Analysis of acyclic nucleoside modifications in siRNAs finds sensitivity at position 1 that is restored by 5′-terminal phosphorylation both in vitro and in vivo

Small interfering RNAs (siRNAs) are short, double-stranded RNAs that use the endogenous RNAi pathway to mediate gene silencing. Phosphorylation facilitates loading of a siRNA into the Ago2 complex and subsequent cleavage of the target mRNA. In this study, 2′, 3′ seco nucleoside modifications, which contain an acylic ribose ring and are commonly called unlocked nucleic acids (UNAs), were evaluated at all positions along the guide strand of a siRNA targeting apolipoprotein B (ApoB). UNA modifications at positions 1, 2 and 3 were detrimental to siRNA activity. UNAs at positions 1 and 2 prevented phosphorylation by Clp1 kinase, abrogated binding to Ago2, and impaired Ago2-mediated cleavage of the mRNA target. The addition of a 5′-terminal phosphate to siRNA containing a position 1 UNA restored ApoB mRNA silencing, Ago2 binding, and Ago2 mediated cleavage activity. Position 1 UNA modified siRNA containing a 5′-terminal phosphate exhibited a partial restoration of siRNA silencing activity in vivo. These data reveal the complexity of interpreting the effects of chemical modification on siRNA activity, and exemplify the importance of using multiple biochemical, cell-based and in vivo assays to rationally design chemically modified siRNA destined for therapeutic use.     

Unlocking G-quadruplex: Effect of unlocked nucleic acid on G-quadruplex stability

G-quadruplexes are common structural motifs in aptamers. UNA or unlocked nucleic acid is the latest nucleic acid modification. We have attempted to evaluate the impact of UNA modification on the structure and stability of G-quadruplex oligonucleotides for application in aptamer design. We show using CD spectroscopy that UNA modifications can cause structural transitions in some cases although they retain the inherent G- quadruplex signature. From UV melting studies we showed a position dependent effect of UNA modifications such that quadruplexes with UNA modified loops are further stabilized whereas UNA modifications in stem of the G-quadruplex significantly destabilize the structure. The impact of UNA modification on different nucleobases is also investigated. From the analysis of UV melting results, thermodynamic profile was computed and it was concluded that all the sequences are stable at 37 °C. Finally, a greater serum stability of the modified oligonucleotides in comparison with unmodified ones is also demonstrated. Overall, the position dependent effect of single UNA substitutions was observed and analysed.     

Improved specificity of gene silencing by siRNAs containing unlocked nucleobase analogs

siRNAs confer sequence specific and robust silencing of mRNA. By virtue of these properties, siRNAs have become therapeutic candidates for disease intervention. However, their use as therapeutic agents can be hampered by unintended off-target effects by either or both strands of the siRNA duplex. We report here that unlocked nucleobase analogs (UNAs) confer desirable properties to siRNAs. Addition of a single UNA at the 5'-terminus of the passenger strand blocks participation of the passenger strand in RISC-mediated target down-regulation with a concomitant increase in guide strand activity. Placement of a UNA in the seed region of the guide strand prevents miRNA-like off-target silencing without compromising siRNA activity. Most significantly, combined substitution of UNA at the 3'-termini of both strands, the addition of a UNA at the 5'-terminus of the passenger strand, and a single UNA in the seed region of the guide strand, reduced the global off-target events by more than 10-fold compared to unmodified siRNA. The reduction in off-target events was specific to UNA placement in the siRNA, with no apparent new off-target events. Taken together, these results indicate that when strategically placed, UNA substitutions have important implications for the design of safe and effective siRNA-based therapeutics.     

SiRNAs conjugated with aromatic compounds induce RISC-mediated antisense strand selection and strong gene-silencing activity

Short interference RNA (siRNA) is a powerful tool for suppressing gene expression in mammalian cells. In this study, we focused on the development of siRNAs conjugated with aromatic compounds in order to improve the potency of RNAi and thus to overcome several problems with siRNAs, such as cellular delivery and nuclease stability. The siRNAs conjugated with phenyl, hydroxyphenyl, naphthyl, and pyrenyl derivatives showed strong resistance to nuclease degradation, and were thermodynamically stable compared with unmodified siRNA. A high level of membrane permeability in HeLa cells was also observed. Moreover, these siRNAs exhibited enhanced RNAi efficacy, which exceeded that of locked nucleic acid (LNA)-modified siRNAs, against exogenous Renilla luciferase in HeLa cells. In particular, abundant cytoplasmic localization and strong gene-silencing efficacy were found in the siRNAs conjugated with phenyl and hydroxyphenyl derivatives. The novel siRNAs conjugated with aromatic compounds are promising candidates for a new generation of modified siRNAs that can solve many of the problems associated with RNAi technology.     

Synergistic effects between analogs of DNA and RNA improve the potency of siRNA-mediated gene silencing

We report that combining a DNA analog (2′F-ANA) with rigid RNA analogs [2′F-RNA and/or locked nucleic acid (LNA)] in siRNA duplexes can produce gene silencing agents with enhanced potency. The favored conformations of these two analogs are different, and combining them in a 1–1 pattern led to reduced affinity, whereas alternating short continuous regions of individual modifications increased affinity relative to an RNA:RNA duplex. Thus, the binding affinity at key regions of the siRNA duplex could be tuned by changing the pattern of incorporation of DNA-like and RNA-like nucleotides. These heavily or fully modified duplexes are active against a range of mRNA targets. Effective patterns of modification were chosen based on screens using two sequences targeting firefly luciferase. We then applied the most effective duplex designs to the knockdown of the eIF4E binding proteins 4E-BP1 and 4E-BP2. We identified modified duplexes with potency comparable to native siRNA. Modified duplexes showed dramatically enhanced stability to serum nucleases, and were characterized by circular dichroism and thermal denaturation studies. Chemical modification significantly reduced the immunostimulatory properties of these siRNAs in human peripheral blood mononuclear cells.     

Locked Nucleic Acids: Promising Nucleic Acid Analogs for Therapeutic Applications

Locked Nucleic Acid (LNA) is a unique nucleic‐acid modification possessing very high binding affinity and excellent specificity toward complementary RNA or DNA oligonucleotides. The remarkable properties exhibited by LNA oligonucleotides have been employed in different nucleic acid‐based therapeutic strategies both in vitro and in vivo. Herein, we highlight the applications of LNA nucleotides for controlling gene expression.     

基于核酸自组装纳米结构的siRNA药物递送研究进展

RNA干扰(RNA interference,RNAi)作为转录后调节机制,可靶向mRNA进行剪切降解从而发挥基因沉默效应.siRNA (small interference RNA)因其高效性和特异性而被广泛应用于药物研究中.目前,研究者们已开发了多种阳离子载体用于siRNA递送.但由于siRNA双链结构具有相对较强的刚性结构,且阴离子电荷密度较低,无法与阳离子载体形成稳定、致密的复合物,使得siRNA的应用仍面临诸多挑战,如细胞摄取率低、靶向特异性差、递送过程不稳定、潜在的细胞毒性以及易诱发免疫反应等.近年来,核酸自组装纳米结构由于其结构灵活且负电荷密度较高而受到广泛关注,有望实现siRNA药物的高效递送和基因沉默.本文综述了近年来基于核酸自组装纳米结构的siRNA递送的研究进展及其应用.     

In vivo screening of modified siRNAs for non-specific antiviral effect in a small fish model: number and localization in the strands are important

Small interfering RNAs (siRNAs) are promising new active compounds in gene medicine but the induction of non-specific immune responses following their delivery continues to be a serious problem. With the purpose of avoiding such effects chemically modified siRNAs are tested in screening assay but often only examining the expression of specific immunologically relevant genes in selected cell populations typically blood cells from treated animals or humans. Assays using a relevant physiological state in biological models as read-out are not common. Here we use a fish model where the innate antiviral effect of siRNAs is functionally monitored as reduced mortality in challenge studies involving an interferon sensitive virus. Modifications with locked nucleic acid (LNA), altritol nucleic acid (ANA) and hexitol nucleic acid (HNA) reduced the antiviral protection in this model indicative of altered immunogenicity. For LNA modified siRNAs, the number and localization of modifications in the single strands was found to be important and a correlation between antiviral protection and the thermal stability of siRNAs was found. The previously published sisiRNA will in some sequences, but not all, increase the antiviral effect of siRNAs. The applied fish model represents a potent tool for conducting fast but statistically and scientifically relevant evaluations of chemically optimized siRNAs with respect to non-specific antiviral effects in vivo.     

A structure-activity relationship of a thrombin-binding aptamer containing LNA in novel sites

In this report, structural characterization, aptamer stability and thrombin of a new modified thrombin-ligand complex binding aptamer (TBA) containing anti-guanine bases and a loop position locked nucleic acid (LNA) are presented. NMR, circular dichroic spectroscopy and molecular modeling were used to characterize the three-dimensional structure of two G-quadruplexes. LNA-modification of the anti-guanosines yields G-quadruplexes that show affinity and inhibitory activity toward thrombin, whereas LNA-modification of a thymine nucleotide in the TGT loop increases the thermal stability of TBA. As assessed by denatured PAGE electrophoresis, all modified aptamers display an increase in environmental stability. The prothrombin time assay and fibrinogen assay showed that the aptamers still had good inhibitory activity, and 15 of them had the longest PT time. Therefore, the LNA modification is well suited to improve the physicochemical and biological properties of the native thrombin-binding aptamer.     

Energetic aspects of locked nucleic acids quadruplex association and dissociation

The design of modified nucleic acid aptamers is improved by considering thermodynamics and kinetics of their association/dissociation processes. Locked Nucleic Acids (LNA) is a promising class of nucleic acid analogs. In this work the thermodynamic and kinetic properties of a LNA quadruplex formed by the TGGGT sequence, containing only conformationally restricted LNA residues, are reported and compared to those of 2'-OMe-RNA (O-RNA) and DNA quadruplexes. The thermodynamic analysis indicates that the sugar-modified quadruplexes (LNA and O-RNA) are stabilized by entropic effects. The kinetic analysis shows that LNA and O-RNA quadruplexes are characterized by a slower dissociation and a faster association with respect to DNA quadruplex. Interestingly, the LNA quadruplex formation process shows a second-order kinetics with respect to single strand concentration and has a negative activation energy. To explain these data, a mechanism for tetramer formation with two intermediate states was proposed.     

锁核酸研究进展

锁核酸(locked nucleic acid,LNA)是一种新型的寡核酸衍生物,结构中β-D-呋喃核糖的2’-O,4’-C位通过缩水作用形成环形的氧亚甲基桥、硫亚甲基桥或胺亚甲基桥,呋喃糖的结构锁定在C3’内型的N构型,形成了刚性的缩合结构。LNA作为一种新的反义核酸,具有与DNA／RNA强大的杂交亲和力、反义活性、抗核酸酶能力、水溶性好及体内无毒性等优点。LNA在基因诊断和基因治疗上有很多优势,如：单链核酸的多态性基因分型、LNA寡聚体具有高效抑制端粒酶活性及LNA修饰的DNA核酶(LNAzymes)高效清除高级结构的RNA等,有良好的应用研究前景。     

Chimeric aptamers in cancer cell-targeted drug delivery

Aptamers are single-stranded structured oligonucleotides (DNA or RNA) that can bind to a wide range of targets (“apatopes”) with high affinity and specificity. These nucleic acid ligands, generated from pools of random-sequence by an in vitro selection process referred to as systematic evolution of ligands by exponential enrichment (SELEX), have now been identified as excellent tools for chemical biology, therapeutic delivery, diagnosis, research, and monitoring therapy in real-time imaging. Today, aptamers represent an interesting class of modern pharmaceuticals which with their low immunogenic potential mimic extend many of the properties of monoclonal antibodies in diagnostics, research, and therapeutics. More recently, chimeric aptamer approach employing many different possible types of chimerization strategies has generated more stable and efficient chimeric aptamers with aptamer–aptamer, aptamer–nonaptamer biomacromolecules (siRNAs, proteins) and aptamer–nanoparticle chimeras. These chimeric aptamers when conjugated with various biomacromolecules like locked nucleic acid (LNA) to potentiate their stability, biodistribution, and targeting efficiency, have facilitated the accurate targeting in preclinical trials. We developed LNA-aptamer (anti-nucleolin and EpCAM) complexes which were loaded in iron-saturated bovine lactofeerin (Fe-blf)-coated dopamine modified surface of superparamagnetic iron oxide (Fe₃O₄) nanoparticles (SPIONs). This complex was used to deliver the specific aptamers in tumor cells in a co-culture model of normal and cancer cells. This review focuses on the chimeric aptamers, currently in development that are likely to find future practical applications in concert with other therapeutic molecules and modalities.     

Synthesis of 2'-O-guanidinopropyl-modified nucleoside phosphoramidites and their incorporation into siRNAs targeting hepatitis B virus

Synthetic RNAi activators have shown considerable potential for therapeutic application to silencing of pathology-causing genes. Typically these exogenous RNAi activators comprise duplex RNA of approximately 21 bp with 2 nt overhangs at the 3' ends. To improve efficacy of siRNAs, chemical modification at the 2'-OH group of ribose has been employed. Enhanced stability, gene silencing and attenuated immunostimulation have been demonstrated using this approach. Although promising, efficient and controlled delivery of highly negatively charged nucleic acid gene silencers remains problematic. To assess the potential utility of introducing positively charged groups at the 2' position, our investigations aimed at assessing efficacy of novel siRNAs containing 2'-O-guanidinopropyl (GP) moieties. We describe the formation of all four GP-modified nucleosides using the synthesis sequence of Michael addition with acrylonitrile followed by Raney-Ni reduction and guanidinylation. These precursors were used successfully to generate antihepatitis B virus (HBV) siRNAs. Testing in a cell culture model of viral replication demonstrated that the GP modifications improved silencing. Moreover, thermodynamic stability was not affected by the GP moieties and their introduction into each position of the seed region of the siRNA guide strand did not alter the silencing efficacy of the intended HBV target. These results demonstrate that modification of siRNAs with GP groups confers properties that may be useful for advancing therapeutic application of synthetic RNAi activators.     

Locked nucleic acid flow cytometry-fluorescence in situ hybridization (LNA flow-FISH): a method for bacterial small RNA detection

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination, cellular identification and gene expression, monitoring viral multiplication in infected cells, and bacterial community analysis and enumeration. Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (T(m)) of these analogs for natural nucleic acids. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes and have been successfully used to detect eukaryotic mRNA and viral RNA in mammalian cells. Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2).     

功能核酸DNA水凝胶的制备与组装

功能核酸DNA水凝胶是一种以DNA为构建单元通过化学反应或物理缠结自组装而成的新型柔性材料,其构建单元中包含1种或多种能够形成功能核酸的特定序列。功能核酸是通过碱基修饰和DNA分子之间的相互作用力组合的一类特定核酸结构,包括核酸适配体、DNA核酶、G-四联体(G-quadruplex,G4)和i-motif结构等。传统上,高浓度的长DNA链是制备DNA水凝胶的必要条件,而核酸扩增方法的引入为DNA水凝胶的组装方式提供了新的可能。因此,对常用于制备DNA水凝胶的多种功能核酸以及核酸的提取、合成和扩增手段进行了详细的介绍。在此基础上,综述了通过化学或物理交联方式组装功能核酸DNA水凝胶的制备方法。最后,提出了DNA纳米材料的组装所面临的挑战和潜在的发展方向,以期为开发高效组装的功能核酸DNA水凝胶提供参考。     

Structural insight into antisense gapmer-RNA oligomer duplexes through molecular dynamics simulations

There is an extensive research carrying out on antisense technology and the molecules entering into clinical trials are increasing rapidly. Phosphorothioate (PS) is a chemical modification in which nonbridged oxygen is replaced with a sulfur, consequently providing resistance against nuclease activity. The 2'-4' conformationally restricted nucleoside has the structural features of both 2'-O-methoxy ethyl RNA (MOE), which shows good toxicity profile, and locked nucleic acid (LNA), which shows good binding affinity towards the target RNA. These modifications have been studied and suggested that they can be a potential therapeutic agents in antisense therapy. Mipomersen (ISIS 301012), which contains the novel nucleoside modification has been used to target to apolipoprotein (Apo B), which reduces LDL cholesterol by 6–41%. In this study, classical molecular dynamics (MD) simulations were performed on six different antisense gapmer/target-RNA oligomer duplexes (LNA-PS-LNA/RNA, RcMOE-PS-RcMOE/RNA, ScMOE-PS-ScMOE/RNA, MOE-PS-MOE/RNA, PS-DNA/RNA and DNA/RNA) to investigate the structural dynamics, stability and solvation properties. The LNA, MOE nucleotides present in respective duplexes are showing the structure of A-form and the PS-DNA nucleotides resemble the structure of B-form helix with respect to some of the helical parameters. Free energy calculations suggest that the oligomer, which contains LNA binds to the RNA strongly than other modifications as shown in experimental results. The MOE modified nucleotide, which although had a lower binding affinity but higher solvent accessible surface area (SASA) compared to the other modifications, may be influencing the toxicity and hence may be used it in Mipomersen, the second antisense molecule which is approved by FDA.

Communicated by Ramaswamy H. Sarma     

Comparison of different antisense strategies in mammalian cells using locked nucleic acids, 2'-O-methyl RNA,phosphorothioates and small interfering RNA

Locked nucleic acids (LNAs) and double-stranded small interfering RNAs (siRNAs) are rather new promising antisense molecules for cell culture and in vivo applications. Here, we compare LNA–DNA–LNA gapmer oligonucleotides and siRNAs with a phosphorothioate and a chimeric 2′-O-methyl RNA–DNA gapmer with respect to their capacities to knock down the expression of the vanilloid receptor subtype 1 (VR1). LNA–DNA–LNA gapmers with four or five LNAs on either side and a central stretch of 10 or 8 DNA monomers in the center were found to be active gapmers that inhibit gene expression. A comparative co-transfection study showed that siRNA is the most potent inhibitor of VR1–green fluorescent protein (GFP) expression. A specific inhibition was observed with an estimated IC₅₀ of 0.06 nM. An LNA gapmer was found to be the most efficient single-stranded antisense oligonucleotide, with an IC₅₀ of 0.4 nM being 175-fold lower than that of commonly used phosphorothioates (IC₅₀ ~70 nM). In contrast, the efficiency of a 2′-O-methyl-modified oligonucleotide (IC₅₀ ~220 nM) was 3-fold lower compared with the phosphorothioate. The high potency of siRNAs and chimeric LNA–DNA oligonucleotides make them valuable candidates for cell culture and in vivo applications targeting the VR1 mRNA.     

siRNA-induced immunostimulation through TLR7 promotes antitumoral activity against HPV-driven tumors in vivo

Oncogene-specific downregulation mediated by RNA interference (RNAi) is a promising avenue for cancer therapy. In addition to specific gene silencing, in vivo RNAi treatment with short interfering RNAs (siRNAs) can initiate immune activation through innate immune receptors including Toll-like receptors, (TLRs) 7 and 8. Two recent studies have shown that activation of innate immunity by addition of tri-phosphate motifs to oncogene-specific siRNAs, or by co-treatment with CpG oligos, can potentiate siRNA antitumor effects. To date, there are no reports on applying such approach against human papillomavirus (HPV)-driven cancers. Here, we characterized the antitumor effects of non-modified siRNAs that can target a specific oncogene and/or recruit the innate immune system against HPV-driven tumors. Following the characterization of silencing efficacy and TLR7 immunostimulatory potential of 15 siRNAs targeting the HPV type 16 E6/E7 oncogenes, we identified a bifunctional siRNA sequence that displayed both potent gene silencing and active immunostimulation effect. In vivo systemic administration of this siRNA resulted in reduced growth of established TC-1 tumors in C57BL/6 mice. Ablation of TLR7 recruitment via 2'O-methyl modification of the oligo backbone reduced these antitumor effects. Further, a highly immunostimulatory, but non-HPV targeting siRNA was also able to exert antitumoral effects although for less prolonged time compared with the bifunctional siRNA. Collectively, our work demonstrates for the first time that siRNA-induced immunostimulation can have antitumoral effects against HPV-driven tumors in vivo, even independent of gene silencing efficacy.