Ewa Biala

Synthesis and Enzymatic Digestion of an RNA Nonamer in Both Enantiomeric Forms

Abstract The d - and l -RNA nonamers of the sequence r(GCUUCGGC)T have been synthesised for X-ray crystallographic purposes. In vitro digestion of the unnatural optical antipode by snake venom phosphodiesterase I takes place at an approximately 1800-fold slower rate than that of the natural d -nonamer. The digestion experiments showed—to our knowledge for the first time—that l -RNA can indeed be cleaved enzymatically when phosphodiesterase I from snake venom is used—as opposed to a number of cellular ribonucleases—which sheds an interesting light on the evolution and possibly structure/function relationship of venom versus cellular degradation enzymes. The broad substrate specificity of this enzyme could be taken advantage of to study and further optimise the resistance towards biodegradation of therapeutic l -RNA aptamers.

The relationship of thermodynamic stability at a G x U recognition site to tRNA aminoacylation specificity.

The G x U pair at the third position in the acceptor helix of Escherichia coli tRNA(Ala) is critical for aminoacylation. The features that allow G x U recognition are likely to include direct interaction of alanyl-tRNA synthetase with distinctive atomic groups and indirect recognition of the structural and stability information encoded in the sequence of G x U and its immediate context. The present work investigates the thermodynamic stability and acceptor activity for a comprehensive set of variant RNAs with substitutions of the G x U pair of E. coli tRNA(Ala). The four RNAs with Watson-Crick substitutions had a lower acceptor activity and a higher stability relative to the G x U RNA. On the other hand, the RNAs with mispair substitutions had a lower stability, but either a higher or a lower acceptor activity. Thus, the entire set of variant RNAs does not exhibit a correlation between thermodynamic stability of the free, unbound tRNA and its acceptor activity. The substantial acceptor activity of tRNAs with particular mispair substitutions may be explained by their ability to assume the conformational preferences of alanyl-tRNA synthetase. Moreover, the G x U pair may provide a point of deformability for the substrate tRNA to adapt to the enzymes active site.

Tetraethylene glycol-derived spacer for oligonucleotide synthesis

Abstract 3,6,9-Trioxaundecane-1, 11-diisocyanate ( 6 ) was synthesised from tetraethylene glycol in 5 steps and 48 % overall yield. Spacer 6 was monofunctionalised with the fully protected adenosyl-3′-O-succinate derivative 7 and linked to aminomethyl polystyrene (50 % crosslinked with divinylbenzene) affording a solid support suitable for oligoribonucleotide synthesis (loading: ∼20 μmol/g). The HPLC analysis of a crude oligoribonucleotide synthesis and the isolated yield of purified oligomer show that this spacer compares well to hexamethylene diamine.

19F NMR of RNA. The Structural and Chemical Aspects of 5-Fluoro-cytidine and-uridine Labelling of Oligoribonucleotides #

Abstract Results of PM3 semiempirical calculation revealed that energy and hydrogen bonds geometry of 1-methyl-5-fluoro-uracil and -cytosine base-pairs with 9-methyl-adenine and -guanine respectively are virtually the same as for the natural bases. Analysis of proton coupling constants proved that the sugar puckering of 5-fluorouridine and 5-fluorocytidine is analogous to non-modified ribonucleosides. 5-Fluorocytidine was regioselectively introduced to oligoribonucleotides, prepared using 2′-O-tert-dimethylsilyl protection, via post-synthetic quantitative ammonolysis of 4-O-methyl-5-fluorouridine derived precursor. #Dedicated to Professor Yoshihisa Mizuno on the occasion of his 75th birthday.

A Tandem Oligonucleotide Approach for SNP-Selective RNA Degradation Using Modified Antisense Oligonucleotides

Antisense oligonucleotides have been studied for many years as a tool for gene silencing. One of the most difficult cases of selective RNA silencing involves the alleles of single nucleotide polymorphisms, in which the allele sequence is differentiated by a single nucleotide. A new approach to improve the performance of allele selectivity for antisense oligonucleotides is proposed. It is based on the simultaneous application of two oligonucleotides. One is complementary to the mutated form of the targeted RNA and is able to activate RNase H to cleave the RNA. The other oligonucleotide, which is complementary to the wild type allele of the targeted RNA, is able to inhibit RNase H cleavage. Five types of SNPs, C/G, G/C, G/A, A/G, and C/U, were analyzed within the sequence context of genes associated with neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, ALS (Amyotrophic Lateral Sclerosis), and Machado-Joseph disease. For most analyzed cases, the application of the tandem approach increased allele-selective RNA degradation 1.5–15 fold relative to the use of a single antisense oligonucleotide. The presented study proves that differentiation between single substitution is highly dependent on the nature of the SNP and surrounding nucleotides. These variables are crucial for determining the proper length of the inhibitor antisense oligonucleotide. In the tandem approach, the comparison of thermodynamic stability of the favorable duplexes WT RNA-inhibitor and Mut RNA-gapmer with the other possible duplexes allows for the evaluation of chances for the allele-selective degradation of RNA. A larger difference in thermodynamic stability between favorable duplexes and those that could possibly form, usually results in the better allele selectivity of RNA degradation.

Thermodynamics of Site-Specific Variant tRNAAla Acceptor Stem Microhairpins

Thermal denaturation studies were carried out on a set of site-specific variants of a 22mer RNA hairpin comprising the aminoacyl acceptor stem sequence of E. coli tRNA(Ala). The pairing thermodynamics were calculated from the melting profiles.

Tetraethylene Glycol-Derived Spacer for Oligonucleotide Synthesis

Abstract 3,6,9-Trioxaundecane-1,11-diisocyanate (6) was synthesised from tetraethylene glycol in 5 steps and 48 % overall yield. Spacer 6 was monofunctionalised with the fully protected adenosyl-3′-O-succinate derivative 7 and linked to aminomethyl polystyrene affording a solid support suitable for oligoribonucleotide synthesis (loading: ∼20 μmol/g). The HPLC analysis of a crude oligoribonucleotide synthesis and the yield of the full-length product show that this spacer compares well to hexamethylene diamine.

Influence of mismatched and bulged nucleotides on SNP-preferential RNase H cleavage of RNA-antisense gapmer heteroduplexes

This study focused on determining design rules for gapmer-type antisense oligonucleotides (ASOs), that can differentiate cleavability of two SNP variants of RNA in the presence of ribonuclease H based on the mismatch type and position in the heteroduplex. We describe the influence of structural motifs formed by several arrangements of multiple mismatches (various types of mismatches and their position within the ASO/target RNA duplex) on RNase H cleavage selectivity of five different SNP types. The targets were mRNA fragments of APP, SCA3, SNCA and SOD1 genes, carrying C-to-G, G-to-C, G-to-A, A-to-G and C-to-U substitutions. The results show that certain arrangements of mismatches enhance discrimination between wild type and mutant SNP alleles of RNA in vitro as well as in HeLa cells. Among the over 120 gapmers tested, we found two gapmers that caused preferential degradation of the mutant allele APP 692 G and one that led to preferential cleavage of the mutant SNCA 53 A allele, both in vitro and in cells. However, several gapmers promoted selective cleavage of mRNA mutant alleles in in vitro experiments only.

The Solution NMR Structure of 2′-O-Methyl CGCGCG RNA Duplex

Abstract Complete assignments of nonexchangeable protons in 1H NMR spectra of 2′-O-methyl-CGCGCG complemented by its analysis of 13C and 31P NMR spectra revealed A-RNA double helical structure in low salt solution.

STUDIES ON OLIGORIBONUCLEOTIDES SYNTHESIS

Abstract Some chemical problems concerning hypermodification and deprotection steps during the synthesis of the anticodon loop of tRNA Met i from the yellow lupine are disscused.