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Conformational properties of RNA backbones containing α-or β-epimers of 4 -C-modified uridines. Close-up views of the modified residue and 5 -and 3 -adjacent nucleotides: (A) uo, (B) Ufme, (C) uob and (D) Ufob. Distances are averages based on multiple strands per crystallographic asymmetric unit (A, B: 2; C, D: 8), and backbone torsion angle ranges for modified residues are shown on the right.
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Chemical modification is a prerequisite of oligonucleotide therapeutics for improved metabolic stability, uptake and activity, irrespective of their mode of action, i.e. antisense, RNAi or aptamer. Phosphate moiety and ribose C2'/O2' atoms are the most common sites for modification. Compared to 2'-O-substituents, ribose 4'-C-substituents lie in pro...
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... distances mentioned here represent average values based on two strands (Ufme, uo), four strands (Ufo), or eight strands (Ufob, uob) unless stated otherwise. Views of 5 -AXU-3 trimers for the uo, Ufme, Ufob and uob modifications are depicted in Figure 3. ...
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... the puckers of residues that bracket 4 -C-modified uridines and those of modified residues, all fall within the C3 -endo range. As well, backbone torsion angles lie in ranges associated with A-form RNA, i.e. sc-, ap, sc+, sc+, ap, sc-(from α to ; Figure 3A, B--see Figure 4 in (16) for a depiction of the Ufo-modified backbone). ...
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... 4 -C-OMe substituent in uo and Ufo as well as the 4 -C-Me group in Ufme assume a pseudo-axial orientation and are directed into the minor groove ( Figures 3A, B and 4A). The distances between methyl carbon and the 5 -and 3 -adjacent phosphorus atoms range from 4.4 ˚ A (Ufme) to 5.8 ˚ A (uo). ...
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... distances between methyl carbon and the 5 -and 3 -adjacent phosphorus atoms range from 4.4 ˚ A (Ufme) to 5.8 ˚ A (uo). But despite the relatively close spacing between the two moieties, e.g. 4 -C-Me and 3 -PO 2 in the case of Ufme (Figure 3B), the orientation of the 4 -substituents in the α-epimers results only in limited protection against attack by a nuclease. This contrasts with the relative orientations of (R)-and (S)-5 -C-Me substituents and phosphates that also result in a close approach but, in addition, methyl groups being inserted between the 5 -and 3 -adjacent phosphates (15). ...
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... the α-epimers, Ufob and Uob with inverted stereochemistry at C4 inside RNA locally give rise to drastic deviations from the canonical RNA geometry. The changes include torsion angles (α to δ), sugar pucker (C2 -exo for Ufob and uob), helical twist (nearly absent between Ufob/uob and the preceding A), very short P-P distances (as tight as 5.1 ˚ A) and a local kink in the backbone (Figures 3C, D, 4). The 4 -substituent is now directed into the major groove and the methyl group is wedged between 5 -and 3 -adjacent phosphates. ...
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... spaced phosphates create unfavorable electrostatics (Figure 4BC), and the lack of a twist at the site of modification leads to a tight contact between Ufob/uob (X) sugar and the adjacent adenine base (O4 [X]. . . N9[A] = 3.3 ˚ A; Figure 3CD). These observations can account for the steep loss in stability as a result of Ufob or uob incorporation into RNA (Table 1). ...
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... latter nucleoside thus exhibits a tendency to adopt the N pucker that matches that of Ufme. The crystal structures of octamers with incorporated Ufo (16), uo ( Figure 3A) and Ufme ( Figure 3B) residues demonstrate that all modified sugars adopt the C3 -endo pucker, consistent with the preference established at the nucleoside level. Therefore, both substituents assume a pseudoaxial orientation and in the case of the 4 -Cα-OMe and -Me groups, that is consistent with the anomeric effect. ...
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... latter nucleoside thus exhibits a tendency to adopt the N pucker that matches that of Ufme. The crystal structures of octamers with incorporated Ufo (16), uo ( Figure 3A) and Ufme ( Figure 3B) residues demonstrate that all modified sugars adopt the C3 -endo pucker, consistent with the preference established at the nucleoside level. Therefore, both substituents assume a pseudoaxial orientation and in the case of the 4 -Cα-OMe and -Me groups, that is consistent with the anomeric effect. ...
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... the β-epimers i.e., Ufob and uob, the NMR data are consistent with a much-diminished tendency for an N-type pucker at the nucleoside level. However, in the crystal structures of modified octamers all 4 -Cβ-modified uridines exhibit a C2 -exo N-type sugar conformation ( Figure 3C,D), a 36 • sector in the pseudorotation phase angle cycle that is adjacent to C3 -endo. ...
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... comparison, it is not surprising that the 4 -Cβ epimers Ufob and uob are strongly destabilizing. The crystal structures of modified octamers reveal tight phosphatephosphate and 4 -Cβ-OMe-phosphate spacings consistent with unfavorable electrostatic and steric effects ( Figure 3C,D). It is noteworthy that Ufob is significantly more destabilizing than uob (−8.9 • C and -6.3 • C, respectively, Table 1). ...
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... interesting difference between the Ufob and uob modifications is the inverted spacing between methyl groups from the 4 -C-substituent and the N -1 and N + 1 phosphates, 3 -and 5 -adjacent nucleotides, (N being the modified nucleotide) in the two structures. Thus, in the uob-modified octamer, the methyl group is positioned closer to the N + 1 phosphate (average 4.3 ˚ A versus 6.8 ˚ A to N -1; Figure 3C), whereas in the Ufob-modified octamer, the methyl group sits nearer to the N -1 phosphate (4.3 ˚ A versus 5.5 ˚ A to N + 1; Figure 3D). It is perhaps surprising that Ufob and uob exhibit stacking and Watson-Crick base pairing despite the inverted stereochemistry at C4 . ...
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... interesting difference between the Ufob and uob modifications is the inverted spacing between methyl groups from the 4 -C-substituent and the N -1 and N + 1 phosphates, 3 -and 5 -adjacent nucleotides, (N being the modified nucleotide) in the two structures. Thus, in the uob-modified octamer, the methyl group is positioned closer to the N + 1 phosphate (average 4.3 ˚ A versus 6.8 ˚ A to N -1; Figure 3C), whereas in the Ufob-modified octamer, the methyl group sits nearer to the N -1 phosphate (4.3 ˚ A versus 5.5 ˚ A to N + 1; Figure 3D). It is perhaps surprising that Ufob and uob exhibit stacking and Watson-Crick base pairing despite the inverted stereochemistry at C4 . ...
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... and Ufme are more effective at stabilizing against SVPD than uo, particularly in combination with the PS modification ( Figure 2). Indeed, Ufme shows Me-P contacts that are tighter than those seen in Ufo and uo (Figure 3). Moreover, those distances are slightly shorter in Ufo than in uo. ...
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... For our LPX study we have employed a chemically stabilized Luc-siRNA with two phosphorothioate bonds on the 3′ terminus of the sense strand and one 3′ terminal phosphorothioate bond on the antisense strand [47]. This can additionally slow down degradation by exonucleases [48]. On the other hand, after a 32 h incubation period with lipoplexes and subsequent chloroquine treatment, a reduction in luciferase signal was not observed (Fig. 9C). ...
Although cationic liposomes are efficient carriers for nucleic acid delivery, their toxicity often hampers the clinical translation. Polyethylene glycol (PEG) coating has been largely used to improve their stability and reduce toxicity. Nevertheless, it has been found to decrease the transfection process. In order to exploit the advantages of cationic liposomes and PEG decoration for nucleic acid delivery, liposomes decorated with tetraArg-[G-1]-distearoyl glycerol (Arg 4-DAG) dendronic oligo-cationic lipid enhancer (OCE) and PEG-lipid have been investigated. Non decorated or OCE-decorated lipoplexes (OCE free-LPX and OCE-LPX, respectively) were obtained by lipid film hydration using oligonucleotide (ON) solutions. PEG and OCE/PEG decorated lipoplexes (PEG-OCE free-LPX and PEG-OCE-LPX, respectively) were obtained by post-insertion of 2 or 5 kDa PEG-DSPE on preformed lipoplexes. The OCE decoration yielded lipoplexes with size of about 240 nm, 84% loading efficiency at 10 N/P ratio, ten times higher than OCE free-LPX, and prevented the ON release when incubated with physiological heparin concentration or with plasma. The PEG decoration reduced the zeta potential, enhanced the lipoplex stability in serum and decreased both hemolysis and cytotoxicity, while it did not affect the lipoplex size and ON loading. With respect to OCE free-LPX, the OCE-LPX remarkably associated with cells and were taken up by different cancer cell lines (HeLa and MDA-MB-231). Interestingly, 2 or 5 kDa PEG decoration did not reduce either the cell interaction or the cell uptake of the cationic lipoplexes. With siRNA as a payload, OCE enabled efficient internalization, but endosomal release was hampered. Post-transfection treatment with the lysosomo-tropic drug chloroquine allowed to identify the optimal time point for endosomal escape. Chloroquine treatment after 12 to 20 h of LPX pre-incubation enabled siRNA mediated target knockdown indicating that this is the time window of endo-lysosomal processing. This indicates that OCE can protect siRNA from lysosomal degradation for up to 20 h, as shown by these rescue experiments.
... Alterations to the phosphodiester backbone or the sugars have been reported to increase aptamer resistance to nuclease degradation. [69][70][71][72] Base substitutions with modified nucleotides can alter the affinity of aptamers for their targets. 73 One hypothesis for the increased systemic stability and delivery of aptamers carrying hydrophobic bases is enhanced association with serum albumin as a protective bloodstream carrier with presumed aptamer dissociation when in contact with tumor cells. ...
... 69,70 Sugar modifications such as locked nucleic acids (LNAs) reportedly increase nuclease stability of the aptamer and certain base pairing interactions. 71,72 Base substitutions with modified nucleotides can alter the affinity of aptamers for their targets. 73 In one example, the addition of a hydrophobic 3,5-bis(trifluoromethyl)benzoyl analog at two terminal and three internal positions reportedly improved tumor specificity, systemic delivery, and stability in vivo. ...
Despite recent advances in the understanding of brain tumor pathophysiology, challenges associated with tumor location and characteristics have prevented significant improvement in neuro-oncology therapies. Aptamers are short, single-stranded DNA or RNA oligonucleotides that fold into sequence-specific, three-dimensional shapes that, like protein antibodies, interact with targeted ligands with high affinity and specificity. Aptamer technology has recently been applied to neuro-oncology as a potential approach to innovative therapy. Preclinical research has demonstrated the ability of aptamers to overcome some obstacles that have traditionally rendered neuro-oncology therapies ineffective. Potential aptamer advantages include their small size, ability in some cases to penetrate the blood-brain barrier, inherent lack of immunogenicity, and applicability for discovering novel biomarkers. Herein, we review recent reports of aptamer applications in neuro-oncology including aptamers found by cell- and in-vivo-SELEX approaches, aptamer-targeted therapeutic delivery modalities, and aptamers in diagnostics and imaging. We further identify crucial future directions for the field that will be important to advance aptamer-based drugs or tools to clinical application in neuro-oncology.
... Interestingly, in the crystal structure of an RNA octamer (PDB ID: 6CY0), the modified 4'-Cβ epimers could be found in the C3'-endo and C2'-exo states. [65,90] However, the incorporation of 2'-fluoro-4'-Cβ-methoxy ribouridine results in high destabilization of all the duplexes: DNA:DNA (ΔT m~À 8.7°C/modification), RNA:RNA (ΔT m~À 4.7°C/modification) and DNA:RNA (ΔT m~À 8.9°C/modification). Fortunately, no perturbation of the global geometry was observed for the modified duplexes in the CD spectroscopy analysis. ...
Ribofuranose sugar conformation plays an important role in the structure and dynamics of functional nucleic acids such as siRNAs, AONs, aptamers, miRNAs, etc. To improve their therapeutic potential, several chemical modifications have been introduced into the sugar moiety over the years. The stability of the oligonucleotide duplexes as well as the formation of stable and functional protein-oligonucleotide complexes are dictated by the conformation and dynamics of the sugar moiety. In this review, we systematically categorise various ribofuranose sugar modifications employed in DNAs and RNAs so far. We discuss different stereoelectronic effects imparted by different substituents on the sugar ring and how these effects control sugar puckering. Using this data, it would be possible to predict the precise use of chemical modifications and design novel sugar-modified nucleosides for therapeutic oligonucleotides that can improve their physicochemical properties.
... Endosomal entrapment of oligonucleotides creates an intracellular depot and provides a continuous supply of siRNA for RISC loading 12 , explaining the long duration of effect. A combination of sugar, backbone, and terminal phosphate modifications [13][14][15][16][17][18] stabilizes the compound in the endosomal compartment and is required to ensure durable gene silencing in vivo. ...
Small interfering RNAs (siRNAs) are a new class of drugs, exhibiting sequence-driven, potent, and sustained silencing of gene expression in vivo . We recently demonstrated that siRNA chemical architectures can be optimized to provide efficient delivery to the CNS. Many genetically-defined neurodegenerative disorders are autosomal dominant favoring selective silencing of the mutant allele. In some cases, successful targeting of the mutant allele requires targeting of a single nucleotide polymorphism (SNP) heterozygosity. Using Huntington’s disease as a model, we demonstrate allele-specific RNAi-based silencing of gene expression in vivo and in neurons differentiated from HD patient-derived iPSCs. A series of in vitro screens, with chemical and thermodynamic optimization, identified compounds with >50-fold selectivity for the mutant HD-causing allele, based on a single nucleotide difference. The optimized compound exhibits selective silencing of mutant huntingtin (HTT) protein in patient derived cells and throughout the HD mouse brain, providing a demonstration of SNP-based allele-specific RNAi silencing of gene expression in vivo in the CNS. The ability to target a disease-causing allele using RNAi-based therapies could be applied to a wide range of dominant CNS disorders, where maintenance of wild-type expression is essential.
... Recently, Manoharan et al. reported the synthesis and properties of C4 0 and C5 0 -modied nucleosides, such as the 4 0 -C-methoxy-modied nucleosides, (R)-, or (S)-5 0 -C-methyl-modied nucleosides. [14][15][16][17][18][19] These nucleosides enhanced the nuclease resistance of oligonucleotides, compared with C2 0 -modied nucleosides without thermally destabilizing RNA duplexes and inhibiting gene silencing activity. Moreover, (R)or (S)-5 0 -C-methylnucleosides improved the stability of oligonucleotides to snake venom phosphodiesterase (SVPD), compared with 4 0 -C-methoxy-nucleoside. ...
... This was due to the 5 0 -C modications close to the phosphate linkages, compared with 4 0 -C modications. 16 Recently, we reported the synthesis of RNA oligomers containing (S)-5 0 -C-aminopropyl-2 0 -O-methyluridine (1). 20 We observed that analog 1 signicantly increased the stability of the RNAs in a buffer containing bovine serum and the binding affinity of the RNAs toward complementary RNA, compared with 4 0 -C-aminopropyl-2 0 -O-methyluridine and (R)-5 0 -C-aminopropyl-2 0 -O-methyluridine. ...
... Next, we measured the T m of the siRNAs containing 2 0 -Omethylnucleosides or (S)-5 0 -C-aminopropyl-2 0 -O-methylnucleosides at the 3 0 -and 5 0 -regions of the passenger strand (siRNA [16][17][18][19][20][21]. As shown in Table 4 , and 21 were calculated as À1.3, À1.6 and À6.0 C, respectively. ...
Herein, we report the synthesis of (S)-5'-C-aminopropyl-2'-O-methyladenosine and (S)-5'-C-aminopropyl-2'-O-methylguanosine phosphoramidites and the properties of small interfering RNAs (siRNAs) containing four (S)-5'-C-aminopropyl-2'-O-methylnucleosides (A, adenosine; U, uridine; G, guanosine; and C, cytidine). The siRNAs containing (S)-5'-C-aminopropyl-nucleosides at the 3'- and 5'-regions of the passenger strand were well tolerated for RNA interference (RNAi) activity. Conversely, the (S)-5'-C-aminopropyl modification in the central region of the passenger strand decreased the RNAi activity. Furthermore, the siRNAs containing three or four consecutive (S)-5'-C-aminopropyl-2'-O-methylnucleosides at the 3'- and 5'-regions of the passenger strand exhibited RNAi activity similar to that of the corresponding 2'-O-methyl-modified siRNAs. Finally, it was observed that (S)-5'-C-aminopropyl modifications effectively improved the serum stability of the siRNAs, compared with 2'-O-methyl modifications. Therefore, (S)-5'-C-aminopropyl-2'-O-methylnucleosides would be useful for improving the serum stability of therapeutic siRNA molecules without affecting their RNAi activities.
... In 2018, Egli and co-workers investigated the potential effects of fluoro and methoxy modifications at C2' and C4' positions in siRNAs [82]. The 2'-F and 2'-O-Me modified oligonucleotides, as expected, were degraded within 1 h of incubation time in SVPD. ...
... The crystal structures of Ufo and Ufob modified 8-mer RNAs bound to human Ago2 showed that the steric conflict arises between Ago-2 amino acids and RNA residues due to the presence of the C4'-O-Me group in both Ufo and Ufob nucleotides [82]. Overall, the results suggested that the Cαepimers showed enhanced serum stability, thermal stability and were compatible with the RNAi machinery. ...
Nucleic acid-based therapeutics that control gene expression have been steadily progressing towards achieving their full clinical potential throughout the last few decades. Rapid progress has been achieved in RNAi-based therapy by optimizing high specificity and gene silencing efficiency using chemically modified siRNAs. Since 2018, four siRNA drugs - patisiran, givosiran, lumasiran, and inclisiran, were approved by the US FDA, providing a testament to the promise of RNAi therapeutics. Despite these promising results, safe and efficient siRNA delivery at the target site remains a major obstacle for efficient siRNA-based therapeutics. In this review, we have outlined the synergistic effects of emerging dual ribose modifications, including 2',4'- and 2',5'-modifications, 5'-E/Z-vinylphosphonate, and northern methanocarbacyclic (NMC) modifications that have contributed to drug-like effects in siRNA. These modifications enhance nuclease stability, prolong gene silencing efficiency, improve thermal stability, and exhibit high tissue accumulation. We also highlight the current progress in siRNA clinical trials. This review will help to understand the potential effects of dual ribose modifications and provides alternative ways to use extensive 2'-modifications in siRNA drugs. Moreover, the minimal number of these dual ribose modifications could be sufficient to achieve the desired therapeutic effect. In future, detailed in vivo studies using these dual ribose modifications could help to improve the therapeutic effects of siRNA. Rational design could further open doors for the rapid progress in siRNA therapeutics. [Figure: see text].
... Oligonucleotide therapeutics (ONTs), including siRNA, miRNA, antisense oligonucleotides (ASO), aptamer-based pharmaceuticals and nucleic acid vaccines, are emerging as a new generation of medicine next to small molecule drugs and antibody therapeutics (1)(2)(3)(4)(5)(6)(7). ONs are primarily manufactured by computer-controlled DNA synthesizer (8) automatically with excellent batch-to-batch consistency and reproducibility, and more than that, they can be readily modified with functionalities for optimization in biological activities (9)(10)(11)(12). The potential of ONTs is tremendous, but their clinical translation is hampered by fast degradation and renal excretion, as unmodified ONs stay in the circulation system for <1 h (13). ...
... Chemical modification is the simplest and most powerful strategy and has led to the success of several ONTs in clinical practice (26,27). Oligonucleotides modified with phosphorothioate backbone or 2 -functionalities have increased stability for their resistance to nuclease degradation (9,12,19,20). The conjugation with functionalities such as cholesterol and GalNAc at the terminals gives ONs as pure biomolecules with improved in vivo stability, cellular uptake and other properties (10,11,13,28). ...
Oligonucleotide (ON) therapeutics are emerging as a new generation of medicine with tremendous potential, but their clinical translation is hampered by inferior stability and short circulation time in the human body. Here, we report a general approach to manipulating the interaction between ONs and albumin by modulating hydrophobicity. A series of DNA aptamer derivatives were designed and prepared by programmable synthesis as an ON library with a gradient of hydrophobic base 'F'. In vitro experiments revealed that the introduction of two F bases at both ends of ONs enhanced the biostability without sacrificing biological activities, while the binding affinity toward albumin was dramatically increased with Kd in the range of 100 nM to 1 μM. In vivo imaging confirmed the immediate formation of the aptamer-albumin complex after the injection, and the circulation time of the aptamer was dramatically elongated owing to the enhanced biostability and retarded renal excretion. The programmable incorporation of the F base provides a general approach to regulating albumin-binding affinity and enhancing the stability of aptamers in vivo, conferring aptamer therapeutics prolonged circulation time to meet clinical requirements.
... The 4 ′ ,5 ′ -olefinic compound 3 was synthesized according to a previously reported method. [18] After epoxidation of 3, the product was successively treated with the N-protected aminoethanol in the presence of ZnCl 2 to give the 4 ′ -C-aminoethoxy derivative 4 in 56% yield. [19] Configuration of the 4 ′ -carbon in 4 was determined by nuclear Overhauser effect spectroscopy (NOESY). ...
We designed and synthesized RNAs containing 4'-C-aminoethoxy-2'-O-methyluridine (4'-AEoU), and compared their properties with those containing 4'-C-aminopropyl-2'-O-methyluridine (4'-APU). The RNA duplexes containing 4'-AEoU showed higher melting temperatures than those containing 4'-APU. The 4'-AEoU modification enhanced the stability of the RNAs in buffer containing serum. Furthermore, it was found that the incorporation of 4'-AEoU into the passenger strand of small interfering RNAs (siRNAs) is tolerated for the RNA interference (RNAi) activity of the siRNAs.
... C4'-modified nucleos(t)ides have found a wide range of biomedical applications, either as probes of nucleic acid structure and function and antiviral agents [1][2][3][4] or for use in oligonucleotide-based therapeutics. [5][6][7][8][9][10] The obvious advantage of C4'-modification on nucleos(t)ides is that it is located at the backbone edge, thus rendering a stereoelectronic effect to the ribose conformation without introducing notable steric clash to the nucleic acid duplexes. [11,12] This feature makes C4'-modification an attractive approach to modulate the structure of nucleic acids, which in turn dictates their functions. ...
Fluoro‐substitution on the ribose moiety (e. g., 2’‐F‐deoxyribonucleotide) represents a popular way to modulate the ribose conformation and, hence, the structure and function of nucleic acids. In the present study, we synthesized 4’‐F‐deoxythymidine (4’‐FT) and introduced it to oligodeoxyribonucleotides (ODNs). Though scission of the glycosylic bond of 4’‐FT followed by strand cleavage occurred to some extent under alkaline conditions, the 4’‐FT‐modified ODNs were rather stable in neutral buffers. NMR studies showed that like 2’‐F‐deoxyribonucleoside, 4’‐FT exists predominantly in the North conformation not only in the nucleoside form but also in the context of ODN strands. Circular dichroism spectroscopy, thermal denaturing and RNase H1 footprinting studies of 4’‐FT‐modified ODN/cDNA and ODN/cRNA duplexes indicated that the North conformation tendency of 4’‐FT is maintained in the duplexes, leading to a local structural perturbation. Collectively, 4’‐F‐deoxyribonucleotide structurally resembles the 2’‐F‐deoxyribonucleotide but imparts less structural perturbation to the duplex than the latter.
... Modifications at the C4' sugar position ( Figure 1H) have long been desirable as a means of modulating the properties of nucleic acids without interfering with Watson-Crick pairing. Incorporations at C4' are close in proximity to both the 3' and 5'-neighboring phosphate groups, allowing for a tailoring of the nuclease resistance [200]. In 2011, Rosenberg demonstrated the [190]. ...
Over the past 25 years, the acceleration of achievements in the development of oligonucleotide-based therapeutics has resulted in numerous new drugs making it to the market for the treatment of various diseases. Oligonucleotides with alterations to their scaffold, prepared with modified nucleosides and solid-phase synthesis, have yielded molecules with interesting biophysical properties that bind to their targets and are tolerated by the cellular machinery to elicit a therapeutic outcome. Structural techniques, such as crystallography, have provided insights to rationalize numerous properties including binding affinity, nuclease stability, and trends observed in the gene silencing. In this review, we discuss the chemistry, biophysical, and structural properties of a number of chemically modified oligonucleotides that have been explored for gene silencing.
