Jan Milecki

THE FIRST EXAMPLE OF SEQUENCE-SPECIFIC NON-UNIFORMLY 13C5 LABELLED RNA : SYNTHESIS OF THE 29MER HIV-1 TAR RNA WITH 13C RELAXATION WINDOW

Abstract A complete synthetic protocol as well as 1H- and 13C-NMR data on the monomer building blocks used for the solid-phase synthesis of specifically 13C-labelled (99 atom % 13C) stem (27A and 43G), bulge (24C) and loop (31U) regions of 29mer HIV-1 TAR RNA hairpin starting from the 13C6- D -glucose are presented. The complex NMR spectra of 13C-labelled monomer building blocks, due to the interaction of various 13C and 1H spins, have been assigned. It has been demonstrated by heteronuclear 2D NMR that the non-uniform labelling of the HIV-1 TAR 29mer RNA achieved herein by chemical synthesis provides an optimal opportunity to perform full T1 and T2 relaxation measurements (the “NMR Relaxation Window”) of each type of sugar-carbons for all four strategically placed 13C-labelled residues in a unique and unprecedented manner because of minimal overlap of 13C resonances compared to uniformly labelled oligo-RNA.

On the Application of t-Butyldimethylsilyl Group in Chemical RNA Synthesis. Part I. 31P NMR Study of 2′-O-t-BDMSi Group Migration During Nucleoside 3′-OH Phosphorylation and Phosphitylation Reactions

Abstract 31P NMR spectroscopy has been used for evaluation of 2′-O-t-BDMSi group migration during reactions of suitably protected 3′-OH ribonucleosides with P(V) and P(III) reagents used in major methodologies for oligoribonucleotide synthesis.

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.

The hydration and unusual hydrogen bonding in the crystal structure of an RNA duplex containing alternating CG base pairs

The X-ray structures of two crystal forms of the RNA duplex [CGCG(5-FC)G]2 are the first crystal structures for an RNA duplex containing alternating CG base pairs with unmodified 2′-hydroxyl groups. The triclinic and the rhombohedral crystal forms (PDB ids 3jxq, 3jxr) differ in the molecular packing, the amount of ordered structure and in molecular details, especially the hydration. The P1 data extend to 1.45 A resolution, while the R32 data reach 1.25 A. In both structures the RNA oligomers take the form of a right-handed half-turn with conformational parameters close to the canonical A-RNA. The duplexes stack coaxially in the crystal lattice. The geometry of C:G vs. 5-FC:G pairing is similar in terms of H-bond geometry. In CG steps, inter-strand guanines are parallel while cytosines are not parallel. In steps GC this motif is reversed. Both structures contain ordered water molecules, forming characteristic hydration network in the major and the minor grooves. In addition to water, the triclinic structure contains four magnesium cations, one of them forming an inner complex with a phosphate. Two types of unusual interactions are observed. In the major groove, the 5-fluorocytidine residues form C–F⋯H–O–H hydrogen bonds with water molecules. In the minor groove, intermolecular contacts include C–H hydrogen bonds (C1′–H1⋯O2′ and C4′–H4′⋯O4′) between two ribose rings.

Synthesis and hybridization properties of oligonucleotide analogues with novel acyclic triazole internucleotide linkages

Herein, we describe synthesis of novel acyclic dinucleotide analogues connected via triazole linkage in CuAAC reaction. Synthesis pathway starting from previously obtained building blocks containing alkyne or azide functional group is described. Further functionalization and application of dinucleotide analogues in DNA phosphoramidite solid-phase synthesis is also explained. Additionally, we have examined the influence of novel modifications on DNA duplex thermodynamic stability.

Highly Efficient Fluorescent Interstrand Photo‐crosslinking of DNA Duplexes Labeled with 5‐Fluoro‐4‐thio‐2′‐O‐methyluridine

The formation of a fluorescent photoadduct between 5‐fluoro‐4‐thiouridine (FSU), in the sequence context 5′‐AFSUA‐3′ and incorporated into a synthetic oligonucleotide either at its 3′‐ or 5′‐end, and one of the thymines of the TAT motif in a complementary target DNA strand led to photo‐crosslinking of the two strands for several oligonucleotide constructs. Enzymatic digestion, MS, UV, and fluorescence spectral analyses of the interstrand crosslinked oligonucleotides revealed the identity of the thymidine that participates in the photo‐crosslinking reaction as well as the diastereomeric structures of the crosslinks. The proposed pathways of interstrand photo‐crosslinking are supported by experiments with isotopically labeled oligonucleotide constructs and visualized by means of molecular dynamics simulations.

The Effect of Stereochemistry on Sodium Ion Complexation in Nucleoside-Metallacarborane Conjugates

Conjugates of purine and pyrimidine nucleosides: thymidine and 2′-deoxyguanosine with cobalt-metallacarborane were studied for their sodium ion complexing properties. Formation of stable complexes of 1 : 1 stoichiometry was proved by ESI MS spectroscopy and 23Na NMR. Equilibrium constants and energies of complex formation were calculated. Complexation of alkali-metals by nucleoside-metallacarborane conjugates may affect the physicochemical and biological properties of the conjugates and should be taken into consideration during biological evaluation of these types of modifications.

Synthesis and cytotoxic activity of novel acyclic nucleoside analogues with functionality in click chemistry

ABSTRACT We describe synthesis of novel acyclic nucleoside analogues which are building blocks for CuAAC reaction and their activity against two types of human cancer cell lines (HeLa, KB). Three of chosen compounds show promising cytotoxic activity. Synthesis pathway starting from simple and easily accessible substrates employing DMT or TBDPS protective groups is described. Adenosine and thymidine analogues containing alkyne moiety and adenosine analogue containing azido group were synthesized. The obtained units showed ability of forming triazole motif under the CuAAC reaction conditions. GRAPHICAL ABSTRACT

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.