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Short oligonucleotides incorporating either 1-mercuri-6-phenylcarbazole, 8-mercuri-6-phenylcarbazole, or 1,8-dimercuri-6-phenylcarbazole C-nucleoside in the middle of the chain have been synthesized and studied for their potential as hybridization probes for sequences containing thiopyrimidine nucleobases. All of these oligonucleotides formed very stable duplexes with complementary sequences pairing the organometallic moiety with either 2- or 4-thiothymine. The isomeric monomercurated oligonucleotides were also able to discriminate between 2- and 4-thiothymine based on the different melting temperatures of the respective duplexes. DFT-optimized structures of the most stable mononuclear Hg

A water‐soluble arylmercury complex has been synthesized, and its ability to catalyze the cleavage of the phosphodiester linkage of the RNA model compound adenylyl‐3′,5′‐(2′,3′‐O‐methyleneadenosine) has been assessed over a pH range of 3–8.5 and a catalyst concentration range of 0–7 mM. In the presence of 1 mM catalyst, the observed pH–rate profile featured a new pH‐independent region between pH 6 and 7, the catalyzed reaction being as much as eight times faster than the background reaction. At pH 7, the acceleration increased linearly from three‐ to 17‐fold upon increasing the catalyst concentration from 1 to 7 mM. The linear dependence indicates a relatively low affinity of the catalyst for the substrate and, hence, the potential for considerable improvement on tethering to an appropriate targeting group, such as an oligonucleotide., Soluble, but not so speedy: The potential of an water‐soluble arylmercury compound to cleave RNA has been explored. The somewhat low catalytic activity compared to free HgII was attributed to the relatively low affinity of the arylmercury catalyst for the RNA model compound. This suggests that improved derivatives could be obtained by covalent tethering to an appropriate targeting group.

Synthetic efforts towards nucleosides, nucleotides, oligonucleotides and nucleic acids covalently mercurated at one or more of their base moieties are summarized, followed by a discussion of the proposed, realized and abandoned applications of this unique class of compounds. Special emphasis is given to fields in which active research is ongoing, notably the use of HgII‐mediated base pairing to improve the hybridization properties of oligonucleotide probes. Finally, this minireview attempts to anticipate potential future applications of organomercury nucleic acids., The organomercury renaissance: Research on the synthesis and applications of organomercury nucleic acids and their constituents is reviewed, with a focus on recent developments, such as efforts to improve the hybridization properties of oligonucleotide probes through HgII‐mediated base pairing of covalently mercurated base moieties.

A highly nucleobase-discriminating metalated nucleoside analogue, 3-fluoro-2-mercuri-6-methylaniline, was incorporated into an oligonucleotide molecular beacon. Fluorescence emission spectra were measured after the addition of four different complementary strands, in which the nucleobase opposite the metalated analogue varies. The fluorescence results showed a clear binding selectivity at room temperature, in the order G>T>C>A. The selectivity is based on the different affinities between the metalated nucleoside analogue and the canonical nucleobases. The synthesized probe is capable of robust discrimination between the two purine as well as the two pyrimidine bases by fluorescence at room temperature, and more sophisticated temperature analysis allows clear separation of every canonical nucleobase. The probe would, hence, be a suitable method for the detection of single nucleotide polymorphisms.

Oligonucleotides incorporating a central C‐nucleoside with either a rigid or flexible benzaldoxime base moiety have been synthesized, and the hybridization properties of their metallacyclic derivatives have been studied by UV melting experiments. In all cases, the metallated duplexes were less stable than their unmetallated counterparts, and the metallacyclic nucleobases did not show a clear preference for any of the canonical nucleobases as a base‐pairing partner. With palladated oligonucleotides, increased flexibility translated to less severe destabilization, whereas the opposite was true for the mercurated oligonucleotides; this reflects the greater difficulties in accommodating a rigid PdII‐mediated base pair than a rigid HgII‐mediated base pair within the base stack of a double helix., Take the strain: A flexible PdII‐mediated base pair of a benzaldoxime palladacycle disrupts oligonucleotide hybridization less than its rigid analogue, whereas the opposite is true for HgII‐mediated base pairs. With a base pair of an incompatible geometry, increased flexibility alleviates some of the strain; with a strain‐free base pair it merely leads to an increased entropic penalty of hybridization.

A phthaloyl-protected aminooxymethyl-C-2'-deoxyriboside building block has been prepared and incorporated in the middle of an oligodeoxyribonucleotide. Removal of the phthaloyl protection followed by on-support oximation with either mercurated or palladated benzaldehydes yielded oligonucleotides bearing the respective benzaldoxime metallacycles.

Covalent metalation of the base moieties affords a new class of modified oligonucleotides. These organometallic oligonucleotides share many properties, notably increased hybridization affinity conferred by metal-mediated base pairing, with oligonucleotides incorporating coordinative transition-metal complexes. They are, however, set apart by their ability to retain the transition-metal ion even at extreme dilution. Such stability towards dissociation would be desirable in DNA nanotechnology and necessary in therapeutic applications. Herein we describe our efforts towards preparation and characterization of covalently mercurated and palladated oligonucleotides, highlighting in particular our recent contribution on the synthesis and potential applications of oligonucleotides incorporating dimercurated artificial nucleobases.1 Introduction2 Synthesis of Covalently Mercurated and Palladated Oligonucleotides3 Hybridization Properties of Covalently Mercurated and Palladated Oligonucleotides4 Outlook

An 11-mer oligonucleotide incorporating a central (2-iodobenzoylamino)methyl residue has been synthesized and palladated by oxidative addition of Pd2 (dba)3 . UV melting profiles of the duplexes formed by the palladated oligonucleotide with its natural complements were biphasic and the higher melting temperatures (Tm ) exhibited considerable hysteresis. CD spectra, in turn, resembled those of canonical B-type double helices. Two-step denaturation, with the "low-Tm " melting involving only canonical base pairs and the "high-Tm " melting involving also dissociation of a PdII -mediated base pair, appears the most likely explanation for the observed UV melting profiles. As the latter step in all cases takes place at a higher temperature than denaturation of natural duplexes of the same length, the putative PdII -mediated base pairs are stabilizing.

A C-nucleoside derivative of phenylpyridine or the respective palladacycle was incorporated at either 3′- or 5′-terminus of a short oligodeoxynucleotide. Hybridization properties of these modified oligonucleotides were studied in a fluorescence-based competition assay in addition to conventional UV melting temperature analysis and compared with those of a previously prepared analogue featuring the modified nucleoside in the middle of the sequence. With the unpalladated phenylpyridine oligonucleotides, UV melting temperature qualitatively correlated with the ability to displace a strand from a double helix in the competition assay, decreasing in the order 5′ > 3′ > middle. Corresponding results on the palladacyclic oligonucleotides were more difficult to interpret but both UV melting and competition experiments revealed a decrease in the duplex stability upon palladation in most cases. On the other hand, dependence of the UV melting temperature on the identity of the canonical nucleobase opposite to the modified nucleobase analogue was much more pronounced with the palladacyclic duplexes than with their unpalladated counterparts. Furthermore, UV melting profiles of the palladacyclic duplexes featured an additional transition at a temperature exceeding the melting temperature of the unmodified part of the duplex. Taken together, these results lend support to the idea of Pd(II)-mediated base pairs that are highly stable but incompatible with the geometry of a double helix.

A C-nucleoside with 6-phenyl-1H-carbazole as the base moiety has been synthesized and incorporated in the middle of an oligonucleotide. Mercuration of this modified residue at positions 1 and 8 gave the first example of an oligonucleotide featuring a monofacial dinuclear organometallic nucleobase. The dimercurated oligonucleotide formed stable duplexes with unmodified oligonucleotides placing either cytosine, guanine, or thymine opposite to the organometallic nucleobase. A highly stabilizing (ΔTm =7.3 °C) HgII -mediated base pair was formed with thymine. According to DFT calculations performed at the PBE0DH level of theory, this base pair is most likely dinuclear, with the two HgII ions coordinated to O2 and O4 of the thymine base.

An oligonucleotide incorporating a palladacyclic nucleobase has been prepared by ligand-directed metalation of a phenylpyridine moiety. This oligonucleotide hybridized with natural counterparts placing any of the canonical nucleobases opposite to the palladacyclic residue. The palladated duplexes had B-type conformation and melting temperatures comparable to those of respective unmodified duplexes with a single mismatch. In the duplexes placing C, G or T (but not A) opposite to the palladacyclic residue, greatly increased absorptivity suggested formation of a PdII -mediated base pair. Absorptivity and ellipticity of these duplexes persisted even at the highest temperatures applicable in Tm and CD experiments (90 °C). Evidently the PdII -mediated base pairs do not dissociate under the experimental conditions.

Double-helical oligonucleotide probes featuring a single-nucleotide gap opposed by one of the canonical nucleobases and flanked by the fluorescent nucleobase analogue pyrrolocytosine have been synthesized and titrated with PdII chelates of dipicolinamide and its N2,N6-dialkylated derivatives. The fluorometric titrations revealed greatly increased affinity of the PdII chelates for the nucleobases opposing the gap compared to the respective free nucleotides in solution. Owing to the constrained environment of the single-nucleotide gap, the relative stabilities of the various PdII mediated base pairs were also significantly different from those previously reported at monomer level.

A 3-fluoro-6-methylaniline nucleoside was synthesized and incorporated into an oligonucleotide, and its ability to form mercury-mediated base pairs was studied. UV melting experiments revealed increased duplex stability with thymine, guanine, and cytosine opposite to the probe and a clear nucleobase-specific binding preference (T > G > C > A). Moreover, the 3-fluoro group was utilized as a spin label that showed distinct 19F NMR resonance shifts depending on the complementary nucleobase, providing more detailed information on Hg(II)-mediated base pairing.

An 11-mer oligonucleotide incorporating a central (2-iodobenzoylamino)methyl residue has been synthesized and palladated by oxidative addition of Pd

Metal-ion-mediated base pairing has been extensively studied in the context of DNA nanotechnology or expansion of the genetic alphabet but only recently with the aim of recognition of natural nucleic acid sequences. This review article focuses on the attempts to utilize metal-ion-carrying oligonucleotides as high-affinity probes for nucleic acids but relevant studies carried out at the monomer level are also discussed. Special emphasis is given to challenges inherent to this approach, such as the limited recognition elements of the canonical nucleobases and the scarcity of suitable metal ions in the intracellular medium. Finally, potential ways to overcome these challenges are proposed.

A multi-nucleating azacrown conjugate ( 5a ) consisting of two 3,5-bis(1,5,9-triazacyclododecan-3-yloxymethyl)benzyl groups attached to 1 and 7 sites of cyclen was prepared and tested as an artificial ribonuclease. The conjugate in the presence of five equivalents of zinc nitrate expectedly showed uridine selectivity comparable to that 1,3,5-tris(1,5,9-triazacyclododecan-3-yl)benzene ( 2 ), a compound known to bind to two adjacent uridine residues and cleave the intervening phosphodiester bond. 5a was, however, unable to discriminate between two and three adjacent uridine residues, but cleaved oligonucleotides containing a UpU and UpUpU site at a comparable rate, even when present at sub-saturating concentrations.

The base-pairing properties of 5-mercuricytosine have been explored at the monomer level by NMR titrations and at the oligonucleotide level by melting temperature measurements. The NMR studies revealed a relatively high affinity for guanine, hypoxanthine, and uridine, that is, bases that are deprotonated upon coordination of Hg(II) . Within an oligonucleotide duplex, 5-mercuricytosine formed Hg(II) -mediated base pairs with thymine and guanine. In the former case, the duplex formed was as stable as the respective duplex comprising solely Watson-Crick base pairs. Based on detailed thermodynamic analysis of the melting curves, the stabilization by the Hg(II) -mediated base pairs may be attributed to a comparatively low entropic penalty of hybridization.

A C-nucleoside featuring picolinamide as the base moiety has been synthesized and incorporated in the middle of a DNA oligonucleotide. The melting temperatures of duplexes formed by this modified oligonucleotide with its unmodified counterparts placing either adenine, cytosine, guanine or thymine opposite to the picolinamide residue were found to be highly dependent on the identity and concentration of transition metal ions in the samples. For example, AgI uniquely promoted pairing with adenine and PdII with guanine. Pairing with cytosine, on the other hand, was retarded by all of the metal ions studied (AgI, CuII, HgII, NiII, PdII and ZnII).

We describe the use of Bayesian analysis methods applied to time-of-flight secondary ion mass spectrometer (TOF-SIMS) spectra. The method is applied to the COmetary Secondary Ion Mass Analyzer (COSIMA) TOF-SIMS mass spectra where the analysis can be broken into subgroups of lines close to integer mass values. The effects of the instrumental dead time are discussed in a new way. The method finds the joint probability density functions of measured line parameters (number of lines, and their widths, peak amplitudes, integrated amplitudes and positions). In the case of two or more lines, these distributions can take complex forms. The derived line parameters can be used to further calibrate the mass scaling of TOF-SIMS and to feed the results into other analysis methods such as multivariate analyses of spectra. We intend to use the method, first as a comprehensive tool to perform quantitative analysis of spectra, and second as a fast tool for studying interesting targets for obtaining additional TOF-SIMS measurements of the sample, a property unique to COSIMA. Finally, we point out that the Bayesian method can be thought of as a means to solve inverse problems but with forward calculations, only with no iterative corrections or other manipulation of the observed data.

Nucleic acids that store and transfer biological information are polymeric diesters of phosphoric acid. Cleavage of the phosphodiester linkages by protein enzymes, nucleases, is one of the underlying biological processes. The remarkable catalytic efficiency of nucleases, together with the ability of ribonucleic acids to serve sometimes as nucleases, has made the cleavage of phosphodiesters a subject of intensive mechanistic studies. In addition to studies of nucleases by pH-rate dependency, X-ray crystallography, amino acid/nucleotide substitution and computational approaches, experimental and theoretical studies with small molecular model compounds still play a role. With small molecules, the importance of various elementary processes, such as proton transfer and metal ion binding, for stabilization of transition states may be elucidated and systematic variation of the basicity of the entering or departing nucleophile enables determination of the position of the transition state on the reaction coordinate. Such data is important on analyzing enzyme mechanisms based on synergistic participation of several catalytic entities. Many nucleases are metalloenzymes and small molecular models offer an excellent tool to construct models for their catalytic centers. The present review tends to be an up to date summary of what has been achieved by mechanistic studies with small molecular phosphodiesters.

A quinone methide precursor featuring a bis-cyclen anchoring moiety has been synthesized and its capacity to alkylate oligonucleotide targets quantified in the presence and absence of divalent metal ions (Zn(2+) , Ni(2+) and Cd(2+) ). The oligonucleotides were designed for testing the sequence and secondary structure specificity of the reaction. Gel electrophoretic analysis revealed predominant alkylation of C-rich bulges, regardless of the presence of divalent metal ions or even the bis-cyclen anchor. This C-selectivity appears to be an intrinsic property of the quinone methide electrophile as reflected by its reaction with an equimolar mixture of the 2'-deoxynucleosides. Only dA-N1 and dC-N3 alkylation products were detected initially and only the dC adduct persisted for detection under conditions of the gel electrophoretic analysis.

4'-(Hydroxymethyl)uridylyl-3',5'-thymidine, an RNA model bearing an extra hydroxymethyl group at the 4'-position of the 3'-linked nucleoside, has been prepared and its cleavage and isomerization reactions studied over a wide pH range (from 0 to 12). Overall, the pH-rate profiles of these reactions were very similar to those of uridylyl-3',5'-uridine (UpU) - only a very modest acceleration was observed under acidic and neutral conditions. Evidently, hydrogen bond assistance by the additional hydroxymethyl function does not play a significant role.

The sensitivity of fluorescence of pyrrolocytosine-containing oligonucleotides to changes in their secondary structure has been harnessed to monitor the hybridization of modified metal-ion-chelating oligonucleotides with their unmodified counterparts. With short double-helical oligonucleotides, quenching of fluorescence correlated well with the length and, hence, Tm of the duplex provided that the pyrrolo-dC residue was incorporated within the duplex. Furthermore, hybridization of the metal-ion-chelating oligonucleotides was greatly enhanced on addition of 1 eq. of Cu2+, as evidenced by both Tm and fluorometric measurements. As an example of a case where interpretation of a conventional UV-melting profile is challenging, application of the fluorometric method was extended to probing hybridization of short metal-ion-carrying oligonucleotides with the trinucleotide bulge motif of TAR RNA models. In this case the results were more ambiguous, presumably due to weak hybridization with the short target sequence.

Buffer catalysis of the cleavage and isomerization of uridylyl-3',5'-uridine (UpU) has been studied over a wide pH range in 80% aq. DMSO. The diminished hydroxide ion concentration in this solvent system made catalysis by amine buffers (morpholine, 4-hydroxypiperidine and piperidine) visible even at relatively low buffer concentrations (10-200 mmol L(-1)). The observed catalysis was, however, much weaker than what has been previously reported for the activated RNA model 2-hydroxypropyl 4-nitrophenyl phosphate (HPNP) in the same solvent system. In the case of morpholine, contribution of both the acidic and the basic buffer constituent was significant, whereas with 4-hydroxypiperidine and piperidine participation of the acidic constituent could not be established unambiguously. The results underline the importance of using realistic model compounds, along with activated ones, in the study of the general acid/base catalysis of RNA cleavage.

The potential of three modified purine bases, namely, 6-(3,5-dimethylpyrazol-1-yl)purine, 2-(3,5-dimethylpyrazol-1-yl)hypoxanthine, and 2-(3,5-dimethylpyrazol-1-yl)adenine, for metal-ion-mediated base pairing within an oligonucleotide environment has been investigated. The respective modified nucleosides were incorporated in the middle of 9-mer 2'-O-methyl oligonucleotides and the hybridization of these modified oligonucleotides with their unmodified counterparts studied by UV and CD spectrometry in the absence and presence of Cu(2+) or Zn(2+). All of the modified oligonucleotides formed more stable duplexes in the presence of divalent metal ions than in the absence thereof, but with different preferences for the complementary oligonucleotide. The oligonucleotide incorporating 2-(3,5-dimethylpyrazol-1-yl)hypoxanthine readily accepted any of the natural nucleobases opposite to this modified base regardless of whether Cu(2+) or Zn(2+) was used as the bridging metal ion. The other two oligonucleotides, on the other hand, were much more discriminating, exhibiting markedly elevated Tm values only in the presence of Cu(2+) and only when certain natural nucleobases were paired with the modified one. The origin of the selectivity (or promiscuity) of the metal-ion-mediated base pairing is discussed in terms of the ability of the modified nucleobases, as well as their natural counterparts, to serve as anionic ligands.

To learn more about the underlying principles of metal-ion-mediated recognition of nucleic acid bases, PdCl(+) complexes of six 2,6-disubstituted pyridines, viz. pyridine-2,6-dicarboxamide, its N(2),N(6)-dimethyl and N(2),N(6)-diisopropyl derivatives, 6-carbamoylpyridine-2-carboxylic acid, 6-aminomethylpyridine-2-carboxamide and its N(2)-methyl derivative, were prepared and their interaction with nucleoside 5'-monophosphate (NMP) was studied by (1)H NMR spectroscopy in D2O at pH7.2. The binding sites within the nucleobases were assigned on the basis of Pd(2+) induced changes in chemical shifts of the base moiety proton resonances. The mole fractions of NMPs engaged in mono- or dinuclear Pd(2+) complexes were determined at various concentrations by comparing the intensities of the aromatic and anomeric protons of the complexed and uncomplexed NMPs. Some of the pyridine complexes showed moderate discrimination between the NMPs.

Four metal-ion-binding nucleosides, viz. 2,6-bis(1-methylhydrazinyl)-9-(β-D-ribofuranosyl)-9H-purine (2a) and its N-acetylated derivative, 2b, 2,4-bis(3,5-dimethyl-1H-pyrazol-1-yl)-5-(β-D-ribofuranosyl)pyrimidine (3), and 2,4-bis(1-methylhydrazinyl)-5-(β-D-ribofuranosyl)pyrimidine (4) have been synthesized. The ability of these nucleosides and the previously prepared 2,6-bis(3,5-dimethyl-1H-pyrazol-1-yl)-9-(β-D-ribofuranosyl)-9H-purine to form Pd2+- and Hg2+-mediated complexes with uridine has been studied by 1H-NMR spectroscopy. To obtain additional support for the interpretation of the NMR data, comparative measurements on the ternary-complex formation between pyridine-2,6-dicarboxamide (5), pyrimidine nucleosides, and K2PdCl4 were carried out.

A dinucleoside-3',5'-phosphodiester model, 5'-amino-4'-aminomethyl-5'-deoxyuridylyl-3',5'-thymidine, incorporating two aminomethyl functions in the 4'-position of the 3'-linked nucleoside has been prepared and its hydrolytic reactions studied over a wide pH range. The amino functions were found to accelerate the cleavage and isomerization of the phosphodiester linkage in both protonated and neutral form. When present in protonated form, the cleavage of the 3',5'-phosphodiester linkage and its isomerization to a 2',5'-linkage are pH-independent and 50-80 times as fast as the corresponding reactions of uridylyl-3',5'-uridine (3',5'-UpU). The cleavage of the resulting 2',5'-isomer is also accelerated, albeit less than with the 3',5'-isomer, whereas isomerization back to the 3',5'-diester is not enhanced. When the amino groups are deprotonated, the cleavage reactions of both isomers are again pH-independent and up to 1000-fold faster than the pH-independent cleavage of UpU. Interestingly, the 2'- to 3'-isomerization is now much faster than its reverse reaction. The mechanisms of these reactions are discussed. The rate accelerations are largely accounted for by electrostatic and hydrogen-bonding interactions of the protonated amino groups with the phosphorane intermediate.

International audience; Two short glycol nucleic acid (GNA) oligonucleotides, having either a terminal or an intrachain nucleobase replaced by the pyridine-2,6-dicarboxamide chelate of Pd2 +, have been synthesized and their hybridization properties studied by melting temperature measurements. In the termini of a double-stranded oligonucleotide, the Pd2 + chelates provided dramatic stabilization of the duplex relative to its metal-free counterpart, in all likelihood owing to formation of Pd2 +-mediated base pairs between pyridine-2,6-dicarboxamide and the opposing nucleobase. In contrast, no stabilization was observed when the Pd2 + chelate was placed in the middle of the chain. Furthermore, the results could not be reproduced by adding a Pd2 + salt in situ to the dilute oligonucleotide solutions but the palladated oligonucleotides had to be synthesized and purified prior to the hybridization studies. This behavior, presumably attributable to the relatively slow ligand-exchange reactions of Pd2 +, differs greatly from what is usually observed with more labile metal ions. The present results offer an explanation for the failure of previous attempts to incorporate Pd2 +-mediated base pairs into oligonucleotides.

A 2,6-bis(3,5-dimethylpyrazol-1-yl)purine ribonucleoside has been prepared and incorporated as a conventionally protected phosphoramidite into a 9-mer 2′-O-methyl oligoribonucleotide. According to 1H NMR spectroscopic studies, this nucleoside forms with Pd2+and uridine a ternary complex that is stable at a micromolar concentration range. CD spectroscopic studies on oligonucleotide hybridization, in turn, suggest that the Pd2+chelate of this artificial nucleoside, when incorporated in a 2′-O-methyl-RNA oligomer, is able to recognize thymine within an otherwise complementary DNA strand. The duplex containing thymidine opposite to the artificial nucleoside turned out to be somewhat more resistant to heating than its counterpart containing 2′-deoxycytidine in place of thymidine, but only in the presence of Pd2+. According to UV-melting measurements, replacement of 2′-O-methyladenosine with the artificial nucleoside markedly enhances hybridization with a DNA target, irrespective of the identity of the opposite base and the presence of Pd2+. With the thymidine containing DNA target, theTmvalue is 2–4°C higher than with targets containing any other nucleoside opposite to the artificial nucleoside, but the dependence on Pd2+is much less clear than in the case of the CD studies.

To assess the extent to which steric constraints may influence the product distribution of the reactions of the large ribozymes, phosphate-branched oligonucleotides of varying length and sequence have been synthesized and their alkaline hydrolysis studied over a wide temperature range. At low temperatures, the branching trinucleoside-3',3',5'-monophosphate moiety is hydrolyzed almost exclusively by P-O3' fission. At higher temperatures, P-O5' fission competes, accounting at most for 22% of the overall reaction. The results suggest that steric constraints imposed by the secondary structure of the reaction site may significantly contribute to the observed regioselectivity of the transesterification reactions catalyzed by the large ribozymes.

An artificial site-selective DNA cutter to hydrolyze single-stranded DNA at a desired site was prepared from Ce(IV)/ethylenediamintetraacetic acid (EDTA) and two ethylenediamine-N,N,N',N'-tetrakis(methylenephosphonic acid)-oligonucleotide conjugates. By using this cutter, the sense strand of a blue fluorescent protein (BFP) gene was selectively cut at a predetermined site in the chromophore-coding region. The upstream fragment obtained by the site-selective scission was ligated with the downstream fragment of the closely related green fluorescent protein (GFP) gene so that the 5'- and 3'-end portions of the chromophore came from the BFP fragment and the GFP fragment, respectively. The recombinant gene was successfully expressed in E. coli and the chimeric chromophore emitted green fluorescence as expected.

Telomere is an emerging target for the treatment of human cancers. Here, we report a structure-based approach to sequence-specific cleaving of human telomeric DNA by G-quadruplex formation. Oligonucleotide with multiphosphonate [DNA-EDTP.Ce(IV)] at the 5' end binds to human telomere DNA by G-quadruplex formation and causes a sequence-specific strand break. These results provide the first proof of concept for targeting the human telomere DNA based on G-quadruplex formation, and this may serve as a starting point for the design of more efficient telomere sequence-specific cleaving reagents by G-quadruplex formation.

2,6-Bis(1,4,7,10-tetraazacyclododecan-1-ylmethyl)pyridine (11a) and 1,3-bis(1,4,7,10-tetraazacyclododecan-1-ylmethyl)benzene (11b) have been shown to accelerate at 50 mmol·L−1 concentration both the cleavage and mutual isomerization of uridylyl-3′,5′-uridine and uridylyl-2′,5′-uridine by up to two orders of magnitude. The catalytically active ionic forms are the tri- (in the case of 11b) tetra- and pentacations. The pyridine nitrogen is not critical for efficient catalysis, since the activity of 11b is even slightly higher than that of 11a. On the other hand, protonation of the pyridine nitrogen still makes 11a approximately four times more efficient as a catalyst, but only for the cleavage reaction. Interestingly, the respective reactions of adenylyl-3′,5′-adenosine were not accelerated, suggesting that the catalysis is base moiety selective.

A phosphate-branched oligonucleotide has been prepared by using an appropriately protected trinucleoside phosphotriester building block in conventional solid-phase synthesis. Hydrolysis of the branched oligonucleotide has been followed over a wide pH range. Comparison of the present results with those previously obtained for simpler analogues indicates that a trinucleoside 3',3',5'-monophosphate, when embedded in an oligonucleotide structure, is stabilized toward hydroxide-ion catalyzed cleavage by more than one order of magnitude, lending some support to the feasibility of existence of phosphate-branched RNA X in biological systems.

Mechanistic studies of the action of catalytic ribonucleic acids, ribozymes, are highly challenging, because even a slight structural change can dramatically affect the chain folding. This, in turn, alters the binding properties of the catalytic core, making identification of the real origin of the observed influence on rate difficult. Unambiguous structure-reactivity correlations based on studies with structurally simplified chemical models may help to distinguish between alternative mechanistic interpretations. The results of such model studies are reviewed. The topics include intramolecular cleavage of RNA phosphodiester bonds by solvent-derived species, general acids/bases and metal ions, effect of molecular environment on their hydrolytic stability and trinucleoside monophosphates as models for large ribozymes.