(PDF) Differential Effects on Allele Selective Silencing of Mutant

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Differential Effects on Allele Selective Silencing of Mutant Huntingtin by Two Stereoisomers of α,β-Constrained Nucleic Acid Michael E. Østergaard,† Béatrice Gerland,‡ Jean-Marc Escudier,‡ Eric E. Swayze,† and Punit P. Seth*,† †

Isis Pharmaceuticals Inc. 2855 Gazelle Court, Carlsbad, California 92011, United States Laboratoire de Synthèse et Physico-Chimie de Molécules d’Intérêt Biologique, UMR CNRS 5068, Université Paul Sabatier, 118 Route de Narbonne, Toulouse F-31062, France

‡

S Supporting Information *

ABSTRACT: We describe the effects of introducing two epimers of neutral backbone α,β-constrained nucleic acid (CNA) on the activity and allele selectivity profile of RNase H active antisense oligonucleotides (ASOs) targeting a single nucleotide polymorphism (SNP) for the treatment of Huntington’s disease (HD). ASOs modified with both isomers of α,β-CNA in the gap region showed good activity versus the mutant allele, but one isomer showed improved selectivity versus the wild-type allele. Analysis of the human RNase H cleavage patterns of α,β-CNA modified ASOs versus matched and mismatched RNA revealed that both isomers support RNase H cleavage on the RNA strand across from the site of incorporation in the ASOan unusual observation for a neutral linkage oligonucleotide modification. Interestingly, ASOs modified with (R)and (S)-5′-hydroxyethyl DNA (RHE and SHE respectively) formed by partial hydrolysis of the dioxaphosphorinane ring system in α,β-CNA also showed good activity versus the mutant allele but an improved selectivity profile was observed for the RHE modified ASO. Our observations further support the profiling of neutral and 5′-modified nucleic acid analogs as tools for gene silencing applications. in patient cells and in the CNS14 of a fully humanized mouse model of HD. 15 ASO A D (Figure 1a) has a 9-base phosphorothioate modified DNA gap region flanked on either ends with 2′,4′-constrained 2′-O-ethyl bridged nucleic acid (cEt)16 and 2′-O-methoxyethyl RNA (MOE)17 nucleotides (Figure 1c). AD is fully matched to the muHTT mRNA but forms a GT wobble base-pair18 with the wt allele (Figure 1b). ASO AD shows ∼5-fold selectivity for the muHTT allele, which could be enhanced to >100-fold by introducing chemical modifications in the gap region.14 To further expand on these observations, we have investigated the effect of introducing two epimeric analogs of α,β-constrained nucleic acid (CNA) X (RC5′,SP) and Y (SC5′,RP) (Figure 1d), in the gap region of AD and examined their ability to modulate ASO potency and allele selectivity. CNA are a family of conformationally restricted nucleic acid analogs with covalent tethers between rotatable bonds of the sugar−phosphate backbone.19 Conformational restraint is exerted by tethering the nonbridging oxygen in the phosphodiester backbone back to the 5′C-position of the nucleoside monomer resulting in the formation of a dioxaphosphorinane ring system.20 α,β-CNA X improves affinity for DNA and RNA complements by constraining torsion angles α and β in the oligonucleotide backbone in the

Huntington’s disease (HD) is an autosomal dominant disease for which there is presently no cure.1 Disease onset typically occurs in the third decade of life and is characterized by extensive neurodegeneration, motor and cognitive disorders, and, eventually, death.2 HD is caused by an expansion of a CAG tract in the huntingtin gene (HTT) which results in the formation of a toxic huntingtin protein (HTT) with an expanded polyglutamine tract.3 However, wild-type HTT is essential during embryogenesis but its physiological roles in the adult central nervous system (CNS) are poorly understood.4 As a result, strategies that partially suppress wild type (wt) and muHTT or selectively suppress muHTT in an allele selective manner are being investigated as potential HD therapeutics.5 Recent reports have described the use of RNase H and RISC active antisense oligonucleotides (ASOs) for developing panallele6−8 as well as allele selective HD therapeutics.9−11 RNase H active ASOs typically have a central gap region of 7−14 deoxynucleotides flanked on either end with 2−5 2′-modified or conformationally restricted nucleotides.12 ASOs that target homologous sequence tracts in the HTT mRNA partially suppress production of wt and mutant HTT and provide a therapeutic benefit in rodent disease models.8 In contrast, ASOs that target the CAG expansion directly,11,13 or target SNPs associated with the CAG expansion,10 can selectively suppress the mutant protein without affecting expression of the wt variant. We recently showed that ASOs targeting SNP rs7685686_A in intron 42 of the HTT gene can selectively suppress muHTT © XXXX American Chemical Society

Received: April 23, 2014 Accepted: July 22, 2014

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Figure 1. (A) Sequence, modification pattern, and Tm of ASO AD versus RNAmu and RNAwt. (B) Structure of AT base-pair and GT wobble basepair. (C) Chemical modifications used in present study. (D) Sequence, position, and structures of DNA analogs investigated for modulating ASO activity and allele selectivity profile.

Table 1. Sequence, Chemical Modification Pattern, Tm versus Matched (RNAmu) and Mismatched RNA (RNAwt), Activity Against muHTT and Fold Selectivity versus wtHTT in Patient Fibroblastsa

a

Orange letters represent MOE, blue represent cEt, and black represent DNA nucleotides. Red letters denote position of modification in the gap region. Base code: T = thymine, C = 5-Me-cytosine, A = adenine, and G = guanine. All ASOs are fully phosphorothioate modified, except AX and AY which have a dioxaphosphorinane ring system and ARHE and ASHE which have a phosphodiester linkage between the TT nucleotides indicated in red. Tm values were measured in 10 mM sodium phosphate buffer (pH 7.2) containing 100 mM NaCl and 0.1 mM EDTA. ΔTm (wt-mu) denotes GT discrimination. ASO IC50 and selectivity were measured in human GM4022 fibroblasts using allele selective qRT-PCR to measure reduction of wt and muHTT mRNA.

We also examined the effect of introducing R5′- and S5′hydroxyethyl DNA (RHE and SHE, respectively), at position 6 of AD (ARHE and ASHE, Figure 1d). These analogs were formed by partial hydrolysis of the dioxaphosphorinane ring system in AX and AY, respectively (Supporting Information Figure S1), during the ammonia treatment used to deprotect and detach the ASOs from solid support. In addition, we included previously evaluated ASOs ARMe and ASMe modified with R5′Me-DNA (RMe) and S5′-Me DNA (SMe), respectively,23 at position 6 of ASO AD respectively, as additional controls.14 Consistent with previously reported Tm data,21,24 X had a stabilizing effect on ASO/RNA duplex thermal stability, while Y was destabilizing (ΔTm +2.7 and +1.3 vs −2.3 °C, respectively, Table 1). In comparison, SMe (ΔTm +1.8 and +1.3 °C) and SHE (ΔTm +1.0 and +0.3 °C) had a slightly stabilizing effect

canonical ranges (gauche(−), trans) observed in B-form DNA duplexes.21 In contrast, analog Y constrains torsion angles α and β in noncanonical ranges (gauche(+), trans) and has a destabilizing effect on duplex thermal stability, while increasing hairpin stability when incorporated in the unpaired moiety.22 Furthermore, to the best of our knowledge, the use of a neutral and backbone restricted nucleic acid analog in the gap region of RNase H ASOs has never been reported previously.

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RESULTS We first examined the effect of replacing the dTdT dinucleotide (Figure 1d) at position 5,6 in control ASO AD with modifications X (AX) and Y (AY) on duplex thermal stability versus complementary and mismatched RNA representing the mutant (RNAmu) and wt (RNAwt) HTT mRNA, respectively. B

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Figure 2. Summary of heteroduplex cleavage patterns using recombinant human RNase H1 in a biochemical assay; ASO activity and selectivity profile in GM4022 human fibroblasts. The arrows denote the sites and intensity of cleavage by recombinant human RNase H1 on the matched and mismatched duplexes representing the mutant and the wild-type HTT mRNA respectively. The presence of the GT wobble pair ablates or reduces cleavage sites a and b for mismatched duplexes of ASOs AD, AX, and AY with RNAwt.

on Tm while RMe (ΔTm −1.7 and −2.5) and RHE (ΔTm −1.2 and −1.8 °C) were destabilizing. All modifications except Y, which behaves similarly to D, showed a modest improvement in discrimination of the GT wobble base-pair with X being the best. The somewhat counterintuitive duplex stabilizing properties of X (RC5′ stabilizing versus RMe and RHE destabilizing) and Y (SC5′ destabilizing versus SMe and SHE stabilizing) can be rationalized by examining the previously described conformational models for X and Y.21 The RC5′ configuration in X is essential for locking torsion angles α and β into the canonical ranges of a B-type duplex for Watson−Crick basepairing. In contrast, the SC5′ configuration in Y causes α to deviate by +120° from the canonical ranges observed in B-type duplexes for effective Watson−Crick base-pairing. However, for the unconstrained analogs RMe/RHE and SMe/SHE, the backbone torsion angles can adapt to position the substituent group in an orientation devoid of steric interactions as previously observed for C5′-substituted analogs of LNA25 and other conformationally restricted nucleic acids.26−28 Most ASOs showed similar potency for reducing muHTT mRNA but had differential effects upon allele selectivity (Table 1, Supporting Information Figure S2). AX, which had slightly higher Tm showed improved activity relative to control ASO AD. Somewhat surprisingly, AY which had a lower Tm (−2.3 and −5 °C relative to AD and AX, respectively) was as active as AD for reducing muHTT mRNA in cell culture, suggesting that introducing slightly destabilizing modifications in the gapregion of AD do not affect ASO activity. ASOs ARMe and ARHE, with the R5′-configured alkyl groups, showed good activity versus the mutant allele. In comparison, ASOs ASMe and ASHE with S5′-configured alkyl groups were slightly less active. The effects of substitution in the gap on ASO allele selectivity were varied (Table 1, Supporting Information Figure S2). ASO AX with the stabilizing modification showed slightly improved (7.2- vs 4.2-fold) selectivity relative to control ASO AD. In contrast, AY showed an improved selectivity profile (27fold). For the 5′-hydroxyethyl series, both isomers showed good allele selectivity but ASO ARHE showed a better overall profile by virtue of improved potency (IC50 0.33 μM, 44-fold selectivity). In the 5′-methyl series, the S-isomer was more selective despite a slight loss in potency. To understand the selectivity observations with the two α,βCNA isomers, we examined the processing of the ASO/RNA heteroduplexes with recombinant RNase H1 in a biochemical

assay.29 We had previously shown that the major RNase H cleavage site on the AD/RNAmu heteroduplex is adjacent to the central AT base-pair representing the SNP site.14 For the mismatched duplex AD/RNAwt, presence of the GT wobble base-pair ablates the major RNase H cleavage site and cleavage is instead shifted to minor sites toward the 5′-end of the ASO gap region (3′-end of the RNA, Figure 2).14 For the AX/RNAmu heteroduplex, we observed a similar cleavage profile with the major cleavage site a adjacent to the SNP base-pair (Figure 2 and Supporting Information Figure S3). Interestingly, we detected minor cleavage sites b and c on the RNA across and in the vicinity of the (RC5′,SP) configured α,β-CNA dinucleotide suggesting that this modification can support RNase H activity. For the AX/RNAwt heteroduplex, the GT base-pair ablates the major cleavage site a and reduces cleavage at site b. However, cleavage increases at site c resulting in a small overall increase in allele selectivity relative to the control ASO AD. For the AY/RNAmu heteroduplex, we observed the major cleavage site a adjacent to the SNP site and another strong cleavage site b across from the (SC5′,RP) configured α,β-CNA dinucleotide. For the AY/RNAwt heteroduplex, the GT base-pair ablates the major cleavage site a, while the combination of the modified nucleotide and the mismatch ablates site b resulting in enhanced allele selectivity.

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DISCUSSION We show that α,β-CNA is a useful modification for allele selective suppression of gene expression when incorporated into the gap region of RNase H ASOs. Both isomers of α,βCNA supported RNase H activity across and in the vicinity of the site of incorporation. However, the (SC5′,RP) configured isomer Y did not support RNase H cleavage of a mismatched duplex. This suggests that the additive local structural perturbations produced by the modification and an adjacent mismatch, ablates RNase H activity. Our observations suggest that this class of modifications could be generally useful for minimizing cleavage of mismatched duplexes by RNase H ASOsan observation with broader implications for improving the selectivity profile of ASOs for therapeutic applications. Our activity and selectivity observations with the 5′-alkyl substituted modifications suggest that S5′-alkyl substituents in the gap region interfere with RNase H activity. Assuming canonical orientation around torsion angle γ, the S5′-alkyl groups are expected to lie in the minor groove, which is an C

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important recognition element for RNase H.30 Interestingly, the improved selectivity observed with the R5′-hydroxyethyl DNA modified ASO ARHE but not with R5′-Me DNA ASO ARMe, suggests that larger R5′-groups can also be useful for improving ASO selectivity without compromising potency. It is conceivable that larger R5′-alkyl groups do not interfere with RNase H1 binding in the minor groove but instead may interfere with secondary interactions with the enzyme. Our observations also suggest that the utility of a nucleic acid modification should not be judged exclusively upon the magnitude of increase in duplex stability observed in Tm experiments. Biological systems are sufficiently complex to discriminate between subtle structural variations and extensive structural activity relationships in systems of interest alone can determine the true utility of a nucleic acid modification for antisense applications.

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(5) Zuccato, C., Valenza, M., and Cattaneo, E. (2010) Molecular mechanisms and potential therapeutical targets in Huntington’s disease. Physiol. Rev. 90, 905−981. (6) Wang, Y.-L., Liu, W., Wada, E., Murata, M., Wada, K., and Kanazawa, I. (2005) Clinico-pathological rescue of a model mouse of Huntington’s disease by siRNA. Neurosci. Res. 53, 241−249. (7) McBride, J. L., Pitzer, M. R., Boudreau, R. L., Dufour, B., Hobbs, T., Ojeda, S. R., and Davidson, B. L. (2011) Preclinical safety of RNAinediated HTT suppression in the rhesus macaque as a potential therapy for Huntington’s disease. Mol. Ther. 19, 2152−2162. (8) Kordasiewicz, H. B., Stanek, L. M., Wancewicz, E. V., Mazur, C., McAlonis, M. M., Pytel, K. A., Artates, J. W., Weiss, A., Cheng, S. H., Shihabuddin, L. S., Hung, G., Bennett, C. F., and Cleveland, D. W. (2012) Sustained therapeutic reversal of Huntington’s disease by transient repression of Huntingtin synthesis. Neuron 74, 1031−1044. (9) Pfister, E. L., Kennington, L., Straubhaar, J., Wagh, S., Liu, W., DiFiglia, M., Landwehrmeyer, B., Vonsattel, J.-P., Zamore, P. D., and Aronin, N. (2009) Five siRNAs targeting three SNPs may provide therapy for three-quarters of Huntington’s disease patients. Curr. Biol. 19, 774−778. (10) Carroll, J. B., Warby, S. C., Southwell, A. L., Doty, C. N., Greenlee, S., Skotte, N., Hung, G., Bennett, C. F., Freier, S. M., and Hayden, M. R. (2011) Potent and selective antisense oligonucleotides targeting single-nucleotide polymorphisms in the Huntington disease gene/allele-specific silencing of mutant huntingtin. Mol. Ther. 19, 2178−2185. (11) Yu, D., Pendergraff, H., Liu, J., Kordasiewicz, H. B., Cleveland, D. W., Swayze, E. E., Lima, W. F., Crooke, S. T., Prakash, T. P., and Corey, D. R. (2012) Single-stranded RNAs use RNAi to potently and allele-selectively inhibit mutant Huntingtin expression. Cell 150, 895− 908. (12) Seth, P. P., Siwkowski, A., Allerson, C. R., Vasquez, G., Lee, S., Prakash, T. P., Wancewicz, E. V., Witchell, D., and Swayze, E. E. (2009) Short antisense oligonucleotides with novel 2′-4′ conformationaly restricted nucleoside analogues show improved potency without increased toxicity in animals. J. Med. Chem. 52, 10−13. (13) Gagnon, K. T., Pendergraff, H. M., Deleavey, G. F., Swayze, E. E., Potier, P., Randolph, J., Roesch, E. B., Chattopadhyaya, J., Damha, M. J., Bennett, C. F., Montailler, C., Lemaitre, M. M., and Corey, D. R. (2010) Allele-selective inhibition of mutant Huntingtin expression with antisense oligonucleotides targeting the expanded CAG repeat. Biochemistry 49, 10166−10178. (14) Ostergaard, M. E., Southwell, A. L., Kordasiewicz, H., Watt, A. T., Skotte, N. H., Doty, C. N., Vaid, K., Villanueva, E. B., Swayze, E. E., Bennett, C. F., Hayden, M. R., and Seth, P. P. (2013) Rational design of antisense oligonucleotides targeting single nucleotide polymorphisms for potent and allele selective suppression of mutant Huntingtin in the CNS. Nucleic Acids Res. 41, 9634−9650. (15) Southwell, A. L., Warby, S. C., Carroll, J. B., Doty, C. N., Skotte, N. H., Zhang, W., Villanueva, E. B., Kovalik, V., Xie, Y., Pouladi, M. A., Collins, J. A., Yang, X. W., Franciosi, S., and Hayden, M. R. (2013) A fully humanized transgenic mouse model of Huntington disease. Hum. Mol. Genet. 22, 18−34. (16) Seth, P. P., Vasquez, G., Allerson, C. A., Berdeja, A., Gaus, H., Kinberger, G. A., Prakash, T. P., Migawa, M. T., Bhat, B., and Swayze, E. E. (2010) Synthesis and biophysical evaluation of 2′,4′-constrained 2′O-methoxyethyl and 2′,4′-constrained 2′O-ethyl nucleic acid analogues. J. Org. Chem. 75, 1569−1581. (17) Teplova, M., Minasov, G., Tereshko, V., Inamati, G. B., Cook, P. D., Manoharan, M., and Egli, M. (1999) Crystal structure and improved antisense properties of 2′-O-(2-methoxyethyl)-RNA. Nat. Struct. Biol. 6, 535−539. (18) Varani, G., and McClain, W. H. (2000) The G × U wobble base pair. A fundamental building block of RNA structure crucial to RNA function in diverse biological systems. EMBO Rep. 1, 18−23. (19) Catana, D.-A., Renard, B.-L., Maturano, M., Payrastre, C., Tarrat, N., and Escudier, J.-M. (2012) Dioxaphosphorinane-constrained nucleic acid dinucleotides as tools for structural tuning of nucleic acids. J. Nucleic Acids 2012, 17.

ASSOCIATED CONTENT

* Supporting Information S

Experimental protocols for oligonucleotide synthesis, thermal denaturation, and cell culture experiments; dose−response curves for allele-selective PCR; analytical data for oligonucleotides; and LCMS traces of RNaseH1 cleavage assay. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: 760-603-2587. Email: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS J-M. Escudier and B. Gerland are grateful to the Agence Nationale de la Recherche for a grant to B. Gerland and for financial support to the CNA project (ANR-11-BS07-012-01). The authors thank J. Nichols, W. Lima, J. Wu, and H. Gaus for assistance with the human RNase H1 assay.

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REFERENCES

(1) Walker, F. O. (2007) Huntington’s disease. Lancet 369, 218−228. (2) de la Monte, S. M., Vonsattel, J. P., and Richardson, E. P., Jr. (1988) Morphometric demonstration of atrophic changes in the cerebral cortex, white matter, and neostriatum in Huntington’s disease. J. Neuropathol. Exp. Neurol. 47, 516−525. (3) MacDonald, M. E., Ambrose, C. M., Duyao, M. P., Myers, R. H., Lin, C., Srinidhi, L., Barnes, G., Taylor, S. A., James, M., Groot, N., MacFarlane, H., Jenkins, B., Anderson, M. A., Wexler, N. S., Gusella, J. F., Bates, G. P., Baxendale, S., Hummerich, H., Kirby, S., North, M., Youngman, S., Mott, R., Zehetner, G., Sedlacek, Z., Poustka, A., Frischauf, A.-M., Lehrach, H., Buckler, A. J., Church, D., DoucetteStamm, L., O’Donovan, M. C., Riba-Ramirez, L., Shah, M., Stanton, V. P., Strobel, S. A., Draths, K. M., Wales, J. L., Dervan, P., Housman, D. E., Altherr, M., Shiang, R., Thompson, L., Fielder, T., Wasmuth, J. J., Tagle, D., Valdes, J., Elmer, L., Allard, M., Castilla, L., Swaroop, M., Blanchard, K., Collins, F. S., Snell, R., Holloway, T., Gillespie, K., Datson, N., Shaw, D., and Harper, P. S. (1993) A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington’s disease chromosomes. Cell 72, 971−983. (4) Nasir, J., Floresco, S. B., O’Kusky, J. R., Diewert, V. M., Richman, J. M., Zeisler, J., Borowski, A., Marth, J. D., Phillips, A. G., and Hayden, M. R. (1995) Targeted disruption of the Huntington’s disease gene results in embryonic lethality and behavioral and morphological changes in heterozygotes. Cell 81, 811−823. D

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(20) Le Clezio, I., Escudier, J.-M., and Vigroux, A. (2003) Diastereoselective synthesis of a conformationally restricted dinucleotide with predefined α and β torsional angles for the construction of α,β-constrained nucleic acids (α,β-CNA). Org. Lett. 5, 161−164. (21) Dupouy, C., Iche-Tarrat, N., Durrieu, M. P., Rodriguez, F., Escudier, J. M., and Vigroux, A. (2006) Watson−Crick base-pairing properties of nucleic acid analogues with stereocontrolled α and β torsion angles (α,β-D-CNAs). Angew. Chem., Int. Ed. Engl. 45, 3623− 3627. (22) Dupouy, C., Millard, P., Boissonnet, A., and Escudier, J.-M. (2010) α,β-D-CNA preorganization of unpaired loop moiety stabilizes DNA hairpin. Chem. Commun. (Cambridge, U.K.) 46, 5142−5144. (23) Saha, A. K., Caulfield, T. J., Hobbs, C., Upson, D. A., Waychunas, C., and Yawman, A. M. (1995) 5′-Me-DNA. A new oligonucleotide analog: Synthesis and biochemical properties. J. Org. Chem. 60, 788−789. (24) Boissonnet, A., Dupouy, C., Millard, P., Durrieu, M.-P., Tarrat, N., and Escudier, J.-M. (2011) α,β-D-CNA featuring canonical and noncanonical α/β torsional angles behaviors within oligonucleotides. New J. Chem. 35, 1528−1533. (25) Seth, P. P., Allerson, C. R., Siwkowski, A., Vasquez, G., Berdeja, A., Migawa, M. T., Gaus, H., Prakash, T. P., Bhat, B., and Swayze, E. E. (2010) Configuration of the 5′-methyl group modulates the biophysical and biological properties of locked nucleic acid (LNA) oligonucleotides. J. Med. Chem. 53, 8309−8318. (26) Pallan, P. S., Yu, J., Allerson, C. R., Swayze, E. E., Seth, P., and Egli, M. (2012) Insights from crystal structures into the opposite effects on RNA affinity caused by the S- and R-6′-methyl backbone modifications of 3′-fluoro hexitol nucleic acid. Biochemistry 51, 7−9. (27) Seth, P. P., Allerson, C. R., Ostergaard, M. E., and Swayze, E. E. (2012) Structural requirements for hybridization at the 5′-position are different in α-L-LNA as compared to β-D-LNA. Bioorg. Med. Chem. Lett. 22, 296−299. (28) Hanessian, S., Schroeder, B. R., Giacometti, R. D., Merner, B. L., Ostergaard, M., Swayze, E. E., and Seth, P. P. (2012) Structure-based design of a highly constrained nucleic acid analogue: Improved duplex stabilization by restricting sugar pucker and torsion angle gamma. Angew. Chem., Int. Ed. Engl. 51, 11242−11245. (29) Lima, W. F., Nichols, J. G., Wu, H., Prakash, T. P., Migawa, M. T., Wyrzykiewicz, T. K., Bhat, B., and Crooke, S. T. (2004) Structural requirements at the catalytic site of the heteroduplex substrate for human RNase H1 catalysis. J. Biol. Chem. 279, 36317−36326. (30) Nowotny, M., Gaidamakov, S. A., Ghirlando, R., Cerritelli, S. M., Crouch, R. J., and Yang, W. (2007) Structure of human RNase H1 complexed with an RNA/DNA hybrid: Insight into HIV reverse transcription. Mol. Cell 28, 264−276.

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