GNA) on Sirna Structure and Function

Impact of Glycol Nucleic Acid (GNA) on siRNA Structure and Function

Authors: Mark K. Schlegel,1 Donald J. Foster,1 Alexander V. Kel’in,1 Ivan Zlatev,1 Anna Bisbe,1 Muthusamy Jayaraman,1 Jeremy G. Lackey,1 Kallanthottathil G. Rajeev,1 Klaus Charissé,1 Joel Harp,2 Pradeep S. Pallan,2 Martin A. Maier,1 Martin Egli,2 Muthiah Manoharan1 Affiliations: 1Alnylam Pharmaceuticals, Cambridge MA, 2Department of Biochemistry, Vanderbilt University, Nashville, TN

Abstract RNA interference, the biological process through which double-stranded small interfering RNA (siRNA) mediates sequence-specific gene silencing, has great potential for therapeutic applications. Chemical modifications of siRNA duplexes are necessary to stabilize these molecules against nuclease degradation, to facilitate their uptake into cells, and to affect formation of active RISC as well as RNAi-mediated target silencing. In fact, thermally destabilizing modifications incorporated at certain positions of the siRNA duplex can lead to an increase in potency by improving strand bias and/or sense strand dissociation during RISC loading.1-5We have investigated the simple three-carbon, acyclic nucleic acid analog, Glycol Nucleic Acid (GNA) within the context of siRNA duplexes. (S)-GNA oligomers form homo-duplexes with structural similarities to a typical RNA A-form duplex and crosspair with RNA, but not DNA, within A/T-rich sequences. The thermal stabilities and nuclease resistance of siRNA duplexes containing (S)- or (R)-GNA were investigated. Structural studies using x-ray crystallography provided further insight into the impact of GNA substitution on RNA duplex structure. In biological studies, we examined the gene silencing activity of GNA-containing siRNA conjugate duplexes.6

Background RNA duplexes containing (S)- and (R)-GNA Impact of single (S)-GNA base pair incorporation • (S)-GNA crosspairs with RNA, but not DNA, within an nucleotides on in vitro siRNA activity A:T rich context • Crosspairing behavior with RNA poorly understood • (S)-GNA duplexes adopt a similar conformation to RNA (A-form) duplexes • Most atom economical nucleic acid analog known • Stereochemically pure monomers synthetically accessible from simple starting materials • Exhibits stable self-pairing • Structures of (S)-GNA homoduplexes well characterized

Figure 7. Positional impact of single (S)-GNA base pair substitution on in vitro silencing activity at a concentration of 10 nM siRNA. The base pair at the indicated position of the guide or passenger strand was substituted with the Figure 4. Crystal structures of RNA duplexes modified with both GNA-T corresponding GNA base pair. stereoisomers. (A) Variations in intrastrand P…P distances as a consequence of the incorporated GNA-T residues (carbon atoms highlighted in green) in an 8-mer RNA duplex. (B) Example of an (S)-GNA-T:RNA-A base pair showing a Gene silencing in mice using (S)-GNA modified rotated nucleobase conformation for the GNA nucleotide (arrow). (C) GNA nucleotides adopt both gauche and anti conformations within the structures. siRNA conjugate duplexes (D) (R)-GNA-T residues distort RNA duplex and pairing geometry to a greater extent than (S)-GNA-T residues. Superimposition of A:U and G:A base pairs flanking (S)-GNA-T(green):RNA-A and (R)-GNA-T(yellow):RNA-A in two 12-mer duplexes reveals a disruption of the neighboring A:U pair in the (R)-GNA-T-modified 12-mer (arrow). (E) Global structures of the RNA duplexes incorporating both (S)- and (R)-isomers of GNA which highlight the phosphate backbones. The two isomers are accommodated differently within the global RNA structure and result in a slight kink in the (R)-isomer-containing duplex (arrow). (F) An (S)-GNA-T residue can seamlessly and with optimal geometry replace an RNA nucleotide at position 7 of the guide strand RNA bound to human Ago 2.14 The RNA strand assumes a kink at that site that is associated with Ile-365 and results in unstacking of the bases of nucleotides 6 and 7.

Crosspairing of (S)-GNA with isoC and isoG RNA nucleotides improves thermal stability

7-10 Figure 1. Background. Bottom Right – backbone-base inclination ( B) and helical twist values for A-form and B-form RNA/DNA, as well as for (S)-GNA. 7 Values for (R)-GNA are extrapolated from the (S)-GNA values by using Figure 8. Knockdown of TTR in mice with (S)-GNA modified siRNA duplexes simple inversion. dosed at 2.5 mg/kg. Error bars represent the SD from each cohort (n=3). Only those comparisons which are statistically significant are shown in the graph; all others are nonsignificant with the exception of all comparisons to PBS which were Thermal modulation of siRNA duplexes using GNA all significant. (A) TTR mRNA levels measured in the liver. (B) TTR protein levels measured in the serum. G = guide strand, P = Passenger strand.

Summary • GNA incorporation resulted in a position-dependent thermal destabilization of the resulting duplex. The extent of destabilization was mostly nucleotide dependent; whereas substitution for an A or U nucleotide resulted in a

significantly smaller TM compared to GNA substitution for G or C nucleotides.

Figure 5. Structures of isocytidine and isoguanosine nucleotides, their potential to form fully complementary base pairs to “rotated” GNA-C or GNA-G, and • Crystal structures of RNA duplexes containing either supporting thermal melting data. (S)- or (R)-GNA exhibit the flexibility of the glycol backbone within the duplex structure, allowing the Impact of single (S)-GNA nucleotide incorporation nucleobases of GNA-T residues to adopt a non- Figure 2. Thermal modulation5 of siRNA conjugate duplexes11 using GNA. 12-13 13 on in vitro siRNA activity canonical base pair with a rotated conformation. The Structures of hAgo2 adapted from PDB file 4W5O and generated using PyMOL. latter result is further supported by crosspairing Potential Impact experiments with isoC and isoG nucleotides. • May improve strand bias (preference for 5’-end Furthermore, (R)-isomer incorporation, preferring of antisense strand to bind to MID domain) a left-handed duplex, resulted in a stronger thermal destabilization6 and a larger perturbation of the overall • May facilitate sense strand dissociation duplex structure.

Thermal Melting (T ) analysis of (S)-GNA-containing m • The incorporation of a single (S)-GNA nucleotide or siRNA duplexes base pair into the seed or supplemental regions of siRNA duplexes resulted in similar levels of TTR mRNA knockdown in vitro.

• Levels of gene silencing were maintained in vivo with an siRNA modified using a single (S)-GNA nucleotide in the Figure 6. Positional impact of single (S)-GNA nucleotide substitution on in vitro passenger or guide strand. Modification using a single silencing activity at a concentration of 10 nM siRNA. The nucleotide at the indicated position of the guide or passenger strand was substituted with the corre- base pair of (S)-GNA trended towards a lower potency sponding GNA nucleotide. and duration of effect.

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