnt-5a mRNA (NM_003352) were constructed, and the biologically active antisense RNA strand was pre-platinated. Platinum adducts were detected and characterized by a combination of gel electrophoresis and MALDI-MS techniques, and the silencing capacity was evaluated in cellular luciferase-expressing systems using HB2 cells. Data show that platination of the antisense strand of the siRNAs results in adducts with protection against hydrolytic cleavage in the proximity of the platination sites, i.e., with altered degradation patterns compared to native RNAs. The MALDI-MS method was successfully used to further identify and characterize platinated RNA, with the naturally occurring platinum isotopic patterns serving as sensitive fingerprints for metalated sites. Expression assays all confirm biological activity of antisense-platinated siRNAs, here with platination sites located outside of the seed region. A significant reduction of silencing capacity was observed as a general trend, however. Of the two complexes studied, oxaliplatin exhibits the larger influence, thus indicating subtle differences between the abilities of cis- and oxaliplatin to interfere with si- and miRNA processing.     

Tandem mass spectrometry of platinated quadruplex DNA

Quadruplexes are higher-order structures formed by G-rich DNA strands that are involved in various processes of cell cycle regulation, such as control of telomere length and participation in gene regulation. Because of these central biological functions, quadruplex DNA represents a promising target for cancer therapy, e.g. by applying organometallic drugs, such as cisplatin. High-resolution electrospray tandem mass spectrometry is evaluated as a technique for exploring structural features of unplatinated and platinated quadruplexes. Results of experiments on tetramolecular, bimolecular and monomolecular quadruplexes provide information about the extent of platination and the binding sites of the drug. The dissociation behavior of the different types of quadruplexes is compared. Tetramolecular quadruplexes were found to weave out a strand end in order to provide a platination site, and their fragmentation is characterized by the release of an unplatinated strand and the formation of a platinated triplex. Partial opening of the structure in combination with the loss of small fragments leads to truncated quadruplex ions. For the bimolecular quadruplexes studied, strand separation is the predominant dissociation pathway. Depending on the loop sequence, cross-linking of the loops by cisplatin is demonstrated. Distinct differences in the product ion spectra of unannealed and annealed monomolecular sequences provide proof of quadruplex formation and show that platination preferentially occurs at the terminal regions.     

Remarkable stability of hairpins containing 2',5'-linked RNA loops.

We report here the results of a comparative study of hairpin loops that differ in the connectivity of phosphodiester linkages (3',5'- versus 2',5'-linkages). In addition, we have studied the effect of changing the stem composition on the thermodynamic stability of hairpin loops. Specifically, we constructed hairpins containing one of six stem duplex combinations, i.e., DNA:DNA ("DD"), RNA:RNA ("RR"), DNA:RNA ("DR"), 2',5'-RNA:RNA ("RR"), 2',5'-RNA:DNA ("RD"), and 2',5'-RNA:2',5'-RNA ("RR"), and one of three tetraloop compositions, i.e., 2',5'-RNA ("R"), RNA ("R"), and DNA ("D"). All hairpins contained the conserved and well-studied loop sequence 5'-...C(UUCG)G...-3' [Cheong et al. Nature 1990, 346, 680-682]. We show that the 2',5'-linked loop C(UUCG)G, i.e.,...C(3'p5')U(2'p5')U(2'p5')C(2'p5')G(2'p5')G(3'p5')..., like its "normal" RNA counterpart, forms an unusually stable tetraloop structure. We also show that the stability imparted by 2',5'-RNA loops is dependent on base sequence, a property that is shared with the regioisomeric 3',5'-RNA loops. Remarkably, we find that the stability of the UUCG tetraloop is virtually independent of the hairpin stem composition (DD, RR, RR, etc.), whereas the native RNA tetraloop exerts extra stability only when the stem is duplex RNA (R:R). As a result, the relative stabilities of hairpins with a 2',5'-linked tetraloop, e.g. ggac(UUCG)gtcc (T(m) = 61.4 degrees C), are often superior to those with RNA tetraloops, e.g. ggac(UUCG)gtcc (T(m) = 54.6 degrees C). In fact, it has been possible to observe the formation of a 2',5'-RNA:DNA hybrid duplex by linking the hybrid's strands to a (UUCG) loop. These duplexes (RD), which are not stable enough to form in an intermolecular complex [Wasner et al. Biochemistry 1998, 37, 7478-7486], were stable at room temperature (T(m) approximately 50 degrees C). Thus, 2',5'-loops have potentially important implications in the study of nucleic acid complexes where structural data are not yet available. Furthermore, they may be particularly useful as structural motifs for synthetic ribozymes and nucleic acid "aptamers".     

Bifunctional binding of cisplatin to DNA: why does cisplatin form 1,2-intrastrand cross-links with ag but not with GA?

The bifunctional binding of the anticancer drug cisplatin to two adjacent nucleobases in DNA is modeled using density functional theory. Previous experimental studies revealed that cisplatin binding to adjacent guanine and adenine is sensitive to nucleobase sequence. Whereas AG 1,2-intrastrand cross-links are commonly observed, the analogous GA adducts are not known. This study focuses on understanding this directional preference by constructing a full reaction profile using quantum chemical simulation methods. Monofunctional and bifunctional cisplatin adducts were generated, and the transition states that connect them were located for the dinucleotides d(pApG) and d(pGpA), assuming that initial platination takes place at the guanine site. Our computer simulations reveal a significant kinetic preference for formation of the AG over the GA adduct. The activation free energies of approximately 23 kcal/mol for AG and approximately 32 kcal/mol for GA suggest that bifunctional closure is approximately 6 orders of magnitude faster for AG than for GA. A strong hydrogen bond between one of the ammine ligands of cisplatin and the 5' phosphate group of the DNA backbone is responsible for the stabilization of the transition state that affords the AG adduct. This interaction is absent in the transition state that leads to the GA adduct because the right-handed helix of the DNA backbone places the phosphate out of reach for the ammine ligand. We found only an insignificant thermodynamic difference between AG and GA adducts and conclude that the preference of AG over GA binding is largely under kinetic control. The puckering of the deoxyribose ring plays an important role in determining the energetics of the bifunctional platination products. Whereas the 3'-nucleoside remains in the native C2'-endo/C3'-exo form of B-DNA, the deoxyribose of the 5'-nucleoside always adopts the C2'-exo/C3'-endo puckering in our simulations. A detailed analysis of the energies and structures of the bifunctional adducts revealed that the observed sugar puckering patterns are necessary for platinum to bind in a relaxed coordination geometry.     

Design of an adenosine analogue that selectively improves the affinity of a mutant U1A protein for RNA

The RNA recognition motif (RRM), one of the most common RNA binding domains, contains three highly conserved aromatic amino acids that participate in stacking interactions with RNA bases. We have investigated the contribution of these highly conserved aromatic amino acids to the affinity of the complex formed between the N-terminal RRM of the U1A protein and stem loop 2 of U1 snRNA. Previously, we found that substitution of one of these conserved aromatic amino acids, Phe56, with Ala resulted in a large destabilization of the complex. Here, we have modified A6, the base in stem loop 2 RNA that stacks with Phe56, to compensate for a portion of the destabilization caused by the Phe56Ala mutation. We have designed two modified adenosines, A-3CPh and A-4CPh, in which a phenyl group is linked to the adenosine such that it may replace the phenyl group that is eliminated by the Phe56Ala mutation in the complex. We have found that incorporation of A-3CPh into stem loop 2 RNA stabilizes the complex formed with Phe56Ala by 0.6 kcal/mol, while incorporation of A-4CPh into stem loop 2 RNA stabilizes this complex by 1.8 kcal/mol. Either base modification destabilizes the wild-type complex by 0.8-0.9 kcal/mol. Experiments with other U1A mutant proteins suggest that the stabilization of the complex between the Phe56Ala U1A protein and stem loop 2 RNA is due to a specific interaction between the Phe56Ala U1A protein and A6-4CPh stem loop 2 RNA.     

Solution Structure of a Hexitol Nucleic Acid Duplex with Four Consecutive T⋅T Base Pairs

The structure of the hexitol nucleic acid (HNA) h(GCGCTTTTGCGC) was determined by NMR spectroscopy. This unnatural nucleic acid was developed as a mimic for A‐RNA. In solution, the studied sequence is forming a symmetric double‐stranded structure with four central consecutive T⋅T wobble pairs flanked by G⋅C Watson‐Crick base pairs. The stem regions adopt an A‐type helical structure. Discrete changes in backbone angles are altering the course of the helix axis in the internal loop region. Two H‐bonds are formed in each wobble pair, and base stacking is preserved in the duplex, explaining the stability of the duplex. This structure elucidation provides information about the influence of a (T)₄ fragment on local helix geometries as well as on the nature of the T⋅T mismatch base pairing in a TTTT tract.     

Guanosine in a Single Stranded Region of Anticodon Stem–Loop tRNA Models is Prone to Oxidatively Generated Damage Resulting in Dehydroguanidinohydantoin and Spiroiminodihydantoin Lesions

Oxidation of RNA hairpin models corresponding to anticodon stem–loop (ASL) of transfer RNA led to RNA damage consisting solely of a unique loop guanine oxidation. Manganese porphyrin/oxone treatment of RNA resulted in dehydroguanidinohydantoin ( DGh ; major) and/or spiroiminodihydantoin ( Sp ) lesions. Ribose damage was not observed. This two‐electron transfer oxidation reaction allowed the identification of guanine oxidation products for further study of RNA species carrying a unique lesion at a single G to investigate their biological impact.     

Cisplatin damage overrides the predefined rotational setting of positioned nucleosomes

Cisplatin and carboplatin are used successfully to treat various types of cancer. The drugs target the nucleosomes of cancer cells and form intrastrand DNA cross-links that are located in the major groove. We constructed two site-specifically modified nucleosomes containing defined intrastrand cis-{Pt(NH3)2}(2+) 1,3-d(GpTpG) cross-links. Histones from HeLa-S3 cancer cells were transferred onto synthetic DNA duplexes having nucleosome positioning sequences. The structures of these complexes were investigated by hydroxyl radical footprinting. Employing nucleosome positioning sequences allowed us to quantify the structural deviation induced by the cisplatin adduct. Our experiments demonstrate that a platinum cross-link locally overrides the rotational setting predefined in the nucleosome positioning sequence such that the lesion faces toward the histone core. Identical results were obtained for two DNA duplexes in which the sites of platination differed by approximately half a helical turn. Additionally, we determined that cisplatin unwinds nucleosomal DNA globally by approximately 24 degree. The intrastrand cis-{Pt(NH3)2}(2+) 1,3-d(GpTpG) cross-links are located in an area of the nucleosome that contains locally overwound DNA in undamaged reference nucleosomes. Because most nucleosome positions in vivo are defined by the intrinsic DNA sequence, the ability of cisplatin to influence the structure of these positioned nucleosomes may be of physiological relevance.     

Molecular dynamics and thermodynamics of protein-RNA interactions: mutation of a conserved aromatic residue modifies stacking interactions and structural adaptation in the U1A-stem loop 2 RNA complex.

Molecular dynamics (MD) simulations and free energy component analysis have been performed to evaluate the molecular origins of the 5.5 kcal/mol destabilization of the complex formed between the N-terminal RNP domain of U1A and stem loop 2 of U1 snRNA upon mutation of a conserved aromatic residue, Phe56, to Ala. MD simulations, including counterions and water, have been carried out on the wild type and Phe56Ala peptide-stem loop 2 RNA complexes, the free wild type and Phe56Ala peptides, and the free stem loop 2 RNA. The MD structure of the Phe56Ala-stem loop 2 complex is similar to that of the wild type complex except the stacking interaction between Phe56 and A6 of stem loop 2 is absent and loop 3 of the peptide is more dynamic. However, the MD simulations predict large changes in the structure and dynamics of helix C and increased dynamic range of loop 3 for the free Phe56Ala peptide compared to the wild type peptide. Since helix C and loop 3 are highly variable regions of RNP domains, this indicates that a significant contribution to the reduced affinity of the Phe56Ala peptide for RNA results from cooperation between highly conserved and highly variable regions of the RNP domain of U1A. Surprisingly, these structural effects, which are manifested as cooperative free energy changes, occur in the free peptide, rather than in the complex, and are revealed only by study of both the initial and final states of the complexation process. Free energy component analysis correctly accounts for the destabilization of the Phe56Ala-stem loop 2 complex, and indicates that approximately 80% of the destabilization is due to the loss of the stacking interaction and approximately 20% is due to differences in U1A adaptation.     

Cisplatin cytotoxicity: a theoretical study of induced mutations

We investigate possible mutations in the genetic code induced by cisplatin with an approach combining molecular dynamics (MD) and hybrid quantum mechanics/molecular mechanics (QM/MM) calculations. Specifically, the impact of platination on the natural tautomeric equilibrium in guanine-cytosine (GC) base pairs is assessed to disclose the possible role played by non-canonical forms in anti-tumour activity. To obtain valuable predictions, the main interactions present in a real DNA environment, namely hydration and stacking, are simultaneously taken into account. According to our results, the Pt-DNA adduct promotes a single proton transfer reaction in GC in the DNA sequence AG[combining low line]G[combining low line]C. Such rare tautomers might play an important role in the cisplatin biological activity since they meet the stability requirements necessary to promote a permanent mutation.     

Effect of thioethers on DNA platination by trans-platinum complexes

Increasing evidence indicates that sulfur-containing molecules can play important roles in the activity of platinum anticancer drugs. Although nuclear DNA is retained to be the ultimate target, these platinum compounds can readily react with a variety of other substrates containing a soft donor atom, such as proteins, peptides, and low molecular weight biomolecules, before reaching DNA. In a recent study it was demonstrated that the DNA platination rate of a trans-geometry antitumor drug was dramatically enhanced by methionine binding, thus suggesting that the thioether could serve as a catalyst for DNA platination. In this work we performed detailed studies on the reactions of a widely investigated and very promising trans-platinum complex having two iminoethers and two chlorido ligands, trans-EE, with methionine (Met) and guanosine 5'-monophosphate (GMP). The results show that in the reaction of trans-EE with methionine the bisadduct is the dominant species in the early stage of the reaction. The reaction is also influenced by chloride concentration: at low NaCl the bis-methionine adduct is formed in preference, whereas the monoadduct is favored at high NaCl concentration. Not only the monomethionine complex, trans-PtCl(E-iminoether)(2)(AcMet), but also the bis-methionine adduct, trans-Pt(E-iminoether)(2)(AcMet)(2), which has already lost both leaving chlorides, can react with GMP to form the ternary platinum complex trans-Pt(E-iminoether)(2)(AcMet)(GMP). The latter reaction discloses the possibility of direct coordination to DNA of a platinum-protein adduct, in which the two carrier ligands remain intact; this is not the case of cis-oriented platinum complexes, like cisplatin, for which formation of a ternary complex is usually accompanied by loss of at least one carrier ligand. Interestingly, isomerization from S to N coordination of one methionine takes place in the bis-methionine complex at neutral pH, while the monoadduct appears to be stable. The shift from S to N coordination of one methionine in the trans-bis-methionine adduct can easily account for the obtainment of the cis isomer in the bis-chelated Pt(Met-S,N)(2) end product.     

A comparative kinetic study of modified Pt(dppf)Cl2 complexes and their interactions with L-cys and L-met

With the success of cisplatin (cis-diamminedichloroplatinum(II)), strong interest has developed in the application of inorganic metal complexes to the treatment of cancer. Research has focused on platinum(II) complexes with a variety of spectator ligands that provide novel physicochemical properties. In this paper we report a kinetic study of 1',1'-bis(diphenylphosphino)ferrocenedichloroplatinum(II) and two related compounds with either an acetate or amide ester substituent attached to the cyclopentadienyl ring. For all compounds the reactivity towards L-cysteine and L-methionine in aqueous solution has been investigated (25 degrees C, I= 0.010 M and pseudo-first-order conditions). For the reactions with l-cysteine and l-methionine the reactions proceeded via a steady-state aquated intermediate to form mono (0.92(2)-3.25(4)) x 10(-3) s(-1)) and bis adducts (0.97(2)-3.67(4)) x 10(-4) s(-1)). For reactions with l-cysteine, direct reactions with the starting complex also contributed (mono adduct: 0.36(2)-1.41(4) M(-1) s(-1), bis adduct: 0.080(1)-0.96(1) M(-1) s(-1)). The attached substituents were found to have a significant effect upon the reaction kinetics, with the substituted complexes found to have increased reactivity. It is proposed that the increased reactivity stems from hydrogen bonding between the substituent and the entering ligand and subsequent outer-sphere complex stabilisation. Evidence in support of this theory was obtained form measurements in dichloromethane with 1-propanethiol as the entering ligand. The reactivity of the dppf containing complexes was also compared to that of cisplatin (mono adduct: (0.170(1)-0.175(1)) x 10(-3) s(-1), bis adduct: (0.183(1)-0.397(1)) x 10(-4) s(-1)) and found to be significantly enhanced.     

GA and AG sequences of DNA react with cisplatin at comparable rates

The sequence selectivity of the antitumor drug cisplatin (cis-[PtCl(2)(NH(3))(2)] (1)) between the 5'-AG-3' and 5'-GA-3' sites of DNA has been a matter of discussion for more than twenty years. In this work, we compared the reactivity of GA and AG sequences of DNA towards the aquated forms of cisplatin (cis-[PtCl(NH(3))(2)(H(2)O)](+) (2), cis-[Pt(NH(3))(2)(H(2)O)(2)](2+) (3), and cis-[Pt(OH)(NH(3))(2)(H(2)O)](+) (4)) using two sets of experiments. In the first, we investigated a DNA hairpin, whose duplex stem contained a TGAT sequence as the single reactive site, and determined the individual rate constants of platination with 2 and 3 for G and A in acidic solution. The rate constants at 20 degrees C in 0.1M NaClO(4) at pH 4.5+/-0.1 were 0.09(4) M(-1)s(-1) (G) and 0.11(3) M(-1)s(-1) (A) for 2, and 9.6(1) M(-1)s(-1) (G) and 1.7(1) M(-1)s(-1) (A) for 3. These values are similar to those obtained previously for an analogous hairpin that contained a TAGT sequence. The monoadducts formed with 2 by both GA purines are extremely long-lived, partly as a result of the slow hydrolysis of the chloro monoadduct at A, and partly because of the very low chelation rate (1.4 x 10(-5)s(-1) at 20 degrees C) of the aqua monoadduct on the guanine. In the second set of experiments, we incubated pure or enriched samples of 1, 2, 3, or 4 for 18-64 h at 25 degrees C with a 19 base pair (bp) DNA duplex, whose radiolabeled top strand contained one GA and one AG sequence as the only reactive sites. Quantification of the number of GA and AG cross-links afforded a ratio of about two in favor of AG, irrespective of the nature of the leaving ligands. These results disagree with a previous NMR spectroscopy study, and indicate that GA sequences of DNA are substantially more susceptible to attack by cisplatin than previously thought.     

Chemical synthesis of an RNA sequence containing 2-thiocytidine (sC): the DY647 labelled anticodon stem and loop sequence of Staphylococcus aureus tRNA (sC32, mnmU34, tA37)

Incorporation of 2-thiocytidine (s²C) into a DY647 labelled 17-mer RNA sequence corresponding to the anticodon stem loop of Staphylococcus aureus tRNA^Arg has been achieved by phosphoramidite chemistry. Key to the success was the use of dilute iodine for the P(III) to P(V) oxidation step plus the choice of the trimethylsilylethyl (tse) ester as protection for the threonyl carboxyl group of t⁶A. In trial experiments with N⁴-benzoyl-2-thiocytidine, the use of dilute t-BuOOH in toluene as an alternative oxidising agent caused substantial desulfurization.