Ion-pair Reversed-phase Liquid Chromatography Research Articles

Analysis of Self-Assembled Micelle Inhibitory RNA (SAMiRNA) Drug Using Ion-Pairing Reversed-Phase Liquid Chromatography Combined with Mass Spectrometry.

Small interfering RNA (siRNA) is known for its ability to silence the expression of specific genes, demonstrating its promising potential as a therapeutic approach. Self-assembled micelle inhibitory RNA (SAMiRNA) is an oligonucleotide duplex developed to overcome the in vivo delivery limitations of siRNA. SAMiRNA has hydrophilic and hydrophobic groups at both ends of a sense strand, forming a spherical nanostructure that enhances the in vivo delivery efficiency. Ion-pairing reversed-phase liquid chromatography (IP-RPLC) is the most commonly used method for the analysis of oligonucleotides. Since SAMiRNA is heavily chemically modified, the behavior of SAMiRNA in IP-RPLC combined with mass spectrometry (MS) is anticipated to differ from that of the conventional siRNA drug. The current investigation using IP-RPLC-MS revealed that a distinct duplex peak along with two minor separate strands of antisense and sense was observed at column temperatures below 35 °C in the IP-RPLC system with a 100 mM ammonium bicarbonate buffer system. At column temperatures higher than 35 °C, however, two fully denatured single strands were observed. The mass spectrum from the chromatographic peak of the SAMiRNA duplex contained signals from the duplex, the antisense, and the sense, probably due to duplex denaturation during the MS ionization process. The current comprehensive analysis results will make a substantial contribution to the future application of IP-RPLC-MS in the analysis of SAMiRNA.

Stability indicating ion-pair reversed-phase liquid chromatography method for modified mRNA

Modified messenger RNA (mRNA) represents a rapidly emerging class of therapeutic drug product. Development of robust stability indicating methods for control of product quality are therefore critical to support successful pharmaceutical development. This paper presents an ion-pair reversed-phase liquid chromatography (IP-RPLC) method to characterise modified mRNA exposed to a wide set of stress-inducing conditions, relevant for pharmaceutical development of an mRNA drug product. The optimised method could be used for separation and analysis of large RNA, sized up to 1000 nucleotides. Column temperature, mobile phase flow rate and ion-pair selection were each studied and optimised. Baseline separations of the model RNA ladder sample were achieved using all examined ion-pairing agents. We established that the optimised method, using 100 mM Triethylamine, enabled the highest resolution separation for the largest fragments in the RNA ladder (750/1000 nucleotides), in addition to the highest overall resolution for the selected modified mRNA compound (eGFP mRNA, 996 nucleotides). The stability indicating power of the method was demonstrated by analysing the modified eGFP mRNA, upon direct exposure to heat, hydrolytic conditions and treatment with ribonucleases. Our results showed that the formed degradation products, which appeared as shorter RNA fragments in front of the main peak, could be well monitored, using the optimised method, and the relative stability of the mRNA under the various stressed conditions could be assessed.

Optimization of elution conditions and comparison of emerging biocompatible columns on the resolving power and detection sensitivity of oligonucleotides by ion-pairing reversed-phase liquid chromatography mass spectrometry

A generic performance comparison strategy has been developed to evaluate the impact of mobile-phase additives (ion-pairing agent / counter ion systems), distinct stationary phases on resulting resolving power, and MS detectability of oligonucleotides and their critical impurities in gradient IP-RPLC. Stationary-phase considerations included particle type (core-shell vs. fully porous particles), particle diameter, and pore size. Separations were carried out at 60°C to optimize mass transfer (C-term). The incorporation of an active column preheater mitigated thermal mismatches, leading to narrower peaks and overcoming peak splitting. Acetonitrile as organic modifier outweighed methanol in terms of peak-capacity generation and yielded a 30% lower back pressure. Performance screening experiments were conducted varying ion-pairing agents and counter ions, while adjusting gradient span achieved an equivalent effective retention window. Hexafluoromethylisopropanol yielded superior chromatographic resolution, whereas hexafluoroisopropanol yielded significantly higher MS detection sensitivity. The 1.7 µm core-shell particle columns with 100 Å pores provided maximum resolving power for small (15–35 mers) oligonucleotides. Sub-min analysis for 15–35 polyT ladders was achieved operating a 50 mm long column at the kinetic performance limits. High-resolution separations between a 21-mer modified RNA sequence oligonucleotides and its related (shortmer and phosphodiester) impurities and complementary strand were obtained using a coupled column set-up with a total length of 450 mm.

Comprehensive Impurity Profiling of mRNA: Evaluating Current Technologies and Advanced Analytical Techniques.

In vitro transcription (IVT) of mRNA is a versatile platform for a broad range of biotechnological applications. Its rapid, scalable, and cost-effective production makes it a compelling choice for the development of mRNA-based cancer therapies and vaccines against infectious diseases. The impurities generated during mRNA production can potentially impact the safety and efficacy of mRNA therapeutics, but their structural complexity has not been investigated in detail yet. This study pioneers a comprehensive profiling of IVT mRNA impurities, integrating current technologies with innovative analytical tools. We have developed highly reproducible, efficient, and stability-indicating ion-pair reversed-phase liquid chromatography and capillary gel electrophoresis methods to determine the purity of mRNA from different suppliers. Furthermore, we introduced the applicability of microcapillary electrophoresis for high-throughput (<1.5 min analysis time per sample) mRNA impurity profiling. Our findings revealed that impurities are mainly attributed to mRNA variants with different poly(A) tail lengths due to aborted additions or partial hydrolysis and the presence of double-stranded mRNA (dsRNA) byproducts, particularly the dsRNA 3'-loop back form. We also implemented mass photometry and native mass spectrometry for the characterization of mRNA and its related product impurities. Mass photometry enabled the determination of the number of nucleotides of different mRNAs with high accuracy as well as the detection of their size variants [i.e., aggregates and partial and/or total absence of the poly(A) tail], thus providing valuable information on mRNA identity and integrity. In addition, native mass spectrometry provided insights into mRNA intact mass, heterogeneity, and important sequence features such as poly(A) tail length and distribution. This study highlights the existing bottlenecks and opportunities for improvement in the analytical characterization of IVT mRNA, thus contributing to the refinement and streamlining of mRNA production, paving the way for continued advancements in biotechnological applications.

Study of nusinersen metabolites in cerebrospinal fluid of children with spinal muscular atrophy using ultra-high-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry

The study aimed to analyze nusinersen metabolites in the cerebrospinal fluid samples using ion-pair reversed-phase ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. Three different sample preparation methods were...

Liquid Chromatography Methods for Analysis of mRNA Poly(A) Tail Length and Heterogeneity.

Messenger RNA (mRNA) is a new class of therapeutic compounds. The current advances in mRNA technology require the development of efficient analytical methods. In this work, we describe the development of several methods for measurement of mRNA poly(A) tail length and heterogeneity. Poly(A) tail was first cleaved from mRNA with the RNase T1 enzyme. The average length of a liberated poly(A) tail was analyzed with the size exclusion chromatography method. Size heterogeneity of the poly(A) tail was estimated with high-resolution ion-pair reversed phase liquid chromatography (IP RP LC). The IP RP LC method provides resolution of poly(A) tail oligonucleotide variants up to 150 nucleotide long. Both methods use a robust ultraviolet detection suitable for mRNA analysis in quality control laboratories. The results were confirmed by the LC-mass spectrometry (LC MS) analysis of the same mRNA sample. The poly(A) tail length and heterogeneity results were in good agreement.

A single-nucleotide resolution capillary gel electrophoresis workflow for poly(A) tail characterization in the development of mRNA therapeutics and vaccines

The 3′ poly(A) tail is an important component of messenger RNA (mRNA). The length of the poly(A) tail has direct impact on the stability and translation efficiency of the mRNA molecule and is therefore considered to be a critical quality attribute (CQA) of mRNA-based therapeutics and vaccines. Various analytical methods have been developed to monitor this CQA. Methods like ion-pair reversed-phase liquid chromatography (IPRP-LC) can be used to quantify the percentage of mRNA with poly(A) tail but fail to provide further information on the actual length of poly(A). High-resolution methods such as liquid chromatography coupled with mass spectrometry (LC-MS) or next generation sequencing (NGS) can separate poly(A) tail length by one nucleotide (n/n + 1 resolution) but are complicated to implement for release testing of manufactured mRNA. In this study, a workflow utilizing capillary gel electrophoresis (CGE) for characterizing the poly(A) tail length of mRNA was developed. The CGE method demonstrated resolution comparable with the LC-MS method. With UV detection and the addition of poly(A) length markers, this method can provide poly(A) tail length information and can also provide quantitation of each poly(A) length, making it a suitable release method to monitor the CQA of poly(A) tail length.

Applicability of supercritical fluid chromatography for oligonucleotide analysis: A proof-of-concept study

We evaluated the suitability of supercritical fluid chromatography (SFC) for oligonucleotide analysis using 4-mer oligonucleotides with various phosphorothioate (PS) contents as model compounds. Column screening showed that the diol-modified column was able to separate sequences with different PS contents. Optimization of the column body and additives allowed us to analyze polar oligonucleotides using SFC. Various sequences were also analyzed using the optimized method. A good peak shape was obtained when the guanine plus cytosine content of the analyte was two or less in the 4-mer oligonucleotides. Furthermore, we found that the retention times of the selected sequences were positively correlated with polar surface areas, indicating that oligonucleotides interact with polar stationary phases. In contrast, more hydrophobic full PS sequences were retained more strongly in the diol column than the full phosphodiester (PO) sequences. This suggests that the diol column has unique selectivity for PO and PS linkages. These results indicate that SFC is potentially applicable to oligonucleotide analysis with a separation mechanism that is different from that of ion-pair reversed-phase liquid chromatography.

High-Throughput Chromatographic Separation of Oligonucleotides: A Proof of Concept Using Ultra-Short Columns.

Ion-pairing reversed-phase liquid chromatography (IP-RPLC) is the reference separation technique for characterizing oligonucleotides (ONs) and their related impurities. The aim of this study was to better understand the retention mechanism of ONs, evaluate the applicability of the linear solvent strength (LSS) retention model, and explore the potential of ultra-short columns having a length of only 5 mm for the separation of model ONs. First, the validity of the LSS model was evaluated for ONs having sizes comprised between 3 and 30 kDa, and the accuracy of retention time predictions was assessed. It was found that ONs in IP-RPLC conditions follow an "on-off" elution behavior, despite a molecular weight lower than that of proteins. For most linear gradient separation conditions, a column length between 5 and 35 mm was found to be appropriate. Ultra-short columns of only 5 mm were therefore explored to speed up separations by considering the impact of the instrumentation on the efficiency. Interestingly, the impacts of injection volume and post-column connection tubing on peak capacity were found to be negligible. Finally, it was demonstrated that longer columns would not improve selectivity or separation efficiency, but baseline separation of three model ONs mixtures was enabled in as little as 30 s on the 5 mm column. This proof-of-concept work paves the way for future investigations using more complex therapeutic ONs and their related impurities.

Reversed Phase-Liquid Chromatography for Recombinant AAV Genome Integrity Assessment.

After decades of research, gene therapy products have reached market maturity in recent years. Recombinant adeno-associated viruses (rAAVs) are one of the most promising gene delivery vehicles and are currently under intense scientific investigation. These next-generation medicines remain very challenging when it comes to designing appropriate analytical techniques for quality control. One critical quality attribute is the integrity of ssDNA incorporated in these vectors. The genome is the active compound driving rAAV therapy and therefore requires proper assessment and quality control. Current techniques for rAAV genome characterization include next-generation sequencing, quantitative polymerase chain reaction, analytical ultracentrifugation (AUC), and capillary gel electrophoresis (CGE), yet each of them presents their limitations or lack of user-friendliness. In this work, we demonstrate for the first time the potential of ion pairing-reverse phase-liquid chromatography (IP-RP-LC) to characterize the integrity of rAAV genomes. The obtained results were supported by two orthogonal techniques, AUC and CGE. IP-RP-LC can be performed above DNA melting temperatures, avoiding the detection of secondary DNA isoforms, and does not require the use of dyes due to UV detection. We demonstrate that this technique is suitable for batch comparability, different rAAV serotypes (AAV2 and AAV8), internal vs external (inside vs outside the capsid) DNA analysis, and contaminated samples. Overall, it is exceptionally user-friendly, needs limited sample preparation, has high reproducibility, and permits fractionation for further peak characterization. All of these factors add significant value of IP-RP-LC to the analytical toolbox of rAAV genome assessment.

Pressure–Enhanced Liquid Chromatography as a Suitable Approach to Improve Selectivity for Large Molecule Separations

This work describes the application of a pressure-enhanced liquid chromatography (PE-LC) setup to tune the separation of various large molecules comprised of nucleic acids (oligonucleotides, messenger ribonucleic acid [mRNA], and deoxyribonucleic acid [DNA]). When adding pressure as a method development parameter, it indeed becomes possible to modify retention, selectivity, and peak width. As an example, the separation of oligonucleotides having sizes comprised between 40 and 100-mer in ion-pairing reversed-phase liquid chromatography (IP-RPLC) was drastically improved by using a stepwise pressure gradient to selectively shift the retention of a peak or group of peaks. Resolution was increased from 1.5 to 11.8 when setting a rapid high pressure step (twofold increased pressure) during the run. On the other hand, it was also possible to improve the separation of erythropoietin (EPO) mRNA and related impurities under ion-exchange chromatography (IEX). However, with this biomolecule, the best separation was achieved by reducing the pressure in the system. Finally, for another sample (DNA ladder) under IEX conditions, the pressure was found to have a limited impact on the overall selectivity. As highlighted in this work, pressure is an additional parameter that can be successfully used to develop LC methods of large biomolecules.

Polybutylene terephthalate-based stationary phase for ion-pair-free reversed-phase liquid chromatography of small interfering RNA. Part 1: Direct coupling with mass spectrometry

Nowadays, ion-pairing reversed-phase liquid chromatography (IP-RPLC) is the dominating generic method for the analysis of nucleic acid related compounds, such as antisense-oligonucleotides (ASO), small-interfering ribonucleic acid (siRNA) or other DNA or RNA type molecules and their conjugates. Despite of its effective performance, the usage of a high concentration of ion-pairing reagent in the eluent in IP-RPLC is unfavorable for the hyphenation with mass spectrometry (MS) which is required for a detailed structural characterization of the analytes and their structurally related impurities. In this work, we tested a polybutylene terephthalate (PBT)-bonded silica-based stationary phase for the separation of generically synthesized Patisiran as siRNA (antisense and sense single strands as well as their annealed double strand) giving some unexpected selectivity without any presence of ion-pairing reagents. Important chromatographic conditions affecting the separation have been investigated and evaluated. Furthermore, MS and tandem MS (MS/MS) characterization was possible without contamination of the MS system with ion-pair agent and related problems.

Analysis of siRNA with Denaturing and Non-Denaturing Ion-Pair Reversed-Phase Liquid Chromatography Methods

In this paper, we describe denaturing and non-denaturing ion-pair reversed-phase liquid chromatography (RPLC) methods for analyzing small interfering ribonucleic acid (siRNA). Drug formulations consisting of one or two siRNA duplexes were analyzed in non-denaturing conditions at a low column temperature to separate intact duplexes from single-stranded oligonucleotide contaminants and quantify them. In a denaturing method, we used an elevated column temperature to ensure the denaturation of siRNA duplexes into their single-stranded oligonucleotide counterparts. The goal of the denaturing LC method was to investigate the impurities in daughter oligonucleotides in siRNA. A column with chemically modified C18 column hardware showed improvements in analytical performance for nucleic acids compared to a conventional C18 stainless-steel column with the same pore size.

Comprehensive Analyses of the Intracellular and in Vivo Disposition of Fab- Small Interfering RNA Conjugate to Identify Key Issues to Improve Its in Vivo Activity.

Comprehensive analyses of intracellular disposition and in vivo pharmacokinetics were performed for small interfering RNA (siRNA) conjugated with the Fab fragment of panitumumab, a fully humanized monoclonal antibody against epidermal growth factor receptor (EGFR). The Fab-siRNA conjugate was internalized into EGFR-expressing cancer cells in an antigen-dependent manner. Intracellular disposition was quantitatively evaluated using fluorescent-labeled panitumumab and confocal microscopy. The majority of internalized panitumumab was suggested to be transferred into lysosomes. In vivo pharmacokinetics were evaluated in EGFR-expressing tumor-bearing mice. Intact Fab-siRNA was measured by immunoprecipitation using anti-Fab antibody followed by quantitative polymerase chain reaction. The Fab portion was measured by a ligand binding assay. Intact Fab-siRNA concentrations rapidly decreased in the plasma and tumor, although the Fab portion concentration remained high, suggesting extensive degradation in the linker-siRNA portion. After incubation of Fab-siRNA in mouse plasma, samples were digested with proteinase K, and extracted siRNA tagged with Fab-derived peptide was subjected to an ion-pair reversed-phase liquid chromatography with mass spectrometry analysis. Results suggested that hydrolysis from the 3' end of the antisense strand of siRNA is the major metabolizing pathway. Based on these findings, endosomal escape and stability in lysosomes, blood, and tumor are key factors to improve to achieve efficient target gene knockdown in tumors, and stabilizing the 3' end of the antisense strand was suggested to be most efficient. Our approaches clearly identified the key issues of Fab-siRNA from a pharmacokinetics aspect, which will be useful for improving the in vivo activity of siRNA conjugated with not only Fab but also other immunoproteins. SIGNIFICANCE STATEMENT: The intracellular and in vivo disposition of Fab-small interfering RNA (siRNA) conjugate was comprehensively investigated using various approaches, including newly developed analytical methods. This study clearly shows that improvements in siRNA stability in lysosomes, blood, and tumor are needed for target gene knockdown in tumors. The major metabolic pathway of Fab-siRNA is 3' exonuclease degradation, suggesting that optimization of the conjugation site to Fab might help improve stability.

Characterization of Impurities in Therapeutic RNAs at the Single Nucleotide Level.

The chemistry of guide RNA (gRNA) affects the performance of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome editing technique. However, the literature is very scarce about the study of gRNA degradation and in particular at the single nucleotide level. In this work, we developed a workflow to characterize the impurities of large RNAs at the single nucleotide level and identified the residues prone to degradation. Our strategy involves (i) the reduction of RNA length, (ii) a chromatographic mode able to capture subtle changes in impurity polarity, and (iii) a streamlined data treatment. To illustrate the approach, stressed gRNA samples were analyzed by coupling an immobilized ribonuclease T1 cartridge to a hydrophilic interaction liquid chromatography (HILIC) column hyphenated with tandem mass spectrometry (MS/MS). Critical findings were made possible by the presented technology. In particular, the desulfurization of phosphorothioate (PS) linkages was the major degradation observed at the single nucleotide level while no change in purity profile could be observed when using conventional ion-pairing reversed-phase (IPRP) liquid chromatography. To our knowledge, this is the first time that several impurity types are screened for a large RNA molecule using an automated online digestion analysis approach.

Application of heart-cutting two-dimensional liquid chromatography-mass spectrometry to the characterization of highly polar impurities in calcium gluconate injection

The separation and characterization of small polar impurities in polar drugs such as calcium gluconate products are always challenging, due to their poor retention on traditional reversed phase (RP) columns. Although ion-pair reversed-phase liquid chromatography (IPRP-LC) and hydrophilic interaction liquid chromatography (HILIC) are commonly used methods for polar compound analysis, both methods have some drawbacks. For example, IPRP-LC is incompatible with mass spectrometry (MS) due to the presence of non-volatile salts in its mobile phase and HILIC has limited sensitivity due to the poor solubility of polar drugs in the organic-rich sample diluents used in HILIC separations. In order to characterize the highly polar impurities in calcium gluconate injections, a heart-cutting two-dimensional liquid chromatography (2D-LC) method coupled with quadrupole time-of-flight mass spectrometry (Q-TOF/MS) was developed in this study. An IPRP-LC method in the first dimension (1D) provided the selectivity for the separation of polar analytes, using a 100% aqueous mobile phase containing phosphate buffer and ion-pair reagent. Heart cuts of target peaks were collected with sample loops and transferred to the second dimension (2D) HILIC column using an organic-rich mobile phase. In order to solve the mobile phase mismatch problem between the two dimensions, a make-up flow module was introduced in the 2D-LC system to dilute the 1D-water-rich fractions with acetonitrile before entering the sample loops. By optimizing the loop size and dilution factor, good retention and peak shape of the highly polar impurities were obtained on the 2D-HILIC column, and the ion suppression effect for MS detection from the ion-pair reagent and non-volatile salt in the 1D-effluent was minimized. A total of five impurities were identified through fragmentation studies by Q-TOF/MS analysis and their fragmentation pathways were proposed. Four of them were further confirmed by reference substances. This study not only provided useful information for quality control of calcium gluconate injections, but also provided an alternative method for polar impurity characterization in pharmaceuticals.

Temperature Dependence of Retention Behavior of Phosphorylated Peptides in Ion-Pair Reversed-Phase Liquid Chromatography

Temperature Dependence of Retention Behavior of Phosphorylated Peptides in Ion-Pair Reversed-Phase Liquid Chromatography

Phosphorothioate oligonucleotides separation in ion-pairing reversed-phase liquid chromatography: effect of temperature

Analysis of diastereomers of phosphorothioate oligonucleotides in ion-pairing reversed-phase liquid chromatography is affected not only by the character and concentration of ion-pairing system, but also by the separation temperature. In this work, eight ion-pairing systems at two concentrations buffered with acetic acid were used with octadecyl column to investigate the effects of temperature (in the range from 20 °C to 90 °C) on retention, diastereomeric separation, resolution of mers of different length and resolution of oligonucleotides with different number of phosphorothioate linkages. It was observed that elevated temperature suppresses the diastereomeric separation and oligonucleotide peaks become narrower. This improves the resolution of n and n-1 mers at elevated temperature. Plots of ln k (k = retention factor) versus reciprocal absolute temperature show that for 100 mM ion-pairing systems the increase in temperature does not lead to simple decrease in oligonucleotides retention as generally observed in reversed-phase liquid chromatography. The aim of this work is to improve chromatographic method for analysis of phosphorothioate oligonucleotides.

Phosphorothioate oligonucleotides separation in ion-pairing reversed-phase liquid chromatography: Effect of ion-pairing system

Ion-pairing reversed-phase liquid chromatography was utilized for the analysis of native and phosphorothioate oligonucleotides of the identical sequence but different amount and position of phosphorothioate modifications. The effects of ion-pairing amines nature (alkyl chain length, hydrophobicity) and concentration on retention, n/n-1 resolution, and diastereomeric separation of phosphorothioate oligonucleotides were investigated using octadecyl column. Eight different ion-pairing agents at two concentrations (10 mM and 100 mM) buffered with acetic acid were investigated. The resolution of n and n-1 mers oligonucleotides improved with hydrophobicity and concentration of ion-pairing amines. Ion-pairing amines with moderate hydrophobicity were most successful in suppressing diastereomeric resolution. However, a partial separation of phosphorothioate oligonucleotide diastereomers was observed with all ion-pairing systems, which resulted in wider peaks compared to phosphorodiester oligonucleotides of the same sequence. This phenomenon complicates the n and n-1 mers separation of oligonucleotides with high degree of phosphorothioate modifications. Separation of oligonucleotides with different number of phosphorothioate modifications was observed, which may be useful for therapeutic oligonucleotide analysis. The aim of the work was to identify the ion-pairing systems useful for chromatographic characterization of phosphorothioate oligonucleotides.

Advancements in the characterisation of oligonucleotides by high performance liquid chromatography-mass spectrometry in 2021: A short review.

The first oligonucleotide therapeutic was approved by the Food and Drug Administration in 1998, and since then, 12 nucleic acids have been commercialised as medicines. To be approved, the oligonucleotides need to be identified and characterised as well as its related impurities. Different methods exist, but the most commonly used is ion-pairing reversed-phase liquid chromatography with tandem mass spectrometry. The separation obtained depends on the mobile phase and column used. Other methods have been developed, notably by using hydrophilic interaction chromatography and two-dimensional high performance liquid chromatography. Furthermore, ion-pairing reversed-phase high performance liquid chromatography ultra-violet spectroscopy detection and mass spectrometry has been optimised for the analysis of methylated nucleobases due to the utilisation of this modification in the drugs. This review covers the recent advancements in the analysis and characterisation of oligonucleotides in 2021 by high performance liquid chromatography mass spectrometry, notably by hydrophilic interaction chromatography and two-dimensional liquid chromatography but also the different parameters that influence the analysis by ion-pairing reversed-phase high performance liquid chromatography, the characterisation of methylated nucleobases, and the recent software developed for oligonucleotides.