ReviewKeynoteChemically modified siRNA: tools and applications
Introduction
It has been ten years since the publication of the seminal paper demonstrating the high potency of long double-stranded RNA in gene knockdown [1]. Shortly thereafter, it was discovered that the same effect could be produced in mammalian cells using synthetic short RNA duplexes [2]. The relatively few years since then have seen an explosion of research into therapeutic applications of RNAi. Several companies have been formed to pursue the technology, and transactions involving these companies have recently been measured in the billions of dollars.
The reason for the excitement is that RNAi allows potent knockdown of virtually any gene. This in turn allows rapid progression from target selection to preclinical trials. siRNA has become the most common tool in functional genomics, and therefore can often also help at the target identification stage. Furthermore, some targets that are not druggable by traditional methods can be targeted by gene knockdown.
In spite of the immense attractiveness of gene knockdown as a therapeutic strategy, siRNA duplexes are not optimal drug-like molecules. RNA is highly vulnerable to serum exo- and endo-nucleases, leading to a short half-life in serum. siRNA duplexes are composed of two strands that can drift apart in a dilute environment-like serum. Because oligonucleotides are polyanions they do not easily cross cell membranes and, because this charge density leads to extensive hydration, they do not easily interact with albumin and other serum proteins, leading to rapid elimination. Unmodified oligonucleotides have limited tissue distribution. And finally, oligonucleotides can have off-target effects, either by stimulating the immune system or by entering other endogenous gene regulation pathways.
A wide variety of chemical modifications have been proposed to address these issues. In this review, we examine the principles of chemical modification of siRNA duplexes. We will briefly look into the toolbox; that is, summarize the possible ways that siRNA duplexes can be modified. Following this, we will review the ways these tools have been applied to move siRNA toward the clinic, including the use of chemical modifications to improve potency, serum stability, specificity and delivery. We will point out the most useful and universal modifications as well as some of the most creative modifications and applications, which stretch our paradigms and open new avenues of research into RNAi-based drugs.
Much excellent work has led to significant growth in understanding the mechanism of RNAi 3, 4, 5 (Fig. 1, also see Glossary of specialist terms). When an exogenous 19–21 bp siRNA is introduced into a mammalian cell the 5′-end is phosphorylated. The duplex is then assembled into the RNA-induced silencing complex (RISC), a multiprotein complex including Argonaute2 (AGO2), Dicer, TRBP (HIV-1 TAR RNA-binding protein) and PACT (a dsRNA-binding protein), as well as other proteins, some of which are yet unknown [3]. The strand with the lower binding affinity at its 5′-end becomes the antisense (guide) strand 6, 7, and the other strand (known as the sense or passenger strand) is cleaved and unwound, to leave a single-stranded RNA associated with Argonaute2 (AGO2), an endonuclease at the heart of RISC that promotes location of complementary mRNA, hybridization and cleavage of the mRNA target. When modifying an siRNA duplex it is important to remember that different modification approaches are required for the sense and antisense strands, because of these very different roles 8, 9.
In most cases it is simply assumed that the RNAi mechanism is unaffected by chemical modification of siRNA duplexes. A few studies using modified siRNA have confirmed this by showing that the cleavage of complementary mRNA occurs between bases 10 and 11, counting from the 5′-end of the guide strand, as is the case for unmodified duplexes 10, 11, 12. However, in principle, this should be verified for each new pattern of modification.
Section snippets
Toolbox
siRNA duplexes have been chemically modified in a wide variety of ways. Some of the results in the literature, however, seem to contradict one another, or to work on one system but not another. This field is still very young, and it will take time for the more robust and universal modifications to be recognized as such. In the meantime, it is useful to have many options so that at least one of the chemistries can be used to modify an siRNA without compromising its potency.
In this section, we
Improving serum stability
Unprotected RNA is very quickly degraded in cells. The fact that siRNA is double-stranded provides it with some degree of protection, but not enough for in vivo use. A nuclease called eri-1 has been found to play a key part in the degradation of siRNA [65], and expression levels of eri-1 inversely correlate with duration of siRNA activity [66]. This and other data suggest that increasing the nuclease resistance of siRNAs can prolong their activity. Chemical modification is the principal
siRNA drugs in clinical trials
Several siRNA drug candidates are now in clinical trials (Table 1), and several more are expected to start soon. At least three of the drugs in clinical trials are unmodified, and most are being delivered locally as naked siRNAs (to the eye, lung and skin). Nevertheless, a recent paper has called into question whether naked siRNAs delivered to the eye are really working via an RNAi mechanism [77]. This includes the siRNA closest to the market, OPKO's Phase III candidate, bevasiranib. However,
Conclusions and future perspectives
We believe that appropriate chemical modifications and delivery technologies will be essential for bringing RNAi to the clinic. Although investigation was initially focused on increasing serum stability, the need for chemical modification is much broader. The areas of specificity and delivery are among the greatest challenges to RNAi therapeutics, and chemistry will certainly be a big part of the solution to both problems.
Several frontiers remain in the field of chemically modified siRNAs. Most
Acknowledgements
We thank Prof. David R. Corey, Prof. Jean-Christophe Leroux and Dr. Muthiah Manoharan for their valuable comments during the preparation of this manuscript.
Glossary
- RNA interference (RNAi)
- An evolutionarily conserved cellular mechanism for gene knockdown found in fungi, plants, and animals, in which double-stranded RNA (dsRNA) triggers the specific cleavage of complementary mRNA molecules via endogenous cellular machinery.
- Short interfering RNAs (siRNAs)
- These triggers of RNAi are dsRNAs that typically contain 19–21 bp and 2-nt 3′-overhangs. siRNAs are naturally produced by Dicer-mediated cleavage of larger dsRNAs, but may also be introduced into cells
JONATHAN K. WATTS Jonathan K. Watts received his BSc in chemistry from Dalhousie University in Halifax, Canada. He has just finished his PhD in the group of Masad Damha at McGill University. He has been awarded a Natural Sciences and Engineering Research Council of Canada (NSERC) postgraduate fellowship, the McGill Tomlinson Fellowship and a postdoctoral fellowship from FQRNT Quebec. His primary research interest is the interface of chemistry and biology in the field of small RNAs.
References (138)
Asymmetry in the assembly of the RNAi enzyme complex
Cell
(2003)Functional siRNAs and miRNAs exhibit strand bias
Cell
(2003)Functional anatomy of a dsRNA trigger: differential requirement for the two trigger strands in RNA interference
Mol. Cell
(2000)Chemical modification of siRNAs to improve serum stability without loss of efficacy
Biochem. Biophys. Res. Commun.
(2006)RNA interference induced by siRNAs modified with 4′-thioribonucleosides in cultured mammalian cells
FEBS Lett.
(2005)- et al.
LNA: a versatile tool for therapeutics and genomics
Trends Biotechnol.
(2003) The RNA-induced silencing complex is a Mg2+-dependent endonuclease
Curr. Biol.
(2004)Biodistribution of phosphodiester and phosphorothioate siRNA
Bioorg. Med. Chem. Lett.
(2004)RNA interference by 2′,5′-linked nucleic acid duplexes in mammalian cells
Bioorg. Med. Chem. Lett.
(2006)ATP requirements and small interfering RNA structure in the RNA interference pathway
Cell
(2001)
RNAi in human cells: basic structural and functional features of small interfering RNA
Mol. Cell
Conjugate for efficient delivery of short interfering RNA (siRNA) into mammalian cells
FEBS Lett.
Steroid and lipid conjugates of siRNAs to enhance cellular uptake and gene silencing in liver cells
Bioorg. Med. Chem. Lett.
Single-stranded antisense siRNAs guide target RNA cleavage in RNAi
Cell
Evidence that siRNAs function as guides, not primers, in the Drosophila and human RNAi pathways
Mol. Cell
Synthetic dsRNA dicer substrates enhance RNAi potency and efficacy
Nat. Biotechnol.
Chemically modified siRNA prolonged RNA interference in renal disease
Biochem. Biophys. Res. Commun.
siRNA and isRNA: two edges of one sword
Mol. Ther.
Design of noninflammatory synthetic siRNA mediating potent gene silencing in vivo
Mol. Ther.
Gene expression analysis in blood cells in response to unmodified and 2′-modified siRNAs reveals TLR-dependent and independent effects
J. Mol. Biol.
2′-O-Methyl-modified RNAs act as TLR7 antagonists
Mol. Ther.
Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA
Immunity
Harnessing in vivo siRNA delivery for drug discovery and therapeutic development
Drug Discov. Today
Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans
Nature
Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells
Nature
Illuminating the silence: understanding the structure and function of small RNAs
Nat. Rev. Mol. Cell Biol.
Perspective: machines for RNAi
Genes Dev.
RNA interference as a gene-specific approach for molecular medicine
Curr. Med. Chem.
Effects on RNA interference in gene expression (RNAi) in cultured mammalian cells of mismatches and the introduction of chemical modifications at the 3′-ends of siRNAs
Antisense Nucl. Acid Drug Dev.
Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs
Nature
Small interfering RNAs containing full 2′-O-methylribonucleotide-modified sense strands display Argonaute2/eIF2C2-dependent activity
RNA
Functional dissection of siRNA sequence by systematic DNA substitution: modified siRNA with a DNA seed arm is a powerful tool for mammalian gene silencing with significantly reduced off-target effect
Nucl. Acids Res.
siRNA function in RNAi: a chemical modification analysis
RNA
RNA interference in mammalian cells by chemically-modified RNA
Biochemistry
Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate
EMBO J
Structural variations and stabilising modifications of synthetic siRNAs in mammalian cells
Nucl. Acids Res.
Improved targeting of miRNA with antisense oligonucleotides
Nucl. Acids Res.
siRNA relieves chronic neuropathic pain
Nucl. Acids Res.
Positional effect of chemical modifications on short interference RNA activity in mammalian cells
J. Med. Chem.
Tolerance for mutations and chemical modifications in a siRNA
Nucl. Acids Res.
Poly-2′-DNP-RNAs with enhanced efficacy for inhibiting cancer cell growth
Oligonucleotides
Sequence, chemical, and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing
Antisense Nucl. Acid Drug Dev.
Fully 2′-deoxy-2′-fluoro substituted nucleic acids induce RNA interference in mammalian cell culture
Chem. Biol. Drug Des.
In vivo activity of nuclease-resistant siRNAs
RNA
Hybrids of RNA and arabinonucleic acids (ANA and 2′F-ANA) are substrates of Ribonuclease H
J. Am. Chem. Soc.
2′-Deoxy-2′-fluoro-β-d-arabinonucleosides and oligonucleotides (2′F-ANA): synthesis and physicochemical studies
Nucl. Acids Res.
Improvements in siRNA properties mediated by 2′-deoxy-2′-fluoro-β-d-arabinonucleic acid (FANA)
Nucl. Acids Res.
2′-Fluoro-4′-thioarabino-modified oligonucleotides: conformational switches linked to siRNA activity
Nucl. Acids Res.
Improving RNA interference in mammalian cells by 4′-thio-modified small interfering RNA (siRNA): effect on siRNA activity and nuclease stability when used in combination with 2′-O-alkyl modifications
J. Med. Chem.
Study of modification pattern-RNAi activity relationships by using siRNAs modified with 4′-thioribonucleosides
ChemBioChem
Cited by (379)
Properties and synergistic effects of a nonionic backbone and aminoalkyl modified nucleosides in RNAs
2024, Bioorganic ChemistryTargeting RNA with synthetic oligonucleotides: Clinical success invites new challenges
2024, Cell Chemical BiologyAsymmetric trichotomous partitioning overcomes dataset limitations in building machine learning models for predicting siRNA efficacy
2023, Molecular Therapy Nucleic AcidsRemodeling the tumor immune microenvironment via siRNA therapy for precision cancer treatment
2023, Asian Journal of Pharmaceutical SciencessiRNA delivery mediated by pH and redox responsive p(DEAEMA-co-HEMA-g-PEGMA) nanogels
2023, Journal of Drug Delivery Science and Technology
JONATHAN K. WATTS Jonathan K. Watts received his BSc in chemistry from Dalhousie University in Halifax, Canada. He has just finished his PhD in the group of Masad Damha at McGill University. He has been awarded a Natural Sciences and Engineering Research Council of Canada (NSERC) postgraduate fellowship, the McGill Tomlinson Fellowship and a postdoctoral fellowship from FQRNT Quebec. His primary research interest is the interface of chemistry and biology in the field of small RNAs.
GLEN F. DELEAVEY Glen F. Deleavey received his BSc in biology–chemistry from the University of New Brunswick in Fredericton, Canada. He is currently a PhD candidate in the group of Masad Damha at McGill University. He has been awarded a postgraduate fellowship from NSERC and a 2007 SCI Merit Award (Canadian section). His research interests lie in the field of chemical biology, with a focus on chemically modified siRNAs.
MASAD J. DAMHA Masad J. Damha received his BSc and PhD degrees from McGill University, the latter in the group of Prof. Kelvin Ogilvie, on synthesis and conformational analysis of RNA and its analogues. After beginning his academic career at the University of Toronto, he returned to McGill in 1992, where he is currently James McGill Professor of Chemistry. His research interests include synthesis of RNA (including novel RNA structures) and the application of oligonucleotide derivatives as therapeutics. He was awarded the 2007 Bernard Belleau Award from the Canadian Society for Chemistry, honoring significant contributions to the field of medicinal chemistry, for the development of 2′F-ANA.