Single strand targeted triplex-formation. Destabilization of guanine quadruplex structures by foldback triplex-forming oligonucleotides.

Oligonucleotides that can hybridize to single-stranded complementary polypurine nucleic acid targets by Watson-Crick base pairing as well as by Hoogsteen base pairing, referred to here as foldback triplex-forming oligonucleotides (FTFOs), have been designed. These oligonucleotides hybridize with target nucleic acid sequences with greater affinity than antisense oligonucleotides, which hybridize to the target sequence only by Watson-Crick hydrogen bonding [Kandimalla, E. R. and Agrawal, S. Gene(1994) 149, 115-121 and references cited therein]. FTFOs have been studied for their ability to destabilize quadruplexes formation by RNA or DNA target sequences. The influence of various DNA/RNA compositions of FTFOs on their ability to destabilize RNA and DNA quadruplexes has been examined. The ability of the FTFOs to destabilize quadruplex structures is related to the structurally and thermodynamically stable foldback triplex formed between the FTFO and its target sequence. Antisense oligonucleotides (DNA or RNA) that can form only a Watson-Crick double helix with the target sequence are unable to destabilize quadruplex structures of RNA and DNA target sequences and are therefore limited in their repertoire of target sequences. The quadruplex destabilization ability of FTFOs is dependent on the nature of the cation present in solution. The RNA quadruplex destabilization ability of FTFOs is -20% higher in the presence of sodium ion than potassium ion. The use of FTFOs, which can destabilize quadruplex structure, opens up new areas for development of oligonucleotide-based therapeutics, specifically, targeting guanine-rich sequences that exist at the ends of pro- and eukaryotic chromosomes and dimerization regions of retroviral RNA.     

Stereospecificity of Oligonucleotide Interactions Revisited: No Evidence for Heterochiral Hybridization and Ribozyme/DNAzyme Activity

A major challenge for the application of RNA- or DNA-oligonucleotides in biotechnology and molecular medicine is their susceptibility to abundant nucleases. One intriguing possibility to tackle this problem is the use of mirror-image (l-)oligonucleotides. For aptamers, this concept has successfully been applied to even develop therapeutic agents, so-called Spiegelmers. However, for technologies depending on RNA/RNA or RNA/DNA hybridization, like antisense or RNA interference, it has not been possible to use mirror-image oligonucleotides because Watson-Crick base pairing of complementary strands is (thought to be) stereospecific. Many scientists consider this a general principle if not a dogma. A recent publication proposing heterochiral Watson-Crick base pairing and sequence-specific hydrolysis of natural RNA by mirror-image ribozymes or DNAzymes (and vice versa) prompted us to systematically revisit the stereospecificity of oligonucleotides hybridization and catalytic activity. Using hyperchromicity measurements we demonstrate that hybridization only occurs among homochiral anti-parallel complementary oligonucleotide strands. As expected, achiral PNA hybridizes to RNA and DNA irrespective of their chirality. In functional assays we could not confirm an alleged heterochiral hydrolytic activity of ribozymes or DNAzymes. Our results confirm a strict stereospecificity of oligonucleotide hybridization and clearly argue against the possibility to use mirror-image oligonucleotides for gene silencing or antisense applications.     

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.     

New approaches to targeting RNA with oligonucleotides: inhibition of group I intron self-splicing

RNA is one class of relatively unexplored drug targets. Since RNAs play a myriad of essential roles, it is likely that new drugs can be developed that target RNA. There are several factors that make targeting RNA particularly attractive. First, the amount of information about the roles of RNA in essential biological processes is currently being expanded. Second, sequence information about targetable RNA is pouring out of genome sequencing efforts at unprecedented levels. Third, designing and screening potential oligonucleotide therapeutics to target RNA is relatively simple. The use of oligonucleotides in cell culture, however, presents several challenges such as oligonucleotide uptake and stability, and selective targeting of genes of interest. Here, we review investigations aimed at targeting RNA with oligonucleotides that can circumvent several of these potential problems. The hallmark of the strategies discussed is the use of short oligonucleotides, which may have the advantage of higher cellular uptake and improved binding selectivity compared to longer oligonucleotides. These strategies have been applied to Group I introns from the mammalian pathogens Pneumocystis carinii and Candida albicans. Both are examples of fungal infections that are increasing in number and prevalence.     

Nuclear extracts promote gene correction and strand pairing of oligonucleotides to the homologous plasmid

We compared strand pairing and gene correction activities between different constructs of oligonucleotides, using homologous supercoiled DNA and eukaryotic nuclear extracts. The RNA-DNA chimeric oligonucleotide was more efficient in strand pairing and gene correction than its DNA-DNA homolog. Single-stranded deoxyoligonucleotides showed similar strand pairing and correction activity to the modified RNA-DNA chimeric oligonucleotides, whereas single-stranded ribooligonucleotides did not show either activity. However, the correlations were not always linear, suggesting that only a fraction of the joint molecules may be processed to cause the final gene correction. Several mammalian extracts with markedly different in vitro activity showed the similar amounts of the joint molecules. These results led us to conclude that strand pairing is a necessary event in gene correction but may not be the rate-limiting step. Furthermore, depletion of HsRad51 protein caused large decreases in both strand-pairing and functional activities, whereas supplementation of HsRad51 produced only a slight increase in the repair activity, indicating that HsRad51 participates in the strand pairing, but subsequent steps define the frequency of gene correction. In addition, we found that the structure and stability of intermediates formed by single-stranded deoxyoligonucleotides and RNA-DNA chimeric oligonucleotides were different, suggesting that they differ in their mechanisms of gene repair.     

Binding enhancement by tertiary interactions and suicide inhibition of a Candida albicans group I intron by phosphoramidate and 2'-O-methyl hexanucleotides

Candida albicans is one of many infectious pathogens that are evolving resistance to current treatments. RNAs provide a large class of targets for new therapeutics for fighting these organisms. One strategy for targeting RNAs uses short oligonucleotides that exhibit binding enhancement by tertiary interactions in addition to Watson-Crick pairing. A potential RNA target in C. albicans is the self-splicing group I intron in the LSU rRNA precursor. The recognition elements that align the 5' exon splice site for a ribozyme derived from this precursor are complex [Disney, M. D., Haidaris, C. G., and Turner, D. H. (2001) Biochemistry 40, 6507-6519]. These recognition elements have been used to guide design of hexanucleotide mimics of the 5' exon that have backbones modified for nuclease stability. These hexanucleotides bind as much as 100000-fold more tightly to a ribozyme derived from the intron than to a hexanucleotide mimic of the intron's internal guide sequence, r(GGAGGC). Several of these oligonucleotides inhibit precursor self-splicing via a suicide inhibition mechanism. The most promising suicide inhibitor is the ribophosphoramidate rn(GCCUC)rU, which forms more trans-spliced than cis-spliced product at oligonucleotide concentrations of >100 nM at 1 mM Mg(2+). The results indicate that short oligonucleotides modified for nuclease stability can target catalytic RNAs when the elements of tertiary interactions are complex.     

A RecA homologue in Ustilago maydis that is distinct and evolutionarily distant from Rad51 actively promotes DNA pairing reactions in the absence of auxiliary factors

Two RecA homologues have been identified to date in Ustilago maydis. One is orthologous to Rad51 while the other, Rec2, is structurally quite divergent and evolutionarily distant. DNA repair and recombination proficiency in U. maydis requires both Rec2 and Rad51. Here we have examined biochemical activities of Rec2 protein purified after overexpression of the cloned gene. Rec2 requires DNA as a cofactor to hydrolyze ATP and depends on ATP to promote homologous pairing and DNA strand exchange. ATPgammaS was found to substitute for ATP in all pairing reactions examined. With superhelical DNA and a homologous single-stranded oligonucleotide as substrates, Rec2 actively promoted formation and dissociation of D-loops. When an RNA oligonucleotide was substituted it was found that R-loops could also be formed and utilized as primer/template for limited DNA synthesis. In DNA strand exchange reactions using oligonucleotides, we found that Rec2 exhibited a pairing bias that is opposite that of RecA. Single-stranded oligonucleotides were activated for DNA strand exchange when attached as tails protruding from a duplex sequence due to enhanced binding of Rec2. The results indicate that Rec2 is competent, and in certain ways even better than Rad51, in the ability to provide the fundamental DNA pairing activity necessary for recombinational repair. We propose that the emerging paradigm for homologous recombination featuring Rad51 as the essential catalytic component for strand exchange may not be universal in eukaryotes.     

Dual specificity antibodies using a double-stranded oligonucleotide bridge.

The covalent conjugation of oligonucleotides to antibody Fab' fragments was optimized by using oligonucleotides modified with a hexaethylene linker arm bearing three amino groups. One oligonucleotide was coupled to antibody of one specificity and a complementary oligonucleotide to antibody of a second specificity. The antibodies were then allowed to hybridize by base pairing of the complementary nucleotide sequences and the generation of bispecific antibody was analyzed on SDS-PAGE and confirmed using BIAcore analysis. The strategy of complementary oligonucleotide-linked bispecific molecules is not limited to antibodies but is applicable to linking any two molecules of different characteristics.     

Thermoprecipitation of streptavidin via oligonucleotide-mediated self-assembly with poly(N-isopropylacrylamide).

A versatile strategy has been developed for selectively and sequentially isolating targets in a liquid-phase affinity separation environment. The strategy uses a recently developed approach for joining together molecules in linkages that are defined by the complementary pairing of oligonucleotides conjugated to the different molecules [Niemeyer, C. M., Sano, T., Smith, C. L., and Cantor, C. R. (1994) Nucleic Acids Res. 22, 5530-9]. In the work presented here, streptavidin was noncovalently coupled with the temperature-responsive poly(N-isopropylacrylamide) [poly(NIPAAM)] through the sequence-specific hybridization of oligonucleotides conjugated to the protein and polymer. A 20-mer oligonucleotide was covalently linked through a heterobifunctional linker to a genetically engineered streptavidin variant that contained a unique cysteine residue at the solvent-accessible site Glu 116. The complementary DNA sequence was conjugated to the end of a linear ester-activated poly(NIPAAM). The two conjugates were allowed to self-assemble in solution via hybridization of their complementary DNA sequences. The streptavidin-poly(NIPAAM) complex could be used to affinity-precipitate radiolabeled biotin or biotinylated alkaline phosphatase above 32 degrees C through the thermally induced phase separation activity of the poly(NIPAAM). The streptavidin-oligo species could then be reversibly separated from the precipitated polymer-oligo conjugate and recycled by lowering the salt concentration, which results in denaturation of the short double-stranded DNA connection. The use of oligonucleotides to couple polymer to streptavidin allows for selective precipitation of different polymers and streptavidin complexes based on the sequence-specific hybridization of their oligonucleotide appendages.     

Antisense masking reveals contributions of mRNA-rRNA base pairing to translation of Gtx and FGF2 mRNAs

We previously showed that a 9-nucleotide sequence from the 5' leader of the Gtx homeodomain mRNA facilitates translation initiation by base pairing to 18S rRNA. These earlier studies tested the Gtx element in isolation; we now assess the physiological relevance of this element in the context of two natural mRNAs that contain this sequence in their 5' leaders, Gtx itself and FGF2 (fibroblast growth factor 2). 2'-O-Methyl-modified RNA oligonucleotides were employed to block mRNA-rRNA base pairing by targeting either the Gtx-binding site in 18S rRNA or Gtx elements in recombinant mRNAs containing the Gtx or FGF2 5' leaders linked to a reporter cistron. Studies in cell-free lysates and transfected COS-7 cells showed that translation of mRNAs containing the Gtx or FGF2 5' leaders was decreased by > 50% when oligonucleotides targeting either the rRNA or mRNA were used. Specificity was demonstrated by showing that translation of the recombinant mRNAs was unaffected by control oligonucleotides. In addition, the specific oligonucleotides did not affect the translation of recombinant mRNAs in which the Gtx elements were mutated. Experiments performed using constructs containing Gtx and FGF2 5' leader and coding sequences ruled out possible effects of the reporter cistron. Furthermore, two-dimensional gel electrophoresis revealed that the oligonucleotides used in this study had little overall effect on the proteomes of cells transfected with these oligonucleotides. This study demonstrates that mRNA-rRNA base pairing affects the expression of two cellular mRNAs and describes a new approach for investigating putative mRNA-rRNA base pairing interactions in mammalian cells.     

Two sides of the coin: affinity and specificity of nucleic acid interactions

During the past decade, synthetic nucleobase oligomers have found wide use in biochemical sciences, biotechnology and molecular medicine, both as research and/or diagnostic tools and as therapeutics. Numerous applications of common and modified oligonucleotides and oligonucleotide mimics rely on their ability to sequence-specifically recognize nucleic acid targets (DNA or RNA) by forming duplexes or triplexes. In general, these applications would benefit significantly from enhanced binding affinities of nucleobase oligomers in the formation of various secondary structures. However, for high-affinity probes, the selectivity of sequence recognition must also be improved to avoid undesirable associations with mismatched DNA and RNA sites. Here, we review recent progress in understanding the molecular mechanisms of nucleic acid interactions and the development of new high-affinity plus high-specificity oligonucleotides and their mimics, with particular emphasis on peptide nucleic acids.     

Studies on the base pairing properties of deoxyinosine by solid phase hybridisation to oligonucleotides.

Extensive analyses of the base-pairing properties of deoxyinosine to A, C, G, and T were carried out by measuring the hybridisation of oligonucleotides with deoxyinosine in various positions to complementary sets of oligonucleotides made as an array on the surface of a glass microscope slide. With deoxyinosine in internal positions, results are consistent with previous studies, showing a preferential order for pairing of I-C > I-A > I-G approximately I-T. With two adjacent deoxyinosines in the centre of the oligonucleotide, the order in duplex yield is CC > CA > AA > AC > GC > GA > CG > TA > TC > CT = AG > AT > GT > TT. With deoxyinosine at the ends of the oligonucleotide, we find that at the 3' end there is the same order in duplex yield as for the deoxyinosine in internal positions, though with lower discrimination between the bases. When hybridisation is carried out in TMACI there is little base pairing discrimination with deoxyinosine, or indeed any of the four natural bases at the 5' end. Changing the cation to Na+ increased discrimination slightly.     

A combined screening and in silico strategy for the rapid design of integrated downstream processes for process and product-related impurity removal

Integrated designs of chromatographic processes for purification of biopharmaceuticals provides potential gains in operational efficiency and reductions of costs and material requirements. We describe a combined method using screening and in silico algorithms for ranking chromatographic steps to rapidly design orthogonally selective integrated processes for purifying protein therapeutics from both process- and product-related impurities. IFN-α2b produced in Pichia pastoris containing a significant product variant challenge was used as a case study. The product and product-related variants were screened on a set of 14 multimodal, ion exchange, and hydrophobic charge induction chromatography resins under various pH and salt linear gradient conditions. Data generated from reversed-phase chromatography of the fractions collected were used to generate a retention database for IFN-α2b and its variants. These data, in combination with a previously constructed process-related impurity database for P. pastoris, were input into an in silico process development tool that generated and ranked all possible integrated chromatographic sequences for their ability to remove both process and product-related impurities. Top-ranking outputs guided the experimental refinement of two successful three step purification processes, one comprising all bind-elute steps and the other having two bind-elute steps and a flowthrough operation. This approach suggests a new platform-like approach for rapidly designing purification processes for a range of proteins where separations of both process- and product-related impurities are needed.     

High sensitivity LC-MS profiling of antibody-drug conjugates with difluoroacetic acid ion pairing

ABSTRACT

Reversed-phase liquid chromatography (RPLC) separations of proteins using optical detection generally use trifluoroacetic acid (TFA) because it is a strong, hydrophobic acid and a very effective ion-pairing agent for minimizing chromatographic secondary interactions. Conversely and in order to avoid ion suppression, analyses entailing mass spectrometry (MS) detection is often performed with a weaker ion-pairing modifier, like formic acid (FA), but resolution quality may be reduced. To gain both the chromatographic advantages of TFA and the enhanced MS sensitivity of FA, we explored the use of an alternative acid, difluoroacetic acid (DFA). This acid modifier is less acidic and less hydrophobic than TFA and is believed to advantageously affect the surface tension of electrospray droplets. Thus, it is possible to increase MS sensitivity threefold by replacing TFA with DFA. Moreover, we have observed DFA ion pairing to concomitantly produce higher chromatographic resolution than FA and even TFA. For this reason, we prepared and used MS-quality DFA in place of FA and TFA in separations involving IdeS digested, reduced NIST mAb and a proprietary antibody-drug conjugate (ADC), aiming to increase sensitivity, resolution and protein recovery. The resulting method using DFA was qualified and applied to two other ADCs and gave heightened sensitivity, resolution and protein recovery versus analyses using TFA. This new method, based on a purified, trace metal free DFA, can potentially become a state-of-the-art liquid chromatography-MS technique for the deep characterization of ADCs.     

A simple and highly sensitive method for sequence determination of 32P-labeled oligonucleotides

A new procedure is described for the sequence determination of oligonucleotides produced by digestion of RNA with pancreatic RNase A. The oligonucleotide is treated with spleen exonuclease and all intermediates are resolved by thin-layer chromatography on polyethyleneimine plates. On the basis of the increase in mobility it can be decided for each successive step whether a Gp- or an Ap-residue has been removed by reference to a calibration grid. The method is very simple and can easily be applied to a large number of samples. An amount of ³²P-radioactivity corresponding to 40 dpm/nucleotide is sufficient for analysis.     

Use of ultrafiltration/diafiltration for the processing of antisense oligonucleotides

Ultrafiltration/diafiltration (UF/DF) has been the hallmark for concentrating and buffer exchange of protein and peptide-based therapeutics for years. Here we examine the capabilities and limitations of UF/DF membranes to process oligonucleotides using antisense oligonucleotides (ASOs) as a model. Using a 3 kDa UF/DF membrane, oligonucleotides as small as 6 kDa are shown to have low sieving coefficients (<0.008) and thus can be concentrated to high concentrations (≤200 mg/mL) with high yield (≥95%) and low viscosity (<15 centipoise), provided the oligonucleotide is designed not to undergo self-hybridization. In general, the oligonucleotide should be at least twice the reported membrane molecular weight cutoff for robust retention. Regarding diafiltration, results show that a small amount of salt is necessary to maintain adequate flux at concentrations exceeding about 40 mg/mL. Removal of salts along with residual solvents and small molecule process-related impurities can be robust provided they are not positively charged as the interaction with the oligonucleotide can prevent passage through the membrane, even for common divalent cations such as calcium or magnesium. Overall, UF/DF is a valuable tool to utilize in oligonucleotide processing, especially as a final drug substance formulation step that enables a liquid active pharmaceutical ingredient.     

A hybridization procedure for the isolation of specific RNA segments applied to the analysis of bacteriophage Qbeta RNA.

A method for the isolation of RNA fragments originating from defined regions of bacteriophage Qbeta RNA minus strands is described. Large RNase T1 oligonucleotides were isolated on a preparative scale from Qbeta RNA. The nucleotide sequences (13 to 26 nucleotides) and map positions of these oligonucleotides were known from previous work (Billeter, M. A. (1978) J. Biol. Chem. 253, 8381-8389). After addition of AMP residues (50 in the average) using terminal adenylate transferase, these pure oligonucleotides were hybridized to 32P-labeled Qbeta RNA minus strands synthesized in vitro. Fragments in the size range of 100 to 500 nucleotides were then generated by partial digestion with RNase T1. Fragments hybridized to such oligonucleotides were recovered by chromatography on poly(U)-Sephadex and then resolved according to their size by polyacrylamide gel electrophoresis. The specificity and reproducibility of the method as well as its suitability for the sequence analysis of Qbeta RNA was verified by using in particular a linker oligonucleotide derived from a Qbeta RNA region near the 3' end. The sequence catalogues of the RNase T1 and RNase A oligonucleotides of two fragments isolated in this way, 202 and 310 nucleotides in length, were established and all fragments isolated were shown to contain a sequence complementary to the linker oligonucleotide.     

Preparation, purification and analyses of thirteen alkali-stable dinucleotides from ribonucleic acid

Of the 16 alkali-stable dinucleotides known to be obtained by hydrolysis of commercial yeast RNA with alkali, 13 were prepared in quantities of the order of 10mg or more. The samples, with only one exception, contain at least 90% of dinucleotide, and spectroscopic constants and nucleotide-sequence determinations, although not conclusive, indicate a high degree of purity of these products. The small dinucleotide fraction in 150g of RNA hydrolysed with alkali (1-2% of the total nucleotides) was separated from the mononucleotides by stepwise ion-exchange chromatography on DEAE-cellulose columns and resolved into seven fractions containing from one to four different dinucleotides by electrophoresis on paper at pH3.0. These fractions were resolved into their constituent dinucleotides by chromatography in ammonium sulphate. Contamination of the products by impurities from the paper was minimized by washing it before using it for chromatography or electrophoresis and, by using a thick grade of paper (Whatman no. 17), it was possible to handle and purify relatively large quantities of nucleotides.