An overview of sugar-modified oligonucleotides for antisense therapeutics

Among the multitude of chemical modifications that have been described over the past two decades, oligonucleotide analogs that are modified at the 2'-position of the furanose sugar have been especially useful for improving the drug-like properties of antisense oligonucleotides (ASOs). These modifications bias the sugar pucker towards the 3'-endo-conformation and improve ASO affinity for its biological target (i.e., mRNA). In addition, antisense drugs incorporating 2'-modified nucleotides exhibit enhanced metabolic stability, and improved pharmacokinetic and toxicological properties. Further conformational restriction of the 2'-substituent to the 4'-position of the furanose ring yielded the 2',4'-bridged nucleic acid (BNA) analogs. ASOs containing BNA modifications showed unprecedented increase in binding affinity for target RNA, while also improved nuclease resistance, in vitro and in vivo potency. Several ASO drug candidates containing 2'-modified nucleotides have entered clinical trials and continue to make progress in the clinic for a variety of therapeutic indications.     

2′-DMAOE RNA: Emerging Oligonucleotides with Promising Antisense Properties

Abstract

Oligonucleotides with modifications at the carbohydrate 2′-position offer potential second-generation drug candidates¹. ISIS 13312, a chimeric compound targeting CMV retinitis, has 2′-O-methoxyethyl² (2′-MOE) modifications at the ends to offer enhanced binding affinity and nuclease resistance is an example of this trend. 2′-MOE modification offers high binding affinity and nuclease resistance presumably due to conformational constraints placed on the linkage by the oxygen-oxygen gauche effect³. On the other hand, 2′-O-aminopropyl modification (2′-AP) exhibits the highest nuclease resistance⁴, due to the presence of a cationic charge at the physiological pH. However, it lacks the binding affinity advantage of MOE due to the lack of oxygen-oxygen gauche effect. To optimize the antisense properties of both 2′-MOE and 2′-AP modifications, we have designed and constructed 2′-O-(aminooxyethyl) modification (2′-AOE)⁵ and 2′-O-(dimethylaminooxy ethyl) modification (2′-DMAOE) and synthesized oligomers having these modifications. 2′-DMAOE oligomers demonstrate higher binding affinity and nuclease resistance than 2′-MOE oligomers and stand out as promising candidates for future antisense oligonucleotide drug development.     

Probing the influence of stereoelectronic effects on the biophysical properties of oligonucleotides: comprehensive analysis of the RNA affinity, nuclease resistance, and crystal structure of ten 2'-O-ribonucleic acid modifications

The syntheses of 10 new RNA 2'-O-modifications, their incorporation into oligonucleotides, and an evaluation of their properties such as RNA affinity and nuclease resistance relevant to antisense activity are presented. All modifications combined with the natural phosphate backbone lead to significant gains in terms of the stability of hybridization to RNA relative to the first-generation DNA phosphorothioates (PS-DNA). The nuclease resistance afforded in particular by the 2'-O-modifications carrying a positive charge surpasses that of PS-DNA. However, small electronegative 2'-O-substituents, while enhancing the RNA affinity, do not sufficiently protect against degradation by nucleases. Similarly, oligonucleotides containing 3'-terminal residues modified with the relatively large 2'-O-[2-(benzyloxy)ethyl] substituent are rapidly degraded by exonucleases, proving wrong the assumption that steric bulk will generally improve protection against nuclease digestion. To analyze the factors that contribute to the enhanced RNA affinity and nuclease resistance we determined crystal structures of self-complementary A-form DNA decamer duplexes containing single 2'-O-modified thymidines per strand. Conformational preorganization of substituents, favorable electrostatic interactions between substituent and sugar-phosphate backbone, and a stable water structure in the vicinity of the 2'-O-modification all appear to contribute to the improved RNA affinity. Close association of positively charged substituents and phosphate groups was observed in the structures with modifications that protect most effectively against nucleases. The promising properties exhibited by some of the analyzed 2'-O-modifications may warrant a more detailed evaluation of their potential for in vivo antisense applications. Chemical modification of RNA can also be expected to significantly improve the efficacy of small interfering RNAs (siRNA). Therefore, the 2'-O-modifications introduced here may benefit the development of RNAi therapeutics.     

Nuclease resistance of oligonucleotides containing the tricyclic cytosine analogues phenoxazine and 9-(2-aminoethoxy)-phenoxazine ("G-clamp") and origins of their nuclease resistance properties.

The tricyclic cytosine analogues phenoxazine and 9-(2-aminoethoxy)-phenoxazine ("G-clamp") are known to significantly enhance the binding affinity of oligonucleotides to their complementary target DNA or RNA strands. To investigate their effect on the nuclease resistance, they were incorporated into model oligomers with a natural phosphodiester backbone, and enzymatic degradation was monitored in an in vitro assay with snake venom phosphodiesterase as the hydrolytic enzyme. In both cases, a single incorporation at the 3'-terminus completely protected the oligonucleotides against 3'-exonuclease attack. Further investigations indicate that the observed high nuclease resistance is not due to the lack of binding affinity to the enzyme's active site, since these modified oligonucleotides were able to inhibit degradation of a natural DNA fragment by bovine intestinal mucosal phosphodiesterase in a dose-dependent manner.     

Interrupted 2'-o,4'-C-aminomethylene bridged nucleic acid modification enhances pyrimidine motif triplex-forming ability and nuclease resistance under physiological condition

Due to instability of pyrimidine motif triplex DNA at physiological pH, triplex stabilization at physiological pH is crucial in improving its potential in various triplex formation-based strategies in vivo, such as regulation of gene expression, mapping of genomic DNA, and gene-targeted mutagenesis. To this end, we investigated the effect of our previously reported chemical modification, 2'-O,4'-C-aminomethylene bridged nucleic acid (2',4'- BNA(NC)) modification, introduced into interrupted and continuous positions of triplex-forming oligonucleotide (TFO) on pyrimidine motif triplex formation at physiological pH. The interrupted 2',4'-BNA(NC) modifications of TFO increased the binding constant of the triplex formation at physiological pH by more than 10-fold, and significantly increased the nuclease resistance of TFO. On the other hand, the continuous 2',4'-BNA(NC) modification of TFO showed lower ability to promote the triplex formation at physiological pH than the interrupted 2',4'-BNA(NC) modifications of TFO, and did not significantly change the nuclease resistance of TFO. Selection of the interruptedly 2',4'-BNA(NC)-modified positions in TFO was more favorable for achieving the higher binding affinity of the pyrimidine motif triplex formation at physiological pH and the higher nuclease resistance of TFO than that of the continuously 2',4'-BNA(NC)-modified positions in TFO. We conclude that the interrupted 2',4'-BNA(NC) modification of TFO could be a key chemical modification to enhance pyrimidine motif triplex-forming ability and nuclease resistance under physiological condition, and may eventually lead to progress in various triplex formation-based strategies in vivo.     

Synthesis of tropane and nortropane analogues with phenyl substitutions as serotonin transporter ligands.

The effects of structural modifications of 2 beta-carbomethoxy-3 beta-phenyl tropane analogues were evaluated on in vitro affinity to the dopamine (DAT) and serotonin (5-HTT) transporters in rat brain tissue. The introduction of a large alkyl group at the 4'-position of the phenyl ring, affording 2 beta-carbomethoxy-3 beta-(4'-alkylphenyl) tropane, diminished the affinity for the DAT whereas moderate 5-HTT affinity was obtained. The introduction of an iodine at the 3'-position of the 4'-alkylphenyl, affording 2 beta-carbomethoxy-3 beta-(3'-iodo-4'-alkylphenyl) tropane, and N-demethylation, affording 2 beta-carbomethoxy-3 beta-(3'-iodo-4'-alkylphenyl) nortropane, improved affinity and specificity for the 5-HTT. It could be assumed from these results that the combination of these three modifications of tropane structure yielded highly selective compounds for the 5-HTT. Of the new compounds synthesized, the most selective cocaine derivative, 2 beta-carbomethoxy-3 beta-(3'-iodo-4'-isopropylphenyl) nortropane (8d) labeled with iodine-123 or carbon-11, could be a potential ligand for exploration of the 5-HT transporter by SPET or PET.     

2'-O-modified oligoribonucleotides with terminal 3'-3'-internucleotide linkage and their derivatives

A high RNA binding affinity and nuclease resistance of 2'-O-modified (2'-O-methyl, 2'-O-tetrahydropyranyl) oligoribonucleotides containing the "inverted" T at the 3'-end have been shown. The synthesis and properties of new photoactivatable perfluoroarylazide derivatives of these oligoribonucleotides are discussed.     

Carbohydrate Modifications in Antisense Oligonucleotide Therapy: New Kids on the Block

Abstract

Chemical modifications to improve the efficacy of an antisense oligonucleotide are designed to increase the binding affinity to target RNA, to enhance the nuclease resistance, and to improve cellular delivery. Among the different sites available for chemical modification in a nucleoside building block, the 2′-position of the carbohydrate moiety¹ has proven to be the most valuable for various reasons: (1) 2′-modification can confer an RNA-like 3′-endo conformation to the antisense oligonucleotide. Such a preorganization for an RNA like conformation^2,3,4,5 greatly improves the binding affinity to the target RNA; (2) 2′-modification provides nuclease resistance to oligonucleotides; (3) 2′-modification provides chemical stability against potential depurination conditions pharmacology evaluations and correlation with pharmacokinetic changes are emerging from these novel chemical modifications. Analytical chemistry of modified oligonucleotides before and after biological administration of antisense oligonucleotides with techniques such as capillary gel electrophoresis (CGE) and mass spectrometry help to determine the purity as well as the in vivo fate of these complex molecules. Large-scale synthesis is becoming a tangible reality for antisense oligonucleotides. Nucleic acid chemists and biologists alike are beginning to understand the structure-biological activity in terms of basic physical-organic parameters such as the gauche effect, the charge effect and conformational constraints. Synthesis of chimeric designer oligonucleotides bringing the attractive features of different modifications to a given antisense oligonucleotide sequence to generate synergistic interactions is forthcoming³⁰. These advances along with the potential availability of complete human genome sequence information promise a bright future for the widespread use of nucleic acid based therapeutics.     

2′-O-MODIFIED OLIGORIBONUCLEOTIDES WITH TERMINAL 3′-3′-INTERNUCLEOTIDE LINKAGE AND THEIR DERIVATIVES

A high RNA binding affinity and nuclease resistance of 2′-O-modified (2′-O-methyl, 2′-O-tetrahydropyranyl) oligoribonucleotides containing the “inverted” T at the 3′-end have been shown. The synthesis and properties of new photoactivatable perfluoroarylazide derivatives of these oligoribonucleotides are discussed.     

Interactions of hairpin oligo-2'-O-methylribonucleotides containing methylphosphonate linkages with HIV TAR RNA

Methylphosphonate-modified oligo-2'-O-methylribonucleotides 15-20 nucleotides (nt) in length were prepared whose sequences are complementary to the 5' and 3' sides of the upper hairpin of HIV trans-acting response element (TAR) RNA. These anti-TAR oligonucleotides (ODNs) form stable hairpins whose melting temperatures (Tm) range from 55 degrees C to 80 degrees C. Despite their rather high thermal stabilities, the hairpin oligo-2'-O-methylribonucleotides formed very stable complexes with TAR RNA, with dissociation constants in the nanomolar concentration range at 37 degrees C. The affinities of the hairpin oligomers for TAR RNA were influenced by the positions of the methylphosphonate linkages. The binding affinity was reduced approximately 17-fold by the presence of two methylphosphonate linkages in the TAR loop complementary region (TLCR) of the oligomer, whereas methylphosphonate linkages outside this region increased binding affinity approximately 3-fold. The configurations of the methylphosphonate linkages in the TLCR also affected binding affinity, with the RpRp isomer showing significantly higher binding than the SpSp isomer. In addition to serving as probes of the interactions between the oligomer and TAR RNA, the presence of the methylphosphonate linkages in combination with the hairpin structure increases the resistance of these oligomers to degradation by exonucleases found in mammalian serum. The combination of high binding affinity and nuclease resistance of the hairpin ODNs containing methylphosphonate linkages suggests their potential utility as antisense compounds.     

NMI Linkage Modification Increases Potency and Stability of H-RAS Antisense Oligonucleotides

Abstract

Phosphorothioate antisense oligodeoxyribonucleotides (PS-ASOs) have proven to be useful first generation antisense tools for in vitro and in vivo uses and now show great promise as human therapeutic agents. However, there are two characteristics of PS-ASOs that make it desirable to continue to attempt to improve their biophysical characteristics through chemical modification. First, PS-ASOs have been reported, at very high concentrations, to have some nonspecific activities, both in vitro and in vivo, usually attributed to their protein binding properties. Second, while significantly more stable than their phosphodiester analogues, the in vivo stability of phosphorothioate oligonucleotides can still be improved. This instability is primarily due to 3′ exonucleases, 5′ exonucleases, and to a lesser degree, endonucleases. There is a strong rationale for exploring backbone modifications that can reduce the P=S content and maintain or increase nuclease resistance of antisense oligonucleotides. One such modification, methylene(methyl)imino (MMI), allows for complete substitution of the phosphate backbone while maintaining high affinity for the target RNA and enhanced nuclease resistance.^1,2 This modification is incorporated into the oligonucleotide as MMI-dimers.     

Alterations in the structure of the ribose moiety of ATP reduce its effectiveness as a substrate for the sarcoplasmic reticulum ATPase

The substrate specificity of the calcium ATPase of isolated sarcoplasmic reticulum vesicles was examined using a series of ribose-modified ATP analogs. Steady state hydrolytic rate measurements using analogs modified at the 2'- and 3'-positions demonstrate that both hydroxyl groups contribute to substrate specificity and high catalytic activity. The 3'-hydroxyl is especially significant in this regard since all analogs modified at the 3'-position were slowly hydrolyzed, if at all, and they did not produce the activation at millimolar concentrations characteristically observed with ATP. In contrast, ATP analogs modified only at the 2'-position were more rapidly hydrolyzed (although at rates less than for ATP), and they did produce activation at millimolar concentrations. These results suggest that neither the catalytic (high affinity) nor the regulatory (low affinity) site of the CaATPase tolerates changes in the hydroxyl substituent at the 3'-position of ATP, whereas steady state rates associated with substrate binding at both types of sites are less affected by changes in the 2'-hydroxyl of ATP.     

Antimessenger oligodeoxyribonucleotides: an alternative to antisense RNA for artificial regulation of gene expression--a review

Synthetic oligodeoxyribonucleotides (oligos) are now widely used as artificial regulators for gene expression both in cell-free media and in cultured cells. We describe the biological consequence of the various chemical modifications that have been introduced into the molecules to improve their resistance against nuclease attack, their affinity for the target mRNA and their uptake by cells. We also describe the rising generation of antimessenger oligos. Covalently linked to reactive groups these molecules direct irreversible modifications of the complementary nucleic acids. We anticipate that these oligos will be targeted to double-stranded nucleic acids to interfere with gene expression at the DNA level.     

Differences in steroid specificity for rat androgen binding protein and the cytoplasmic receptor.

Two proteins in the rat, androgen binding protein (ABP) and the cytoplasmic receptor (CR), have high affinity and limited capacity for binding androgens. To determine the structural requirements for binding with high affinity, each protein was partially purified and the ability of over 100 steroids to compete with [3H]dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one) for binding sites was assessed. The results indicate marked differences in the steroid specificities of the two proteins. Some alterations of dihydrotestosterone at C-2 or C-2 and C-3 increase binding to ABP two to four-fold. Similarly, the affinity of 17 beta-hydroxy-7 alpha-methyl-4-estren-3-one for ABP increases two-fold when a double bond is created at C-14. Addition of a methyl group in the alpha position at C-7 or C-17, or an ethinyl group at C-17 cause little change in affinity; however, modifications at C-11 and C-17 beta, and deletion of the methyl group at C-10 significantly impair binding to ABP. Binding to the CR is maintained or increased by deletion of the methyl group at C-10. Binding is lessened by modifications at C-3 and C-17 beta. Most alterations at C-2, C-7, C-11, and C-17 alpha have only minor effects on binding to the CR. These studies should provide a molecular basis for predicting the effects of specific structural modifications. When some modifications at C-2 or C-2 and C-3 are combined with changes at C-17 beta, the resulting steroids retain very high affinity for ABP and very limited binding to the CR. Such steroids may provide a means for assessing the function of ABP.     

The ups and downs of nucleic acid duplex stability: structure-stability studies on chemically-modified DNA:RNA duplexes.

In an effort to discover novel oligonucleotide modifications for antisense therapeutics, we have prepared oligodeoxyribonucleotides containing more than 200 different modifications and measured their affinities for complementary RNA. These include modifications to the heterocyclic bases, the deoxy-ribose sugar and the phosphodiester linkage. From these results, we have been able to determine structure-activity relationships that correlate hybridization affinity with changes in oligonucleotide structure. Data for oligonucleotides containing modified pyrimidine nucleotides are presented. In general, modifications that resulted in the most stable duplexes contained a heteroatom at the 2'-position of the sugar. Other sugar modifications usually led to diminished hybrid stability. Most backbone modifications that led to improved hybridization restricted backbone mobility and resulted in an A-type sugar pucker for the residue 5'to the modified internucleotide linkage. Among the heterocycles, C-5-substituted pyrimidines stood out as substantially increasing duplex stability.     

Synthesis and biophysical properties of 5′-thio-2′,4′-BNA/LNA oligonucleotide

Phosphorothioate modification of oligonucleotides is one of the most promising chemical modifications in nucleic acid therapeutics. Structurally similar 5′-thio or phosphorothiolate-modified nucleotides, in which the 5′-bridging oxygen atom is replaced with a sulfur atom, are attracting attention and gaining importance in oligonucleotide-based research. In our present study, we synthesized 5′-thio-2′,4′-BNA/LNA monomers bearing thymine or 5-methylcytosine nucleobase. The 5′-thio-2′,4′-BNA/LNA monomers were successfully incorporated into target oligonucleotides, and their nuclease stability and binding affinity with complementary strands were evaluated.     

Towards next generation antisense oligonucleotides: mesylphosphoramidate modification improves therapeutic index and duration of effect of gapmer antisense oligonucleotides

The PS modification enhances the nuclease stability and protein binding properties of gapmer antisense oligonucleotides (ASOs) and is one of very few modifications that support RNaseH1 activity. We evaluated the effect of introducing stereorandom and chiral mesyl-phosphoramidate (MsPA) linkages in the DNA gap and flanks of gapmer PS ASOs and characterized the effect of these linkages on RNA-binding, nuclease stability, protein binding, pro-inflammatory profile, antisense activity and toxicity in cells and in mice. We show that all PS linkages in a gapmer ASO can be replaced with MsPA without compromising chemical stability and RNA binding affinity but these designs reduced activity. However, replacing up to 5 PS in the gap with MsPA was well tolerated and replacing specific PS linkages at appropriate locations was able to greatly reduce both immune stimulation and cytotoxicity. The improved nuclease stability of MsPA over PS translated to significant improvement in the duration of ASO action in mice which was comparable to that of enhanced stabilized siRNA designs. Our work highlights the combination of PS and MsPA linkages as a next generation chemical platform for identifying ASO drugs with improved potency and therapeutic index, reduced pro-inflammatory effects and extended duration of effect.     

7-Substituted 2-phenyl-benzofurans as ER beta selective ligands

A series of 2-(4-hydroxy-phenyl)-benzofuran-5-ols with relatively lipophilic groups in the 7-position of the benzofuran was prepared and the affinity and selectivity for ER beta was measured. Many of the analogues were found to be potent and selective ER beta ligands. Additional modifications at the benzofuran 4-position as well as at the 3'-position of the 2-phenyl group were found to further increase selectivity. Such modifications led to compounds with <10 nM potency and >100-fold selectivity for ER beta.     

Synthesis of 3'-acetamidoadenosine derivatives as potential A3 adenosine receptor agonists

On the basis of high binding affinity of 3'-aminoadenosine derivatives 2b at the human A3 adenosine receptor (AR), 3'-acetamidoadenosine derivatives 3a-e were synthesized from 1,2:5,6-di-O-isopropylidene-D-glucose via stereoselective hydroboration as a key step. Although all synthesized compounds were totally devoid of binding affinity at the human A3AR, our results revealed that 3'-position of adenosine can only be tolerated with small size of a hydrogen bonding donor like hydroxyl or amino group in the binding site of human A3AR.     

Modifying the specificity of an RNA backbone contact.

The interaction between the MS2 bacteriophage coat protein homodimer and its cognate RNA hairpin is facilitated by 21 different RNA-protein contacts. In one of these contacts, the 2'-hydroxyl group at ribose -5 of the RNA acts as a hydrogen bond donor to Glu63 in one subunit of the protein. Previous experiments showed that substitution of ribose -5 with deoxyribose resulted in a 24-fold decrease in binding affinity between RNA and protein. Using a protein where the two MS2 monomers were fused to increase stability, the contribution of this contact to the overall binding affinity was investigated by site-directed mutagenesis. When Glu63 was substituted with glutamine, aspartate, or alanine, the binding affinity of the hairpin for the protein was weakened by 12 to 100-fold, similar to that observed with deoxyribose at position -5. However, the specificity of the three mutant proteins for RNAs with various modifications at the 2'-position of ribose -5 differed dramatically. While the Glu63Asp protein resembled the wild-type protein in preferring the 2'-hydroxyl group over a proton or a bulky 2'-substituent, both the Glu63Ala and Glu63Gln proteins preferred bulky 2'-substituents over the 2'-hydroxyl group by more than 100-fold. These experiments emphasize the ease with which the specificity of a protein-nucleic acid interaction can be changed at thermodynamically important sites.