A large-scale chemical modification screen identifies design rules to generate siRNAs with high activity, high stability and low toxicity.

The use of chemically synthesized short interfering RNAs (siRNAs) is currently the method of choice to manipulate gene expression in mammalian cell culture, yet improvements of siRNA design is expectably required for successful application in vivo. Several studies have aimed at improving siRNA performance through the introduction of chemical modifications but a direct comparison of these results is difficult. We have directly compared the effect of 21 types of chemical modifications on siRNA activity and toxicity in a total of 2160 siRNA duplexes. We demonstrate that siRNA activity is primarily enhanced by favouring the incorporation of the intended antisense strand during RNA-induced silencing complex (RISC) loading by modulation of siRNA thermodynamic asymmetry and engineering of siRNA 3'-overhangs. Collectively, our results provide unique insights into the tolerance for chemical modifications and provide a simple guide to successful chemical modification of siRNAs with improved activity, stability and low toxicity.

Five- and six-membered conformationally locked 2',4'-carbocyclic ribo-thymidines: synthesis, structure, and biochemical studies.

Two unusual reactions involving the 5-hexenyl or the 6-heptenyl radical cyclization of a distant double bond at C4' and the radical center at C2' of the ribofuranose ring of thymidine have been used as key steps to synthesize North-type conformationally constrained cis-fused bicyclic five-membered and six-membered carbocyclic analogues of LNA (carbocyclic-LNA-T) and ENA (carbocyclic-ENA-T) in high yields. Their structures have been confirmed unambiguously by long range 1H-13C NMR correlation (HMBC), TOCSY, COSY, and NOE experiments. The carbocyclic-LNA-T and carbocyclic-ENA-T were subsequently incorporated into the antisense oligonucleotides (AONs) to show that they enhance the Tm of the modified AON/RNA heteroduplexes by 3.5-5 degrees C and 1.5 degrees C/modification for carbocyclic-LNA-T and carbocyclic-ENA-T, respectively. Whereas the relative RNase H cleavage rates with carbocyclic-LNA-T, carbocyclic-ENA-T, aza-ENA-T, and LNA-T modified AON/RNA duplexes were found to be very similar to that of the native counterpart, irrespective of the type and the site modification in the AON strand, a single incorporation of carbocyclic-LNA and carbocyclic-ENA into AONs leads to very much more enhanced nuclease stability in the blood serum (stable >48 h) as compared to that of the native (fully degraded <3 h) and the LNA-modified AONs (fully degraded <9 h) and aza-ENA ( approximately 85% stable in 48 h). Clearly, remarkably enhanced lifetimes of these carbocyclic-modified AONs in the blood serum may produce the highly desired pharmacokinetic properties because of their unique stability and consequently a net reduction of the required dosage. This unique quality as well as their efficient use as the AON in the RNase H-promoted cleavage of the target RNA makes our carbocyclic-LNA and carbocyclic-ENA modifications excellent candidates as potential antisense therapeutic agents.

Comparison of the RNase H cleavage kinetics and blood serum stability of the north-conformationally constrained and 2'-alkoxy modified oligonucleotides.

The RNase H cleavage potential of the RNA strand basepaired with the complementary antisense oligonucleotides (AONs) containing North-East conformationally constrained 1',2'-methylene-bridged (azetidine-T and oxetane-T) nucleosides, North-constrained 2',4'-ethylene-bridged (aza-ENA-T) nucleoside, and 2'-alkoxy modified nucleosides (2'-O-Me-T and 2'-O-MOE-T modifications) have been evaluated and compared under identical conditions. When compared to the native AON, the aza-ENA-T modified AON/RNA hybrid duplexes showed an increase of melting temperature (DeltaTm = 2.5-4 degrees C per modification), depending on the positions of the modified residues. The azetidine-T modified AONs showed a drop of 4-5.5 degrees C per modification with respect to the native AON/RNA hybrid, whereas the isosequential oxetane-T modified counterpart, showed a drop of approximately 5-6 degrees C per modification. The 2'-O-Me-T and 2'-O-MOE-T modifications, on the other hand, showed an increased of Tm by 0.5 C per modification in their AON/RNA hybrids. All of the partially modified AON/RNA hybrid duplexes were found to be good substrates for the RNase H mediated cleavage. The Km and Vmax values obtained from the RNA concentration-dependent kinetics of RNase H promoted cleavage reaction for all AON/RNA duplexes with identical modification site were compared with those of the reference native AON/RNA hybrid duplex. The catalytic activities (Kcat) of RNase H were found to be greater (approximately 1.4-2.6-fold) for all modified AON/RNA hybrids compared to those for the native AON/RNA duplex. However, the RNase H binding affinity (1/Km) showed a decrease (approximately 1.7-8.3-fold) for all modified AON/RNA hybrids. This resulted in less effective (approximately 1.1-3.2-fold) enzyme activity (Kcat/Km) for all modified AON/RNA duplexes with respect to the native counterpart. A stretch of five to seven nucleotides in the RNA strand (from the site of modifications in the complementary modified AON strand) was found to be resistant to RNase H digestion (giving a footprint) in the modified AON/RNA duplex. Thus, (i) the AON modification with azetidine-T created a resistant region of five to six nucleotides, (ii) modification with 2'-O-Me-T created a resistant stretch of six nucleotides, (iii) modification with aza-ENA-T created a resistant region of five to seven nucleotide residues, whereas (iv) modification with 2'-O-MOE-T created a resistant stretch of seven nucleotide residues. This shows the variable effect of the microstructure perturbation in the modified AON/RNA heteroduplex depending upon the chemical nature as well as the site of modifications in the AON strand. On the other hand, the enhanced blood serum as well as the 3'-exonuclease stability (using snake venom phosphodiesterase, SVPDE) showed the effect of the tight conformational constraint in the AON with aza-ENA-T modifications in that the 3'-exonuclease preferentially hydrolyzed the 3'-phosphodiester bond one nucleotide away (n + 1) from the modification site (n) compared to all other modified AONs, which were 3'-exonuclease cleaved at the 3'-phosphodiester of the modification site (n). The aza-ENA-T modification in the AONs made the 5'-residual oligonucleotides (including the n + 1 nucleotide) highly resistant in the blood serum (remaining after 48 h) compared to the native AON (fully degraded in 2 h). On the other hand, the 5'-residual oligonucleotides (including the n nucleotide) in azetidine-T, 2'-O-Me-T, and 2'-O-MOE-T modified AONs were more stable compared to that of the native counterpart but more easily degradable than that of aza-ENA-T containing AONs.

Conformationally constrained 2'-N,4'-C-ethylene-bridged thymidine (aza-ENA-T): synthesis, structure, physical, and biochemical studies of aza-ENA-T-modified oligonucleotides.

The 2'-deoxy-2'-N,4'-C-ethylene-bridged thymidine (aza-ENA-T) has been synthesized using a key cyclization step involving 2'-ara-trifluoromethylsufonyl-4'-cyanomethylene 11 to give a pair of 3',5'-bis-OBn-protected diastereomerically pure aza-ENA-Ts (12a and 12b) with the fused piperidino skeleton in the chair conformation, whereas the pentofuranosyl moiety is locked in the North-type conformation (7 degrees < P < 27 degrees, 44 degrees < phi m < 52 degrees). The origin of the chirality of two diastereomerically pure aza-ENA-Ts was found to be due to the endocyclic chiral 2'-nitrogen, which has axial N-H in 12b and equatorial N-H in 12a. The latter is thermodynamically preferred, while the former is kinetically preferred with Ea = 25.4 kcal mol-1, which is thus far the highest observed inversion barrier at pyramidal N-H in the bicyclic amines. The 5'-O-DMTr-aza-ENA-T-3'-phosphoramidite was employed for solid-phase synthesis to give four different singly modified 15-mer antisense oligonucleotides (AONs). Their AON/RNA duplexes showed a Tm increase of 2.5-4 degrees C per modification, depending upon the modification site in the AON. The relative rates of the RNase H1 cleavage of the aza-ENA-T-modified AON/RNA heteroduplexes were very comparable to that of the native counterpart, but the RNA cleavage sites of the modified AON/RNA were found to be very different. The aza-ENA-T modifications also made the AONs very resistant to 3' degradation (stable over 48 h) in the blood serum compared to the unmodified AON (fully degraded in 4 h). Thus, the aza-ENA-T modification in the AON fulfilled three important antisense criteria, compared to the native: (i) improved RNA target affinity, (ii) comparable RNase H cleavage rate, and (iii) higher blood serum stability.

Non-identical electronic characters of the internucleotidic phosphates in RNA modulate the chemical reactivity of the phosphodiester bonds.

We here show that the electronic properties and the chemical reactivities of the internucleotidic phosphates in the heptameric ssRNAs are dissimilar in a sequence-specific manner because of their non-identical microenvironments, in contrast with the corresponding isosequential ssDNAs. This has been evidenced by monitoring the delta H8(G) shifts upon pH-dependent ionization (pK(a1)) of the central 9-guaninyl (G) to the 9-guanylate ion (G-), and its electrostatic effect on each of the internucleotidic phosphate anions, as measured from the resultant delta 31P shifts (pKa2) in the isosequential heptameric ssRNAs vis-à-vis ssDNAs: [d/r(5'-Cp1Ap2Q1p3Gp4Q2p5Ap6C-3'): Q1 = Q2 = A (5a/5b) or C (8a/8b), Q(1) = A, Q(2) = C (6a/6b), Q1 = C, Q2 = A (7a/7b)]. These oligos with single ionizable G in the centre are chosen because of the fact that the pseudoaromatic character of G can be easily modulated in a pH-dependent manner by its transformation to G- (the 2'-OH to 2-O- ionization effect is not detectable below pH 11.6 as evident from the N(1-Me)-G analog), thereby modulating/titrating the nature of the electrostatic interactions of G to G- with the phosphates, which therefore constitute simple models to interrogate how the variable pseudoaromatic characters of nucleobases under different sequence context (J. Am. Chem. Soc., 2004, 126, 8674-8681) can actually influence the reactivity of the internucleotide phosphates as a result of modulation of sequence context-specific electrostatic interactions. In order to better understand the impact of the electrostatic effect of the G to G- on the tunability of the electronic character of internucleotidic phosphates in the heptameric ssRNAs 5b, 6b, 7b and 8b, we have also performed their alkaline hydrolysis at pH 12.5 at 20 degrees C, and have identified the preferences of the cleavage sites at various phosphates, which are p2, p3 and p4 (Fig.3). The results of these alkaline hydrolysis studies have been compared with the hydrolysis of analogous N(1-Me)-G heptameric ssRNA sequences 5c, 7c and 8c under identical conditions in order to establish the role of the electrostatic effect of the 9-guanylate ion (and the 2'-OH to 2-O- ionization) on the internucleotidic phosphate. It turned out that the relative alkaline hydrolysis rate at those particular phosphates (p2, p3 and p4) in the N(1-Me)-G heptamers was reduced from 16-78% compared to those in the native counterparts [Fig. 4, and ESI 2 (Fig. S11)]. Thus, these physico-chemical studies have shown that those p2, p3 and p4 phosphates in the native heptameric RNAs, which show pKa2 as well as more deshielding (owing to weaker 31P screening) in the alkaline pH compared to those at the neutral pH, are more prone to the alkaline hydrolysis because of their relatively enhanced electrophilic character resulting from weaker 31P screening. This screening effect originates as a result of the systematic charge repulsion effect between the electron cloud in the outermost orbitals of phosphorus and the central guanylate ion, leading to delocalization of the phosphorus p(pi) charge into its dpi orbitals. It is thus likely that, just as in the non-enzymatic hydrolysis, the enzymatic hydrolysis of a specific phosphate in RNA by general base-catalysis in RNA-cleaving proteins (RNase A, RNA phosphodiesterase or nuclease) can potentially be electrostatically influenced by tuning the transient charge on the nucleobase in the steric proximity or as a result of specific sequence context owing to nearest-neighbor interactions.

Synthesis and structure of novel conformationally constrained 1',2'-azetidine-fused bicyclic pyrimidine nucleosides: their incorporation into oligo-DNAs and thermal stability of the heteroduplexes.

[structures: see text] The synthesis of novel 1',2'-aminomethylene bridged (6-aza-2-oxabicyclo[3.2.0]heptane) "azetidine" pyrimidine nucleosides and their transformations to the corresponding phosphoramidite building blocks (20, 39, and 42) for automated solid-phase oligonucleotide synthesis is reported. The novel bicyclonucleoside "azetidine" monomers were synthesized by two different strategies starting from the known sugar intermediate 6-O-benzyl-1,2:3,4-bis-O-isopropylidene-D-psicofuranose. Conformational analysis performed by molecular modeling (ab initio and MD simulations) and NMR showed that the azetidine-fused furanose sugar is locked in a North-East conformation with pseudorotational phase angle (P) in the range of 44.5-53.8 degrees and sugar puckering amplitude (phi(m)) of 29.3-32.6 degrees for the azetidine-modified T, U, C, and 5-Me-C nucleosides. Thermal denaturation studies of azetidine-modified oligo-DNA/RNA heteroduplexes show that the azetidine-fused nucleosides display improved binding affinities when compared to that of previously synthesized North-East sugar constrained oxetane fused analogues.

Facile preparation of the oxetane-nucleosides.

Efficient and practical large scale synthesis of suitably protected 1',2'-oxetane locked purine and pyrimidine nucleosides for incorporation in oligo-DNA or -RNA by solid-phase synthesis is reported. A high regio and stereoselectivity with preferential formation of the beta-anomer in the glycosylation reaction, using the Vorbrüggen procedure, was achieved by a convergent synthetic procedure with orthogonal protection strategy using either 1,2-di-O-acetyl-3,4-O-isopropylidene-6-O-(4-toluoyl)-d-psicofuranose or 2-O-acetyl-6-O-benzyl-1,3,4-tri-O-(4-toluoyl)-d-psicofuranose as the glycosyl donor.

Significant pKa perturbation of nucleobases is an intrinsic property of the sequence context in DNA and RNA.

The pH titration and NMR studies (pH 6.6-12.5) in the heptameric isosequential ssDNA and ssRNA molecules, [d/r(5'-CAQ1GQ2AC-3', with variable Q1/Q2)], show that the pKa of the central G residue within the heptameric ssDNAs (DeltapKa = 0.67 +/- 0.03) and ssRNAs (DeltapKa = 0.49 +/- 0.02) is sequence-dependent. This variable pKa of the G clearly shows that its pseudoaromatic character, hence, its chemical reactivity, is strongly modulated and tuned by its sequence context. In contradistinction to the ssDNAs, the electrostatic transmission of the pKa of the G moiety to the neighboring A or C residues in the heptameric ssRNAs (as observed by the response of the aromatic marker protons of As or Cs) is found to be uniquely dependent upon the sequence composition. This demonstrates that the neighboring As or Cs in ssRNAs have variable electrostatic efficiency to interact with the central G/G-, which is owing to the variable pseudoaromatic characters (giving variable chemical reactivities) of the flanking As or Cs compared to those of the isosequential ssDNAs. The sequence-dependent variation of pKa of the central G and the modulation of its pKa transmission through the nearest-neighbors by variable electrostatic interaction is owing to the electronically coupled nature of the constituent nucleobases across the single strand, which demonstrates the unique chemical basis of the sequence context specificity of DNA or RNA in dictating the biological interaction, recognition, and function with any specific ligand.