B. Ravindra Babu

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.

Improved silencing properties using small internally segmented interfering RNAs

RNA interference is mediated by small interfering RNAs (siRNAs) that upon incorporation into the RNA-induced silencing complex (RISC) can target complementary mRNA for degradation. Standard siRNA design usually feature a 19–27 base pair contiguous double-stranded region that is believed to be important for RISC incorporation. Here, we describe a novel siRNA design composed of an intact antisense strand complemented with two shorter 10–12 nt sense strands. This three-stranded construct, termed small internally segmented interfering RNA (sisiRNA), is highly functional demonstrating that an intact sense strand is not a prerequisite for RNA interference. Moreover, when using the sisiRNA design only the antisense strand is functional in activated RISC thereby completely eliminating unintended mRNA targeting by the sense strand. Interestingly, the sisiRNA design supports the function of chemically modified antisense strands, which are non-functional within the context of standard siRNA designs. This suggests that the sisiRNA design has a clear potential of improving the pharmacokinetic properties of siRNA in vivo.

Sensitive SNP Dual-Probe Assays Based on Pyrene-Functionalized 2′-Amino-LNA: Lessons To Be Learned†

A homogenous fluorescence dual‐probe assay containing 2′‐N‐(pyren‐1‐ylmethyl)‐2′‐amino‐LNA (locked nucleic acid) building blocks has been developed for effective mismatch‐sensitive nucleic acid detection. The pyrene units, which are connected to the rigid bicyclic furanose derivative of 2′‐amino‐LNA through a short linker, are positioned at the 3′ and 5′ ends of a dual‐probe system. Whereas hybridization with complementary DNA/RNA results in very strong excimer signals, as the pyrene units are in close proximity to one another in the ternary complex, exposure to most singly mismatched DNA/RNA targets results in significantly lower excimer emission intensity. The mechanism that underlies this excellent optical discrimination of singly mismatched targets is clarified by comparison of the thermal‐denaturation profiles and fluorescence properties of the dual probe and a covalently linked analogue. Optical discrimination of singly mismatched targets arises from a decrease in excimer emission intensity due to a failure to form a ternary complex (a decrease in thermal stability) and/or local mismatch‐induced changes in the helix geometry, depending on the position of the mismatched base pair. The devised dual‐probe assay constitutes a simple and sensitive system for the detection of single‐nucleotide polymorphism and highlights that conformational restriction combined with the use of short probes conveys favorable properties to dual‐probe constructs.

Conformationally locked aryl C-nucleosides: synthesis of phosphoramidite monomers and incorporation into single-stranded DNA and LNA (locked nucleic acid)

Synthesis of a series of LNA-type β-configured C-aryl nucleosides, i.e., 2′-O,4′-C-methylene-β-D-ribofuranosyl derivatives containing phenyl, 4-fluoro-3-methylphenyl, 1-naphthyl, 1-pyrenyl and 2,4,5-trimethylphenyl groups as aglycons, has been accomplished. The key synthetic step consisted of stereoselective Grignard reactions of the cyclic aldehyde 11 followed by cyclization to give the bicyclic core structure with a locked N-type furanose conformation as confirmed by NOE experiments on the di-O-p-methoxybenzyl derivatives 13a–13e and an X-ray crystallographic study of the phenyl derivative 14a. The phosphoramidite approach was used for automated incorporation of the LNA-type β-configured C-aryl monomers 17a–17e into short DNA and 2′-OMe-RNA/LNA strands. It is shown that universal hybridization can be obtained with a conformationally restricted monomer as demonstrated most convincingly for the pyrene LNA monomer 17d, both in a DNA context and in an RNA-like context. Increased binding affinity of oligonucleotide probes for universal hybridization can be induced by combining the pyrene LNA monomer 17d with affinity-enhancing 2′-OMe-RNA/LNA monomers.

Locked nucleoside analogues expand the potential of DNAzymes to cleave structured RNA targets

BackgroundDNAzymes cleave at predetermined sequences within RNA. A prerequisite for cleavage is that the DNAzyme can gain access to its target, and thus the DNAzyme must be capable of unfolding higher-order structures that are present in the RNA substrate. However, in many cases the RNA target sequence is hidden in a region that is too tightly structured to be accessed under physiological conditions by DNAzymes.ResultsWe investigated how incorporation of LNA (locked nucleic acid) monomers into DNAzymes improves their ability to gain access and cleave at highly-structured RNA targets. The binding arms of DNAzymes were varied in length and were substituted with up to three LNA and α-L-LNA monomers (forming LNAzymes). For one DNAzyme, the overall cleavage reaction proceeded fifty times faster after incorporation of two α-L-LNA monomers per binding arm (kobs increased from 0.014 min-1 to 0.78 min-1).ConclusionThe data demonstrate how hydrolytic performance can be enhanced by design of LNAzymes, and indicate that there are optimal lengths for the binding arms and for the number of modified LNA monomers.

Locked Nucleic Acids and Intercalating Nucleic Acids in the Design of Easily Denaturing Nucleic Acids: Thermal Stability Studies

Intercalating nucleic acids (INA®s) with insertions of (R)‐1‐O‐(1‐pyrenylmethyl)glycerol were hybridized with locked nucleic acids (LNAs). INA/LNA duplexes were found to be less stable than the corresponding DNA/LNA duplexes when the INA monomer was inserted as a bulge close to the LNA monomers in the opposite strand. This property was used to make “quenched” complements that possess LNA in hairpins and in duplexes and are consequently more accessible for targeting native DNA. The duplex between a fully modified 13‐mer LNA sequence and a complementary INA with six pyrene residues inserted after every second base as a bulge was found to be very unstable (Tm=30.1 °C) in comparison with the unmodified double‐stranded DNA (Tm=48.7 °C) and the corresponding duplexes of LNA/DNA (Tm=81.6 °C) and INA/DNA (Tm=66.4 °C). A thermal melting experiment of a mixture of an LNA hairpin, with five LNA nucleotides in the stem, and its complementary DNA sequence gave a transition with an extremely low increase in optical density (hyperchromicity). When two INA monomers were inserted into the stem of the LNA hairpin, the same experiment resulted in a significant hyperchromicity comparable with the one obtained for the corresponding DNA/DNA duplex.

LNA and α-L-LNA: Towards Therapeutic Applications

Abstract LNA and α-L-LNA are promising candidates for the development of efficient oligonucleotide-based therapeutic agents. Here, we report dose-dependent inhibition of HIV-1 Tat-dependent trans activation by a 12-mer chimeric α-L-LNA/DNA oligomer. This oligomer exhibits a dose-dependency similar to that of the corresponding 12-mer chimeric LNA/2′-O-Me-RNA oligomer. In addition, we show that incorporation of α-L-LNA or LNA monomers into each of the two binding arms of a “10–23” DNAzyme markedly increases cleavage of the target RNA.

Optimized DNA targeting using N,N-bis(2-pyridylmethyl)-β-alanyl 2′-amino-LNA

Incorporation of N,N-bis(2-pyridylmethyl)-β-alanyl 2′-amino-LNA (bipyridyl-functionalized 2′-amino locked nucleic acid) monomers into DNA strands enables high-affinity targeting of complementary DNA with excellent Watson–Crick selectivity in the presence of divalent metal ions. Positioning of bipyridyl-functionalized 2′-amino-LNA monomers in two complementary DNA strands in a “3′-end zipper” constitution allows modulation of duplex stability, i.e., a strong stabilizing effect with one equivalent of divalent metal ion per bipyridyl pair, or a strong destabilizing effect with an excess of divalent metal ions.

Novel (phenylethynyl)pyrene-LNA constructs for fluorescence SNP sensing in polymorphic nucleic acid targets.

We describe fluorescent oligonucleotide probes labeled with novel (phenylethynyl)pyrene dyes attached to locked nucleic acids. Furthermore, we prove the utility of these probes for the effective detection of single‐nucleotide polymorphisms in natural nucleic acids. High‐affinity hybridization of the probes and excellent fluorescence responses to single‐base mismatches in DNA/RNA targets are demonstrated in model dual‐probe and doubly labeled probe formats. This stimulated us to develop two diagnostic systems for the homogeneous detection of a drug‐resistance‐causing mutation in HIV‐1 protease cDNA and RNA gene fragments. Target sequences were obtained by analysis of 200 clinical samples from patients currently receiving anti‐HIV/AIDS combination therapy at the Russian Federal AIDS Center. Using these fluorescent oligonucleotides, we were able to detect the target mutation despite all the challenges of the natural targets, that is, the presence of additional mutations, neighboring sequence variation, and low target concentration, which typically reduce binding and effectiveness of sensing by fluorescent oligonucleotides.

Oligodeoxynucleotides containing α-L-ribo configured LNA-type C-aryl nucleotides

Synthesis of 2′-O,4′-C-methylene-α-L-ribofuranosyl derivatives containing phenyl and 1-pyrenyl aglycons, i.e., novel α-L-ribo configured LNA-type C-aryl nucleosides, has been accomplished. Key synthetic steps included stereoselective Grignard reactions on tetrahydrofuran aldehyde 12, configurational inversion of the resulting alcohol 13 into alcohol 15, and concomitant Mitsonobu cyclization furnishing the desired bicyclic furanosyl skeleton with a locked conformation. The phosphoramidite derivatives 19a and 19b were used for automated synthesis of 9-mer DNA and α-L-LNA oligonucleotides containing the α-L-LNA-type C-aryl monomers αLPhL and αLPyL containing a phenyl and pyrenyl aglycon, respectively. Thermal denaturation studies showed universal base pairing behavior for the pyrenyl monomer αLPyL when incorporated into a DNA or an α-L-LNA oligonucleotide.