Influence of oligonucleotides structures for separation of diastereomers by capillary electrophoresis method using polyvinylpyrrolidone 1,300,000.

In the field of oligonucleotides drug discovery, phosphorothioate (PS) modification has been recognized as an effective tool to overcome the nuclease digestion, and generates 2n of possible diastereomers, where n equals the number of PS linkages. However, it is also well known that differences in drug efficacy and toxicity are caused by differences in stereochemistry of oligonucleotides. Therefore, the development of a high-resolution analytical method that enables stereo discrimination of oligonucleotides is desired. Under this circumstance, capillary electrophoresis (CE) using polyvinylpyrrolidone (PVP) is considered as one of the useful tools for the separation analysis of diastereomers. In this study, we evaluated the several oligonucleotides with the structural diversities in order to understand the separation mechanism of the diastereomers by CE. Especially, five kinds of 2'-moieties were deeply examined by CE with PVP 1,300,000 polymer solution. We found that different trend of the peak shapes and the peak resolution were observed among these oligonucleotides. For example, the better peak resolution was observed in 6 mer PS3-DNA compared to the rigid structure of 6 mer PS3-LNA. As for this reason, the computational simulation revealed that difference of accessible surface area caused by the steric structure of thiophosphate in each oligonucleotide is one of the key attributes to explain the separation of the diastereomers. In addition, we achieved the separation of sixteen peak tops of the diastereomers in 6 mer PS4-DNA, and the complete separation of fifteen diastereomers in 6 mer PS4-RNA. These knowledge for the separation of the diastereomers by CE will be expected to the quality control of the oligonucleotide drugs.

"Bind, cleave and leave": multiple turnover catalysis of RNA cleavage by bulge-loop inducing supramolecular conjugates.

Antisense sequence-specific knockdown of pathogenic RNA offers opportunities to find new solutions for therapeutic treatments. However, to gain a desired therapeutic effect, the multiple turnover catalysis is critical to inactivate many copies of emerging RNA sequences, which is difficult to achieve without sacrificing the sequence-specificity of cleavage. Here, engineering two or three catalytic peptides into the bulge-loop inducing molecular framework of antisense oligonucleotides achieved catalytic turnover of targeted RNA. Different supramolecular configurations revealed that cleavage of the RNA backbone upon sequence-specific hybridization with the catalyst accelerated with increase in the number of catalytic guanidinium groups, with almost complete demolition of target RNA in 24 h. Multiple sequence-specific cuts at different locations within and around the bulge-loop facilitated release of the catalyst for subsequent attacks of at least 10 further RNA substrate copies, such that delivery of only a few catalytic molecules could be sufficient to maintain knockdown of typical RNA copy numbers. We have developed fluorescent assay and kinetic simulation tools to characterise how the limited availability of different targets and catalysts had restrained catalytic reaction progress considerably, and to inform how to accelerate the catalytic destruction of shorter linear and larger RNAs even further.

Bio-orthogonal chemistry enables solid phase synthesis and HPLC and gel-free purification of long RNA oligonucleotides.

Solid phase synthesis of RNA oligonucleotides which are over 100-nt in length remains challenging due to the complexity of purification of the target strand from failure sequences. This work describes a non-chromatographic strategy that will enable routine solid phase synthesis of long RNA strands.

Oligonucleotides Containing 1-Aminomethyl or 1-Mercaptomethyl-2-deoxy-d-ribofuranoses: Synthesis, Purification, Characterization, and Conjugation with Fluorophores and Lipids.

Oligonucleotide conjugates are widely used as therapeutic drugs, gene analysis, and diagnostic tools. A critical step in the biologically relevant oligonucleotide conjugates is the design and synthesis of functional molecules that connect oligonucleotide with ligands. Here, we report the synthesis and application for oligonucleotide functionalization of novel tethers based on aminomethyl and mercaptomethyl sugar derivatives. Starting from a common cyano sugar precursor, three novel phosphoramidites have been prepared in the two α- and ß-anomeric forms. The mercaptomethyl sugar was protected with the S-acetyl group, while two different protecting groups have been developed for the aminomethyl sugar. These two protecting groups are orthogonal, as they can be removed independently using photolysis or ammonolysis. This combination allowed the introduction of two different ligands in a single oligonucleotide.

Impact of stationary-phase pore size on chromatographic performance using oligonucleotide separation as a model.

A combined experimental and theoretical study was performed to understand how the pore size of packing materials with pores 60-300 Å in size affects the separation of 5-50-mer oligonucleotides. For this purpose, we developed a model in which the solutes were described as thin rods to estimate the accessible surface area of the solute as a function of the pore size and solute size. First, an analytical investigation was conducted in which we found that the selectivity increased by a factor of 2.5 when separating 5- and 15-mer oligonucleotides using packing with 300 Å rather than 100 Å pores. We complemented the analytical investigation by theoretically demonstrating how the selectivity is dependent on the column's accessible surface area as a function of solute size. In the preparative investigation, we determined adsorption isotherms for oligonucleotides using the inverse method for separations of a 9- and a 10-mer. We found that preparative columns with a 60 Å-pore-size packing material provided a 10% increase in productivity as compared with a 300 Å packing material, although the surface area of the 60 Å packing is as much as five time larger.

A Multi-Endpoint Approach to Base Excision Repair Incision Activity Augmented by PARylation and DNA Damage Levels in Mice: Impact of Sex and Age.

Investigation of processes that contribute to the maintenance of genomic stability is one crucial factor in the attempt to understand mechanisms that facilitate ageing. The DNA damage response (DDR) and DNA repair mechanisms are crucial to safeguard the integrity of DNA and to prevent accumulation of persistent DNA damage. Among them, base excision repair (BER) plays a decisive role. BER is the major repair pathway for small oxidative base modifications and apurinic/apyrimidinic (AP) sites. We established a highly sensitive non-radioactive assay to measure BER incision activity in murine liver samples. Incision activity can be assessed towards the three DNA lesions 8-oxo-2'-deoxyguanosine (8-oxodG), 5-hydroxy-2'-deoxyuracil (5-OHdU), and an AP site analogue. We applied the established assay to murine livers of adult and old mice of both sexes. Furthermore, poly(ADP-ribosyl)ation (PARylation) was assessed, which is an important determinant in DDR and BER. Additionally, DNA damage levels were measured to examine the overall damage levels. No impact of ageing on the investigated endpoints in liver tissue were found. However, animal sex seems to be a significant impact factor, as evident by sex-dependent alterations in all endpoints investigated. Moreover, our results revealed interrelationships between the investigated endpoints indicative for the synergetic mode of action of the cellular DNA integrity maintaining machinery.

Retention and diffusion characteristics of oligonucleotides in a solid phase with polymer grafted anion-exchanger.

In the chromatographic separation process of oligonucleotides (ONs), mechanistic understanding of their binding and diffusion processes is of significant importance to determine operating conditions in a fast and robust way. In this work, we determined the number of binding sites and the diffusivities of ONs in a polymer grafted anion exchange chromatography through linear gradient experiments (LGE) being carried out at selected four to five gradient slopes. Synthetic poly (T)s with length ranging from 3 to 90-mer were employed as a model of an antisense oligonucleotide with typical lengths of 10 - 30 bases. Comparison of the retention was also conducted between the grafted anion exchanger with a conventional ligand and an anion monolith disk. For the ONs up to 50 bases, the number of binding sites determined can be correlated with the length of ONs, and the grafted resin showed a better diffusion and narrower peak width compared to the nongrafted one. The retention behavior became similar for porous media when the longer ONs (> 50mer) were applied. The results obtained suggest that antisense ONs can be separated with grafted ligands without sacrificing mass transfer properties.

Multiple heart-cutting mixed-mode chromatography-reversed-phase 2D-liquid chromatography method for separation and mass spectrometric characterization of synthetic oligonucleotides.

Until today, ion-pair reversed-phase chromatography is still the dominating method for analytical characterization of synthetic oligonucleotides. Its hyphenation with mass spectrometry, however, has some drawbacks such as ion-suppression in electrospray ionization. To overcome this problem, we present in this work a multiple heart-cutting (MHC) two-dimensional liquid chromatography (2D-LC) method with ultra-violet (UV) and electrospray ionization (ESI) mass spectrometry (MS) detection. A reversed-phase/weak anion-exchange (RP/WAX) stationary phase in the first dimension (1D) provides the selectivity for separation of structurally closely related oligonucleotide sequences and deletions (shortmers), respectively, using a mixed pH/triethylammonium phosphate buffer gradient at constant organic modifier content. Heart cuts of the oligonucleotide peaks are transferred to the second dimension (2D) via a multiple heart-cutting valve which is equipped with two loop decks. The 2D RP column is used for desalting via a diverter valve. Active solvent modulation enables to refocus the oligonucleotide peak into a sharp zone by 2D RP entirely free of non-volatile buffer components and ion-pair agents. Oligonucleotides can thus be sensitively detected by ESI-QTOF-MS under MS-compatible conditions.

Oligonucleotides: Current Trends and Innovative Applications in the Synthesis, Characterization, and Purification.

Oligonucleotides (ONs) are gaining increasing importance as a promising novel class of biopharmaceuticals. Thanks to their fundamental role in gene regulation, they can be used to develop custom-made drugs (also called N-to-1) able to act on the gene expression at pre-translational level. With recent approvals of ON-based therapeutics by the Food and Drug Administration (FDA), a growing demand for high-quality chemically modified ONs is emerging and their market is expected to impressively prosper in the near future. To satisfy this growing market demand, a scalable and economically sustainable ON production is needed. In this paper, the state of the art of the whole ON production process is illustrated with the aim of highlighting the most promising routes toward the auspicated market-size production. In particular, the most recent advancements in both the upstream stage, mainly based on solid-phase synthesis and recombinant technology, and the downstream one, focusing on chromatographic techniques, are reviewed. Since ON production is projected to expand to the large scale, automatized multicolumn countercurrent technologies will reasonably be required soon to replace the current ones based on batch single-column operations. This consideration is supported by a recent cutting-edge application of continuous chromatography for the ON purification.

Effect of Pressure Increase on Macromolecules' Adsorption in Ion Exchange Chromatography.

In this study a new method for evaluating the pressure effect on separations of oligonucleotides and proteins on an anion exchange column was developed. The pressure rise of up to 500 bar was attained by coupling restriction capillaries to the column outlet to minimize differences in pressure over the column. Using pH transient measurements it was demonstrated that no shift in ion exchange equilibria occurs due to a pressure increase. Results from isocratic and gradient separations of oligonucleotides (model compounds) were evaluated by stoichiometric displacement and linear gradient elution model, respectively. Both elution modes demonstrated that for smaller oligonucleotides the number of binding sites remained unchanged with pressure rise while an increase for large oligonucleotides was observed, indicating their alignment over the stationary phase. From the obtained model parameters and their pressure dependencies, a thermodynamic description was made and compared between the elution modes. A complementary pattern of a linear increase of partial molar volume change with a pressure rise was established. Furthermore, estimation of the pressure effect was performed for bovine serum albumin and thyroglobulin that required gradient separations. Again, a raise in binding site number was found with pressure increase. The partial molar volume changes of BSA and Tg at the maximal investigated pressure and minimal salt concentration were -31.6 and -34.4 cm3/mol, respectively, indicating a higher rigidity of Tg. The proposed approach provides an insight into the molecule deformation over a surface at high pressures under nondenaturing conditions. The information enables a more comprehensive UHPLC method development.

Broadly applicable oligonucleotide mass spectrometry for the analysis of RNA writers and erasers in vitro.

RNAs are post-transcriptionally modified by dedicated writer or eraser enzymes that add or remove specific modifications, respectively. Mass spectrometry (MS) of RNA is a useful tool to study the modification state of an oligonucleotide (ON) in a sensitive manner. Here, we developed an ion-pairing reagent free chromatography for positive ion detection of ONs by low- and high-resolution MS, which does not interfere with other types of small compound analyses done on the same instrument. We apply ON-MS to determine the ONs from an RNase T1 digest of in vitro transcribed tRNA, which are purified after ribozyme-fusion transcription by automated size exclusion chromatography. The thus produced tRNAValAAC is substrate of the human tRNA ADAT2/3 enzyme and we confirm the deamination of adenosine to inosine and the formation of tRNAValIACin vitro by ON-MS. Furthermore, low resolution ON-MS is used to monitor the demethylation of ONs containing 1-methyladenosine by bacterial AlkB in vitro. The power of high-resolution ON-MS is demonstrated by the detection and mapping of modified ONs from native total tRNA digested with RNase T1. Overall, we present an oligonucleotide MS method which is broadly applicable to monitor in vitro RNA (de-)modification processes and native RNA.

Open-tubular nanoelectrochromatography (OT-NEC): gel-free separation of single stranded DNAs (ssDNAs) in thermoplastic nanochannels.

Electrophoresis or electrochromatography carried out in nanometer columns (width and depth) offers some attractive benefits compared to microscale columns. These advantages include unique separation mechanisms that are scale dependent, fast separation times, and simpler workflow due to the lack of a need for column packing and/or wall coatings to create a stationary phase. We report the use of thermoplastics, in this case PMMA, as the substrate for separating single-stranded DNAs (ssDNAs). Electrophoresis nanochannels were created in PMMA using nanoimprint lithography (NIL), which can produce devices at lower cost and in a higher production mode compared to the fabrication techniques required for glass devices. The nanochannel column in PMMA was successful in separating ssDNAs in free solution that was not possible using microchip electrophoresis in PMMA. The separation could be performed in <1 s with resolution >1.5 when carried out using at an electric field strength of 280 V/cm and an effective column length of 60 µm (100 nm × 100 nm, depth and width). The ssDNAs transport through the PMMA column was driven electrokinetically under the influence of an EOF. The results indicated that the separation was dominated by chromatographic effects using an open tubular nano-electrochromatography (OT-NEC) mode of separation. Interesting to these separations was that no column packing was required nor a wall coating to create the stationary phase; the separation was affected using the native polymer that was UV/O3 activated and an aqueous buffer mobile phase.

Nuclear and Cytoplasmatic Quantification of Unconjugated, Label-Free Locked Nucleic Acid Oligonucleotides.

Methods for the quantification of antisense oligonucleotides (AONs) provide insightful information on biodistribution and intracellular trafficking. However, the established methods have not provided information on the absolute number of molecules in subcellular compartments or about how many AONs are needed for target gene reduction for unconjugated AONs. We have developed a new method for nuclear AON quantification that enables us to determine the absolute number of AONs per nucleus without relying on AON conjugates such as fluorophores that may alter AON distribution. This study describes an alternative and label-free method using subcellular fractionation, nucleus counting, and locked nucleic acid (LNA) sandwich enzyme-linked immunosorbent assay to quantify absolute numbers of oligonucleotides in nuclei. Our findings show compound variability (diversity) by which 247,000-693,000 LNAs/nuclei results in similar target reduction for different compounds. This method can be applied to any antisense drug discovery platform providing information on specific and clinically relevant AONs. Finally, this method can directly compare nuclear entry of AON with target gene knockdown for any compound design and nucleobase sequence, gene target, and phosphorothioate stereochemistry.

LC-MS quantification of oligonucleotides in biological matrices with SPE or hybridization extraction.

Aim: Quantitative LC-MS analysis of oligonucleotides (OGNs) in biological matrices is needed to support candidate selection of new therapeutic OGNs. Methodology & results: A set of 20 single stranded antisense oligonucleotides (ASO) and five siRNAs were extracted from plasma and tissue homogenates. Anion Exchange (AEX) SPE was selected as generic extraction approach, resulting in recoveries from plasma >70%. Extraction from tissue homogenates showed often more variation and lower recoveries. A proof of concept of a novel tailored hybridization extraction is demonstrated for two 16-mer reference OGNs. Conclusion: Two methods for extraction of OGNs were investigated and applied for quantitative analysis. The AEX-SPE is considered a more generic approach preferred when multiple compounds are evaluated. Hybridization extraction has great potential but critical reagents per analyte are needed.

Methods to Study Trinucleotide Repeat Instability Induced by DNA Damage and Repair.

Trinucleotide repeat (TNR) instability (expansion and deletion) is associated with more than 42 human neurodegenerative diseases and cancer and mediated by DNA replication, repair, recombination, and gene transcription. Somatic TNR instability is involved in the progression of TNR expansion diseases and can be modulated by DNA damage repair and gene transcription. Recent studies from our group and others have shown that DNA base damage and its repair play an active role in modulating TNR instability and are responsible for somatic age-dependent CAG repeat expansion in neurons of Huntington's disease mice induced by oxidative DNA damage. However, it remains to be elucidated how DNA damage, non-B form DNA structures, and DNA repair enzymes and cofactors can coordinate to regulate somatic TNR instability. Understanding the molecular mechanisms underlying DNA damage and repair-mediated somatic TNR instability is critically important for identification of new therapeutic targets for treatment and prevention of TNR-related diseases. Here we describe the methods to study the locations and distribution of DNA base lesions and their effects on TNR instability through DNA base excision repair in in vitro reconstituted human systems.

A review on native and denaturing purification methods for non-coding RNA (ncRNA).

Recently, non-coding RNA (ncRNA) became the centerpiece of human genome research. Modern ncRNA-based research has revolutionized disease diagnosis and therapeutics. However, decoding structural/functional information of ncRNA requires large amount of pure RNA, and hence effective RNA preparation and purification protocols. This review focuses on purification schemes of synthetic oligonucleotides, particularly liquid chromatographic (LC) techniques as improved alternatives to urea-polyacrylamide gel electrophoresis (urea-PAGE) purification. Moreover, the review summarizes the shortcomings of urea-PAGE purification method and details the chromatographic purification such as affinity, ion-exchange (IE) or size-exclusion (SE) chromatography. Specifically, we discuss denaturing and native RNA purification schemes with newest developments. In short, the review evaluates nucleic acid purification schemes required for various structural analyses.

Diastereomers of a mono-substituted phosphoryl guanidine trideoxyribonucleotide: Isolation and properties.

Recently, a new type of nucleic acid analogues with modified phosphate group, namely, phosphoryl guanidine oligonucleotides, has been described. In the present work, we assess the difference between diastereomers of a mono-substituted phosphoryl guanidine oligonucleotide and analyze their resistance to nuclease digestion. Individual diastereomers ('fast' and 'slow') of a trideoxynucleotide d (TpCp*A) were isolated by reverse-phase HPLC. Snake venom phosphodiesterase digestion showed that the native trideoxynucleotide was fully degraded after 30 min, whereas both 'fast' and 'slow' diastereomers of d (TpCp*A) were not completely digested even after 7 days. UV and CD spectra revealed similarities in the structure of the diastereomers. Structural analysis by 1D and 2D NMR spectroscopy also uncovered significant similarity in the properties of Rp and Sp diastereomers. Structural analysis of nuclear Overhauser effect spectroscopy (NOESY) data and restrained molecular dynamics methods showed very flexible single-stranded oligonucleotide structures. Detailed computational analysis of restraint penalty energies via restrained molecular dynamics simulations with the 2D NMR interproton distance data allowed us to conclude that most likely, the 'fast' isomer is the Sp diastereomer, and the 'slow' isomer is the Rp diastereomer.

Effect of pressure on the retention of macromolecules in ion exchange chromatography.

Shorter analysis times and greater resolving power are contributing factors for transfer of separation methods from an HPLC to a UHPLC system when performing analysis in biopharmaceutical or clinical research. The effect of pressure on separations in reversed phase chromatography is well described, however such investigations on ion exchange columns were previously not conducted. In this study we describe the effect of pressure on retention properties of proteins, oligonucleotides and plasmid DNA in ion exchange chromatography. Different column inlet pressures were obtained by coupling restriction capillaries with column outlet and performing separations at a constant temperature and mobile phase flow rate. Macromolecules were separated in isocratic mode as well as with various linear gradients of salt concentration at a constant pH value. The measured retention time increase was up to 80% for isocratic and 20% for gradient separations for a 500 bar increase in pressure. The effect of pressure was validated on a separate instrument after few months from initial experiments. The influence of pressure on retention properties seems to be dependent on the size, shape and flexibility of the macromolecule and causes different retention shifts when separating a sample with diverse analytes. Such changes in retention time can sometimes exceed the criteria set by European Pharmacopoeia (Ph. Eur.) for the allowable method adjustment and are thus considered to be a result of a different separation method. Therefore, the pressure effect that follows method transfer from HPLC to UHPLC conditions should not be neglected even for gradient separations in ion exchange chromatography, as the resulting retention change may cause revalidation of the separation method.

Covalent Fluorophore Labeling of Oligonucleotides and Generation of Other Oligonucleotide Bioconjugates.

Oligonucleotide conjugates have already reached considerable importance in life science research and oligonucleotide drug development. Since the preparation of oligonucleotide conjugates depends critically on the chemical nature of the used ligand and linker, there is no general and universal procedure. Here, we present a detailed, quick, and facile protocol for attaching fluorescent dyes or cross-linkers of variable chemical stability to oligonucleotides at 3'- or 5'-aminoalkyl handles. Purification and removal of educts and side-products and structural verification by gel electrophoresis and mass spectrometry are presented. Aspects for adapting this protocol for other reaction sites at the oligonucleotide are discussed. We highlight important issues for generating oligonucleotide conjugates with other molecules, including peptide, proteins, and small molecules for receptor-targeting applications. The methodology is suitable for oligonucleotides with various modifications, including stabilized antisense, siRNAs, and miRNAs.

Synthetic Oligonucleotides in SPECT/CT In Vivo Imaging: Chemical Modifications, In111 Complex Formation, Incorporation into Drug Delivery Systems.

Here in we describe a solid phase synthesis of oligonucleotides bearing unnatural moiety appropriate for complex formation with In111 as well as their deprotection, isolation, and purification. We also present methods for oligonucleotides/In111 complex formulation with single and double stranded oligonucleotides of RNA nature and give an example of preparation method for one supramolecular drug delivery system (DDS) consisting of radiolabeled siRNA and positively charged peptide.