Edouard Zamaratski

A critical survey of the structure-function of the antisense oligo/RNA heteroduplex as substrate for RNase H

The aim of this review is to draw a correlation between the structure of the DNA/RNA hybrid and its properties as a substrate for the RNase H, as well as to point the crucial structural requirements for the modified AONs to preserve their RNase H potency. The review is divided into the following parts: (1) mechanistic considerations, (2) target RNA folding-AON folding-RNase H assistance in AON/RNA hybrid formation, (3) carbohydrate modifications, (4) backbone modifications, (5) base modifications, (6) conjugated AONs, (7) importance of the tethered chromophore in AON for the AON/RNA hybrid interactions with the RNase H. The structural changes in the AON/RNA hybrid duplexes brought by different modifications of the sugar, backbone or base in the antisense strand, and the effect of these changes on the RNase H recognition of the modified substrates have been addressed. Only those AON modifications and the corresponding AON/RNA hybrids, which have been structurally characterized by spectroscopic means and functionally analyzed by their ability to elicit RNase H potency in comparison with the native counterpart have been presented here.

Synthesis of 5′-polyarene-tethered oligo-DNAs and the thermal stability and spectroscopic properties of their duplexes and triplexes

Abstract Eleven different planar hydroxy alkylated polyarenes 1–11 with different geometry, bulk and electronic characteristics were synthesised, and used for tethering to the 5′-phosphate of a 9-mer and a 18-mer DNA sequences through solid-phase synthesis. The 5′-polyarene-tethered 9-mers 30–40 were tested for their ability to form stable duplexes with four complementary target DNA-strands 25–28 of different length. The 5′-polyarene-tethered 18-mers 44–54 were tested for their ability to form stable triplexes witha 24-mer duplex target 41+42 . The Tm measurements of duplexes at low salt and pH 7.3 showed that the angular nitro phenanthrene and phenanthrene conjugates 31 and 30 gave the highest duplex stabilisations with targets 25 (ΔTm 13.8°C and 11.8°C) and 26 (ΔTm 12.3°C and 11.9°C). With the mismatch sequence 28, only 30 and 31 gave a high ΔTm of 11.6°C and 10.8°C respectively, while lower ΔTm values were observed for other conjugates (ΔTm ∼4.0–5.0°C). The Tm measurements of triplexes between 43–54 and duplex target 41+42 at low salt and pH 7.3, 6.5 and 6.0 without Mg2+ showed that the nitro phenanthrene conjugate 45 gave the best triplex stabilisation (ΔTm 4.1–5.4°C). The stabilisation of nitro phenanthrene conjugate 45 compared to phenanthrene conjugate 44 increased more remarkably when Mg2+ was present: 45 (ΔTm 15°C), 44 (ΔTm 10°C). These results imply that the electron density of the chromophore influences the π-π stacking interactions between the chromophore and nucleobases, and thereby influencing the duplex and triplex stability. Fluorescence measurements on single strand to double strand transition indicated that the 5′-tethered polyarenes are stacked only on the neighbouring nucleobases of the opposite strand. In case of 5′-9-N-ethylphenazinium conjugate 36, a comparative NMR and fluorescence measurement has unambiguously shown that the tethered phenazinium ion is indeed intercalated betweenthe nucleotides of the opposite target strand 26. Thermodynamic calculations showed the most stable ΔG∘ (298K) for 30, 31(+targets 25, 26, 28) and 35, 36(+targets 25, 26) compared to the blank 29 (ΔΔG∘ (298K)−10kJ mol−1). The non-palindromic target 27 was shown by Tm measurements to form a stable tertiary structure, which was very little affected by addition of any 5′-tethered conjugate, thereby showing the importance of the tertiary structures of an in vivo antisense target and its implication in regard to its bioavailablity to complementary antisense probes. The thermal stability and spectroscopic properties of the duplexes and triplexes resulting from different polyarene-tethered oligo-DNAs are reported. The importance of finding a correct antisense target which does not form tertiary structure of its own is also highlighted.

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.

Transmission of the conformational information in the antisense:RNA hybrid duplex influences the pattern of the RNase H cleavage reaction †

A set of four hybrid duplexes formed by antisense oligos, each containing one North-form (3%-endo) locked 1-(1%,3%-O-anhydro-b-D-psicofuranosyl)thymine block T6 at different sites and a matched 15mer RNA, were subjected to the RNase H cleavage reaction, showing how the transmission of the local conformation owing to a single North-locked sugar steers the conformational changes of the four neighbouring nucleotides.

The 3 '-modified antisense oligos promote faster hydrolysis of the target RNA by RNase H than the natural counterpart

Abstract We have examined the antisense potency of the hybrid duplexes of fully-matched 3′-, 5′ and interior-chromophore tethered antisense oligos (AON) and three target RNAs (11mer and two 17mers) against RNase H, and found them to be better substrates compared to the native DNA/RNA hybrid. These target RNAs were chosen for complexation with AONs because they have very different folding characteristics as evident from the temperature- and concentration-dependent UV and CD spectroscopy. The differences in the tertiary structures of the target RNAs have been exploited here to investigate the kinetic availability of the single-stranded region accessible for the complexation with the AON during the RNase H promoted cleavage. It has emerged that the cleavage rate of the target RNA in the hybrid is independent of the complexity of the folding of the target RNA, thereby suggesting that (i) The kinetic accessibility of the single strand region in all three RNA targets, ( 11 ), ( 12 ) and ( 13 ), by AONs are very similar, and indeed not rate-limiting, although sequence specificities are non-identical in the 11mer and 17mers RNAs. (ii) The rate of conversion of the folded RNA structures to the single-stranded form, and subsequently its kinetic accessibility to drive the hybrid AON/RNA duplex formation is much faster than the RNase H promoted cleavage rate of the RNA moiety in the hybrid. (iii) This also means that the RNase H promoted cleavage rate of the hybrid is the slowest (i.e. the rate-determining).

Conformation-specific cleavage of antisense oligonucleotide-RNA duplexes by RNase H

The North-form (3′-endo) constrained 1-(1′,3′-O-anhydro-β-D-psicofuranosyl)thymine block, , was systematically incorporated at various sites, one at a time, into a set of four antisense oligonucleotides (AONs). The hybrids of these AONs with a matched 15mer RNA target were subjected to the RNase H cleavage reaction, and compared with that of the native counterpart, in order to probe how far the local influence of a single North-locked sugar is transmitted in steering conformational changes in the neighbouring nucleotides. It was found that the introduction of a single North-sugar locked nucleotide in the AONs makes up to four of the neighbouring nucleotides at the 5′-end of the modification site resistant to the RNase H cleavage reaction. This suggests that a stretch of 5-nucleotides, including the nucleotide, in the AON strand adopts a North-type conformation, giving a local RNA/RNA type hybrid structure instead of a regular DNA/RNA type duplex structure. Although these 5-nucleotide regions were completely resistant to RNase H promoted hydrolysis, they could serve as the binding site for the enzyme. Interestingly, none of these local adaptations of the RNA/RNA type structure were observable by CD spectroscopy, showing it to be an unsuitable means of monitoring any subtle alteration of the local structure. This work, therefore, constitutes an example of how the engineered conformation of a substrate can be used to exploit the stereochemical sensitivity of an enzyme to map local microscopic conformational changes. The other implication of this work is that it provides a new tool to gather local structural information, which may help to optimize the number of constrained residues which need to be incorporated to induce the antisense strand to adopt either A- or B-type geometry in the hybrid duplex, with or without the loss of RNase H recognition and/or cleavage properties.

Synthesis of phenazine-tethered arabino and xylofuranosyl oligonucleotide conjugates: The thermal stability and fluorescence properties of their duplexes (DNA-DNA & DNA-RNA) & triplexes

Abstract The synthesis of phenazine (Pzn) tethered ara-U and xylo-U incorporated oligonucleotides, and their properties as DNA-DNA, DNA-RNA duplexes and as triplexes are reported. 2′-O-Pzn-tethered ara-U amidite (11) and Pzn-linked amidite block (4) as well as CPG solid supports functionalised with 2′-O-Pzn-tethered ara-U (10a – 10c) and 3′-O-Pzn-tethered xylo-U succinates (22a – 22c) were used in the solid-phase DNA synthesis to prepare modified 9mers 25 – 29, 31, 41 – 45 (for DNA-DNA & DNA-DRA duplex studies) and 18mers 35 – 40, 46 – 50 (for the DNA triplex studies). Thermal melting experiments with the resulting duplexes and triplexes showed that all oligo-DNAs, except for the middle-modified ones (31 & 40), have enhanced affinity to the DNA and RNA targets (23&52) as well as for the DNA duplex target 32·33. Pzn-tethered ara-U block was more efficient at the 3′-terminal of oligonucleotides than at the 5′-end in the duplexes and especially in triplexes, where it provided a dramatic improvement in the stability (ΔTm = 16.1°C). Employment of Pzn-tethered ara-U block at the 3′-end together with the Pzn-tethered dT block at the 5′-end of the oligo-DNAs (29, 38) provided the best duplex and triplex stabilisation to give a highest ΔTm of 14.4°C for DNA-DNA duplexes, ΔTm of 11.7°C for DNA-RNA duplexes and ΔTm of 19.6°C for triplexes. All DNA-DNA and DNA-RNA duplexes as well as DNA triplexes, formed by the oligos modified with 2′-O-Pzn-tethered ara-U blocks showed geater stability than those formed by the oligos modified with xylo-analogs with the same length of the linker arms. For both ara- and xylo-configurations, the best DNA-DNA & DNA-RNA duplex stabilisation was provided by the short ethyl linkers, and increasing the length of the linker led to considerable destabilisation of the duplexes. In case of triplexes, longer linker arms were required to obtain better stabilisation. Hexyl linker provided the highest triplex stabilisation for the oligonucleotides modified with Pzn-tethered ara-U block (ΔTm = 16.5°C) and butyl linker was found to be most suitable for the oligo-DNAs modified with Pzn-tehtered xylo-U block (ΔTm = 12.3°C). Fluorescence studies showed that Pzn behaves as a weak exterior binder upon DNA-DNA or DNA-RNA duplex or DNA triplex formation which accounts for moderate changes in the fluorescent properties of the Pzn moiety (ΔF for DNA-DNA and DNA-RNA duplexes = ±0.2, ΔF for triplexes = 1.4 – 2.5). Employment of Pzn at both 3′ and 5′ ends of the oligonucleotides (as in 27, 29, 38 & 39) provided the greatest duplex and triplex stabilisation so far, and led to more effective interaction between the Pzn moieties and the double and triple helixes (ΔF for DNA-DNA and DNA-RNA duplexes = 4, ΔF for triplexes = 5).

The First Example of a Hoogsteen Basepaired DNA Duplex in Dynamic Equilibrium with a Watson-Crick Basepaired Duplex—A Structural (NMR), Kinetic and Thermodynamic Study

Abstract A single-point substitution of the O4′ oxygen by a CH2 group at the sugar residue of A 6 (i.e. 2′-deoxyaristeromycin moiety) in a self-complementary DNA duplex, 5′- d(C1G2C3G4A5A6T7T8C9G10C11G12)2 −3, has been shown to steer the fully Watson-Crick basepaired DNA duplex (1A), akin to the native counterpart, to a doubly A 6:T7 Hoogsteen basepaired (1B) B-type DNA duplex, resulting in a dynamic equilibrium of (1A)→←(1B): Keq = k1/k-1 = 0.56±0.08. The dynamic conversion of the fully Watson-Crick basepaired (1A) to the partly Hoogsteen basepaired (1B) structure is marginally kinetically and thermodynamically disfavoured [k1 (298K) = 3.9± 0.8 sec−1; δH°‡ = 164±14 kJ/mol;-TδS°‡ (298K) = −92 kJ/mol giving a δG298°‡ of 72 kJ/mol. Ea (k1) = 167±14 kJ/mol] compared to the reverse conversion of the Hoogsteen (1B) to the Watson-Crick (1A) structure [k-1 (298K) = 7.0±0.6 sec-1, δH°‡ = 153±13 kJ/mol;-TδS°‡ (298K) = −82 kJ/mol giving a δG298°‡ of 71 kJ/mol. Ea (k-1) = 155±13 kJ/mol]. A comparison of δG298°‡ of the forward (k1) and backward (k-1) conversions, (1A)→←(1B), shows that there is ca 1 kJ/mol preference for the Watson-Crick (1A) over the double Hoogsteen basepaired (1B) DNA duplex, thus giving an equilibrium ratio of almost 2:1 in favour of the fully Watson-Crick basepaired duplex. The chemical environments of the two interconverting DNA duplexes are very different as evident from their widely separated sets of chemical shifts connected by temperature-dependent exchange peaks in the NOESY and ROESY spectra. The fully Watson-Crick basepaired structure (1A) is based on a total of 127 intra, 97 inter and 17 cross-strand distance constraints per strand, whereas the double A 6:T7 Hoogsteen basepaired (1B) structure is based on 114 intra, 92 inter and 15 cross-strand distance constraints, giving an average of 22 and 20 NOE distance constraints per residue and strand, respectively. In addition, 55 NMR-derived backbone dihedral constraints per strand were used for both structures. The main effect of the Hoogsteen basepairs in (1B) on the overall structure is a narrowing of the minor groove and a corresponding widening of the major groove. The Hoogsteen basepairing at the central A 6:T7 basepairs in (1B) has enforced a syn conformation on the glycosyl torsion of the 2′- deoxyaristeromycin moiety, A 6, as a result of substitution of the endocyclic 4′-oxygen in the natural sugar with a methylene group in A 6. A comparison of the Watson-Crick basepaired duplex (1A) to the Hoogsteen basepaired duplex (1B) shows that only a few changes, mainly in α, σ and γ torsions, in the sugar-phosphate backbone seem to be necessary to accommodate the Hoogsteen basepair.

The recognition and cleavage of RNA in the antisense oligo–RNA hybrid duplexes by RNase H

Abstract The different extent of the target RNA cleavage at t 99.9% by RNase H in the AON–RNA duplexes, at the RNA saturation condition by antisense oligo, is due to different recognition and catalytic properties of RNase H toward the hybrids owing to different substituents at the 3′-end of the AONs, not owing to the different thermodynamic stabilities of the antisense oligonucleotide (AON)–RNA duplexes.

Design, synthesis and in vitro biological evaluation of oligopeptides targeting E. coli type I signal peptidase (LepB).

Type I signal peptidases are potential targets for the development of new antibacterial agents. Here we report finding potent inhibitors of E. coli type I signal peptidase (LepB), by optimizing a previously reported hit compound, decanoyl-PTANA-CHO, through modifications at the N- and C-termini. Good improvements of inhibitory potency were obtained, with IC50s in the low nanomolar range. The best inhibitors also showed good antimicrobial activity, with MICs in the low μg/mL range for several bacterial species. The selection of resistant mutants provided strong support for LepB as the target of these compounds. The cytotoxicity and hemolytic profiles of these compounds are not optimal but the finding that minor structural changes cause the large effects on these properties suggests that there is potential for optimization in future studies.