Tuomas Lönnberg

5-Mercuricytosine: An Organometallic Janus Nucleobase.

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.

Metal Ion Chelates as Surrogates of Nucleobases for the Recognition of Nucleic Acid Sequences: The Pd(2+) Complex of 2,6-Bis(3,5-dimethylpyrazol-1-yl)purine Riboside.

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, the T m value 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.

Understanding Catalysis of Phosphate‐Transfer Reactions by the Large Ribozymes

Large ribozymes are unique among catalytic RNA molecules in that their reactions involve intermolecular nucleophilic attack on an RNA phosphodiester linkage. Crystal structures of near-atomic resolution are now available for the group I and group II self-splicing introns and the RNA subunit of RNase P. The structural data agrees well with the earlier models proposed on the basis of biochemical studies and the evidence at hand suggests that all of the large ribozymes utilize a mechanism in which coordination of Mg(II) ions reduces the negative charge on the scissile phosphodiester linkage, as well as assists both the nucleophilic attack and the departure of the leaving group.

Oligonucleotides Incorporating Palladacyclic Nucleobase Surrogates

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.

Fluorescence probing of metal-ion-mediated hybridization of oligonucleotides

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.

Pd2 +-mediated base pairing in oligonucleotides

Two short glycol nucleic acid (GNA) oligonucleotides, having either a terminal or an intrachain nucleobase replaced by the pyridine-2,6-dicarboxamide chelate of Pd(2+), have been synthesized and their hybridization properties studied by melting temperature measurements. In the termini of a double-stranded oligonucleotide, the Pd(2+) chelates provided dramatic stabilization of the duplex relative to its metal-free counterpart, in all likelihood owing to formation of Pd(2+)-mediated base pairs between pyridine-2,6-dicarboxamide and the opposing nucleobase. In contrast, no stabilization was observed when the Pd(2+) chelate was placed in the middle of the chain. Furthermore, the results could not be reproduced by adding a Pd(2+) 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 Pd(2+), 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 Pd(2+)-mediated base pairs into oligonucleotides.

Nucleic acids through condensation of nucleosides and phosphorous acid in the presence of sulfur

Summary Short phosphorothioate oligonucleotides have been prepared by refluxing an equimolar mixture of thymidine and triethylammonium phosphite in toluene in the presence of elemental sulfur. Desulfurization and subsequent digestion of the products by P1 nuclease revealed that nearly 80% of the internucleosidic linkages thus formed were of the canonical 3´,5´-type.

Thio Effects as a Tool for Mechanistic Studies of the Cleavage of RNA Phosphodiester Bonds: The Chemical Basis

Replacement of one of the phosphorus-bound oxygen atoms with sulfur has extensively been used for elucidation of mechanistic details of the cleavage of RNA phosphodiester bonds by ribozymes. Since sulfur atom is larger, less electronegative, and more readily polarizable than oxygen, this substitution affects in many ways metal ion binding and the ease of formation and breakdown of the phosphorane intermediate/transition state obtained by the attack of the entering hydroxyl group on the phosphorus atom. The factors that may be altered by thio substitution include the geometry of the phosphorane intermediate, relative apicophilicities of the ligands, the leaving group property, hydrogen bonding, solvation, and the affinity to metal ions. Experimental studies and theoretical calculations on various model systems have been undertaken to obtain a solid chemical basis for the mechanistic interpretations based on thio effects in ribozyme catalysis. The results of such studies are surveyed in this chapter.

Fluorescent Oligonucleotide Probes for Screening High‐Affinity Nucleobase Surrogates

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.

2,6-Bis(1,4,7,10-tetraazacyclododecan-1-ylmethyl)pyridine and Its Benzene Analog as Nonmetallic Cleaving Agents of RNA Phosphodiester Linkages.

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.