Retention Of Oligonucleotides Research Articles

Separation and identification of oligonucleotides impurities and degradation products by reversed phase ultra-high performance liquid chromatography using phenyl-bonded stationary phases without ion pairs - A step towards sustainability

This manuscript discusses the development of a reversed-phase ultra high-performance liquid chromatography (RP UHPLC) method based on phenyl-bonded stationary phases without ion-pairs for the separation and identification of oligonucleotides. The elimination of ion-pair reagents makes the proposed protocol as more compliant to the principles of green chemistry, compared to the traditional ion-pair reversed-phase liquid chromatography methods (IP RP LC). In detail, three phenyl-based stationary phases were tested, namely a C18/AR (a C18 stationary phase with the addition of aromatic groups), a Phenyl-hexyl, and a Diphenyl. Generally, the retention of oligonucleotides increases with the increase of salt concentration and the decrease of the pH, thus confirming the significant impact of van der Waals interactions, salting-out effect, and π-electrons interactions in the retention mechanism. The highest retention and best peak symmetry were observed for the C18/AR stationary phase, while the lowest retention for the Phenyl-hexyl, with retention influenced by the type of salt in the mobile phase. The obtained methods using C18/AR stationary phases allow for the effective separations of positional isomers and for identifying impurities and degradation products using RP UHPLC Q-TOF-MS in a comparatively short time. The application of RP UHPLC Q-TOF-MS provides reasonable selectivity for the resolution of 33 impurities and two degradation products. Both groups of compounds are mainly 3′N and 5′N-shortmers, but in the case of impurities, modifications of cyclic phosphate and phosphate groups were also identified. Nevertheless, Diphenyl and Phenyl-Hexyl may be applied to separate modified oligonucleotides with higher salt concentrations. The proposed separation methods without ion-pair reagents contribute to a more sustainable approach in oligonucleotide analysis.

Passing Behavior of Oligonucleotides through a Stacked DNA Nanochannel with Featured Path Design.

DNA nanotechnology has emerged as a useful tool for constructing artificial channels penetrating the lipid bilayer. In this work, we introduce a stacked DNA origami nanochannel device characterized by a width-variable pathway, consisting of narrow entrance and exit channels coupled with a wide, modifiable lumen. This design modulates the translocation behavior of oligonucleotides, revealing distinct stages of signal patterns in the recorded current traces. The observed prolonged dwell times indicate oligonucleotide retention, specifically due to the transition from the wide lumen to the narrower exit channel, while variations in current recovery between events suggested intermediate channel states between conducting and blocking. Further, by incorporating sequence-specific overhangs within the channel lumen, we achieved unique asymmetric current profiles during ATP aptamer translocation events. Featured stages also highlighted the aptamer binding dynamics and ATP-induced release. The distinguished oligonucleotide passing behaviors afforded by the stacked DNA origami channel with interior decoration demonstrated the strategic and profitable attempts at DNA nanochannel engineering for nanodevice development and applications.

Impact of ion-pairing systems choice on diastereomeric selectivity of phosphorothioated oligonucleotides in reversed-phase liquid chromatography

Ion-pairing reversed-phase liquid chromatography was utilized for the analysis of native and phosphorothioated oligonucleotides differing in the length (2–6mers and 21mer) and the number and position of phosphorothioate modifications. We investigated the influence of counterion (acetate vs. hexafluoroisopropanol) on the adsorption of eleven alkylamines on the stationary phases. A stronger adsorption of charged alkylamines on octadecyl- and phenyl-based stationary phases led to greater retention of oligonucleotides, and the adsorption of alkylamines was promoted with greater concentration of hexafluoroisopropanol in the mobile phase. Selected amines (triethylamine, dipropylamine, hexylamine) were used to study the resolution of n and n-x mers (main peak and its impurities shortened at 5´end), and diastereomeric separation of phosphorothioated oligonucleotides. The results confirmed a crucial role of alkylamine and counterion choice on the diastereomeric separation. The increasing hydrophobicity of alkylamine led to diminished diastereomeric selectivity which produced narrower phosphorothioated oligonucleotides peaks and led to improved n/n-x separation. Using hexafluoroisopropanol instead of acetate as counterion further enhances this effect (except for 100 mM concentration of hexafluoroisopropanol in combination with highly hydrophobic hexylamine). The elevated column temperature led to suppression of the diastereomeric resolution and improved resolution of n and n-x mers oligonucleotides. Baseline separation of oligonucleotides with different number of phosphorothioate linkages was achieved; this may be useful for therapeutic oligonucleotide analysis.

Selectivity limits of and opportunities for ion pair chromatographic separation of oligonucleotides

Here it was investigated how oligonucleotide retention and selectivity factors are affected by electrostatic and non-electrostatic interactions in ion pair chromatography. A framework was derived describing how selectivity depends on the electrostatic potential generated by the ion-pair reagent concentration, co-solvent volume fraction, charge difference between the analytes, and temperature. Isocratic experiments verified that, in separation problems concerning oligonucleotides of different charges, selectivity increases with increasing surface potential and analyte charge difference and with decreasing co-solvent volume fraction and temperature. For analytes of the same charge, for example, diastereomers of phosphorothioated oligonucleotides, selectivity can be increased by decreasing the co-solvent volume fraction or the temperature and has only a minor dependency on the ion-pairing reagent concentration. An important observation is that oligonucleotide retention is driven predominantly by electrostatic interaction generated by the adsorption of the ion-pairing reagent. We therefore compared classical gradient elution in which the co-solvent volume fraction increases over time versus gradient elution with a constant co-solvent volume fraction but with decreasing ion-pair reagent concentration over time. Both modes decrease the electrostatic potential. Oligonucleotide selectivity was found to increase with decreasing ion-pairing reagent concentration. The two elution modes were finally applied to two different model antisense oligonucleotide separation problems, and it was shown that the ion-pair reagent gradient increases the selectivity of non-charge–based separation problems while maintaining charge-difference–based selectivity.

Ultra-High-Performance Reversed-Phase Liquid Chromatography Hyphenated with ESI-Q-TOF-MS for the Analysis of Unmodified and Antisense Oligonucleotides

The reversed-phase ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry method was developed during the study and successfully applied to separation and analysis of unmodified and antisense oligonucleotides. It was shown that oligonucleotide separation strongly depends on the stationary phase type. The type, pH and salt content in the mobile phase influence also their chromatographic behavior and mass spectrometry sensitivity: increasing the apparent pH causes retention decrease and increase of sensitivity, while increasing the salt concentration produces greater oligonucleotide retention and lower sensitivity. Utilization of octadecyl stationary phase with aryl rings in reversed-phase mode allows successful separation of various modified and unmodified oligonucleotides, differing in length or sequence. The developed method has numerous advantages over current liquid chromatography methods: resolution is comparable to ion pair chromatography; oligonucleotides are analyzed with salts, which allow easy purification and separation without losing column performance, whereas columns run in the presence of ion pair reagents degrade more rapidly. Moreover, the differences in mass spectrometry sensitivity between reversed-phase liquid chromatography and ion pair chromatography were not significant for the studied compounds. The developed method is an effective analytical tool for the determination of oligonucleotide impurities.

Analysis of antisense oligonucleotides with the use of ionic liquids as mobile phase modifiers.

The main goal of this study was the investigation of the impact of several ionic liquids, commonly used as free silanol suppressors, on the retention and separation of phosphorothioate oligonucleotides. Three various stationary phases (octadecyl, octadecyl with embedded polar groups and pentafluorophenyl) as well as ionic liquids with the concentration range of 0.1–7 mM were used for this purpose. The results obtained during this study showed that the increase in concentration of ionic liquids results in increasing retention of the oligonucleotides. Such an effect was observed regardless of the stationary phase used. Moreover, elongation of the alkyl chain in the structure of ionic liquids caused an increase of antisense oligonucleotide retention factors. The results obtained during retention studies confirmed that addition of ionic liquids to the mobile phase influences antisense oligonucleotide retention in a way similar to the case of commonly used ion pair reagents such as amines. A method of oligonucleotide separation was also developed. The best selectivity was obtained for the octadecyl stationary phase since separation of mixtures of antisense oligonucleotides and their metabolites differing in sequence length was successful. It has to be pointed out that ionic liquids were used for the first time as mobile phase additives for oligonucleotide analysis.

Alkylamine ion-pairing reagents and the chromatographic separation of oligonucleotides

Alkylamines are commonly used to improve both chromatographic and mass spectral performance of electrospray ionization liquid chromatography mass spectrometry based methods for the analysis of oligonucleotides. Recently several new alkylamines have been introduced to enhance the electrospray mass spectral response for oligonucleotides; however, the chromatographic properties of these new alkylamines have not been rigorously assessed. We have investigated the retention, peak width, resolution and general chromatographic performance of fifteen different alkylamines for the separation of a model DNA, RNA and an antisense therapeutic oligonucleotide. Eleven of the fifteen alkylamines were shown to provide similar chromatographic performance across all three classes of oligonucleotides. Based on these findings, a model for the mechanism of retention of oligonucleotides using alkylamines and hexafluoroisopropanol mobile phases is proposed. Depending on the concentrations of alkylamines and pH adjustment, oligonucleotides can be retained by micellar chromatography and not the generally held ion-pairing mechanism. This conclusion is supported by light scattering, transmission electron microscopy and ion mobility experiments detecting three micron aggregates in the mobile phase at concentrations that are routinely used for LC–MS analysis of oligonucleotides. These aggregates are not detected at lower alkylamine concentrations where the retention mechanism follows an ion-pairing mechanism. The formation of these aggregates appears to be dependent on the pH of the mobile phase.

A novel strategy for retention prediction of nucleic acids with their sequence information in ion-pair reversed phase liquid chromatography

In this work, retention behaviors of oligonucleotides and double-stranded deoxyribonucleic acids (dsDNAs) have been investigated in ion-pair reversed-phase liquid chromatography (IP-RPLC). We demonstrated that classic linear solvent strength (LSS) model is applicable for describing isocratic retention of oligonucleotides and dsDNAs, which indicated that nucleic acids share the similar retention mechanism as other common small molecules in IP-RPLC. The separation of nucleic acids in IP-RPLC is driven by both hydrophobic and electrostatic interactions. We defined the parameter lnkw/S obtained from LSS model in IP-RPLC as chromatographic hydrophobic and electrostatic interaction index (CHEI). CHEI of a nucleic acid has been revealed to correlate well with its gradient retention time. Notably, we proposed a strategy for retention prediction based on CHEI and base sequence information of nucleic acids. Corresponding to base locus, each base sequence is converted to a featured locus vector consisting of zeros and ones. CHEI prediction models were established by support vector regression (SVR) algorithm with locus vectors. Predicted CHEI values have been applied to predict retention times under desired gradient elution runs. This protocol is easy to grasp and worth pursuing further development for more precise retention prediction performance of nucleic acids in IP-RPLC.

Application of phenyl-based stationary phases for the study of retention and separation of oligonucleotides.

The main goal of our work was to apply three different phenyl-bonded stationary phases in ion pair chromatography for the analysis of synthetic oligonucleotides of various sequences. The influence of the stationary phase structure and the impact of ion-pairing reagent concentration on the retention of oligonucleotides were tested. Moreover the influence of oligonucleotide sequence on their interactions with phenyl-based stationary phases was also investigated. Such complex studies for analysis of oligonucleotides with these adsorbents were done for the first time. Investigations were implemented in the Quantitative Structure Retention Relationships analysis in order to improve the discussion on the retention mechanism of analyzed compounds. The retention of oligonucleotides was the lowest for polar embedded phenyl stationary phase, however its selectivity was high and allowed for complete separation of studied compounds in the shortest time. It was shown that the low retention factor value was observed for oligonucleotides forming secondary structures, such as hairpin loops. Moreover obtained data showed that except for electrostatic and hydrophobic interactions, π-π also influences on the retention mechanism. These interactions cause higher retention factor values for phenyl-based stationary phases compared to octadecyl ones.

The impact of ion-pairing reagents on the selectivity and sensitivity in the analysis of modified oligonucleotides in serum samples by liquid chromatography coupled with tandem mass spectrometry

Present study highlights the usage of various ion-pairing agents and their impact on the process of separation and ionization of oligonucleotides in the fortified human serum samples. What is more, retention studies involved different stationary phases, including: octadecyl, phenyl, pentafluorophenyl groups and ligands with embedded polar groups. It was proved that retention of oligonucleotides strongly depends on the alkyl chain branching in the structure of ion pairing reagent. Furthermore ion-pairing agents build of straight alkyl chain are more easily adsorbed on the stationary phase modified with octadecyl groups, while branching of alkyl chain caused more effective adsorption of studied compounds at phenyl groups compared to octadecyl ones. The lowest limit of quantification values were obtained for 5mMN,N-dimethylbutylamine, while the highest values are characteristic for hexylamine. Moreover it was shown that a 2-fold increase of ion-pairing agent concentration results in higher LOQ. The greatest sensitivity was obtained for 2.5mMN,N-dimethylbutylamine/150mM hexafluoroisopropanol. On the other hand Hypersil GOLD aQ column was the most appropriate in terms of time and separation effectiveness. The developed method was successfully used for the determination of studied oligonucleotides and their metabolites in human serum samples. The compounds were separated in just 3.5min with high sensitivity (0.09–0.16ng).

Retention of nucleic acids in ion-pair reversed-phase high-performance liquid chromatography depends not only on base composition but also on base sequence.

The study on nucleic acid retention in ion-pair reversed-phase high-performance liquid chromatography mainly focuses on size-dependence, however, other factors influencing retention behaviors have not been comprehensively clarified up to date. In this present work, the retention behaviors of oligonucleotides and double-stranded DNAs were investigated on silica-based C18 stationary phase by ion-pair reversed-phase high-performance liquid chromatography. It is found that the retention of oligonucleotides was influenced by base composition and base sequence as well as size, and oligonucleotides prone to self-dimerization have weaker retention than those not prone to self-dimerization but with the same base composition. However, homo-oligonucleotides are suitable for the size-dependent separation as a special case of oligonucleotides. For double-stranded DNAs, the retention is also influenced by base composition and base sequence, as well as size. This may be attributed to the interaction of exposed bases in major or minor grooves with the hydrophobic alky chains of stationary phase. In addition, no specific influence of guanine and cytosine content was confirmed on retention of double-stranded DNAs. Notably, the space effect resulted from the stereostructure of nucleic acids also influences the retention behavior in ion-pair reversed-phase high-performance liquid chromatography.

Different approaches to quantitative structure-retention relationships in the prediction of oligonucleotide retention

Quantitative structure-retention relationships studies were performed for cholesterol and alkylamide stationary phases, which were previously applied in the analysis of nucleotides and oligonucleotides. An octadecyl column was also tested. Twenty-four oligonucleotides of various sequences and length were chosen; next, their structural descriptors were determined with the use of quantum-mechanics method. The sequence features were related mainly to their surface area, hydrophobicity, and the nature of nucleobases. Moreover, for the first time models employing experimentally derived descriptors (the sum of retention factor for individual nucleotides) were developed in the quantitative structure-retention relationship studies of these compounds. The retention of oligonucleotides for alkylamide and cholesterol stationary phases may be effectively predicted with the use of quantitative structure-retention relationship models based only on molecularly modeled descriptors, as well as with models employing experimentally derived descriptors. Therefore, we recommend the first approach, since descriptors may be easily and quickly calculated. However, oligonucleotide retention prediction for octadecyl phases gives better results, when individual nucleotide retention factors are known and utilized for the creation of a mathematical model.

115 Detection of organic carcinogens in water by nanophotonic method

anti-Benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (anti-BPDE), a reactive metabolite of the environmental carcinogen benzo[a]pyrene, predominantly binds to deoxyguanine in DNA and forms four stereoisomeric adducts. Here we developed an improved method for simultaneous analysis and purification of four stereoisomeric adducts in short oligonucleotides using reversed-phase high-performance liquid chromatography, providing a selection strategy of stationary phase for analysis and separation of polyaromatic hydrocarbon–DNA adducts. This work demonstrates that secondary retention of oligonucleotides on C18 stationary phases induced by exposed silanol heavily affects the separation of four stereosiomeric adducts on C18 stationary phases, and the silicone polymer monolayer coating for completely capping exposed silica or silanol greatly reduces such secondary retention, thereby displaying a much better resolution of the four stereoisomeric adducts. We further demonstrate that aromatic group (phenyl)-based stationary phase can significantly improve stereoisomeric separation of four anti-BPDE–deoxyguanosine (dG) adducts in short oligonucleotides over nonaromatic C18 stationary phase due to enhancement of the selective interaction with aromatic anti-BPDE moiety in oligonucleotides. The developed method was also used for purification and preparation of anti-BPDE-oligoncleotide adducts.

A New Combination Strategy as Applied in Predicting Chromatographic Retention Times of Oligonucleotides at a Range of Temperatures from 30 °C to 80 °C

AbstractWe develop a new combination strategy to predict retention times of oligonucleotides in ion‐pair reversed‐phase high performance liquid chromatography. The key step of the strategy is to use score of generalized base properties (SGBP) combined with auto cross covariance (ACC) to resolve the feature representation of nucleic acids. This representation characterize physicochemical, quantumchemical, topological, spatial structural features, etc., and their neighboring effect between bases at a certain distance apart in a sequence. The next step is to use the variables selected by genetic algorithm (GA) to construct prediction models of retention times of oligonucleotides based on support vector machine (SVM). Accordingly, GA‐SVM models give different prediction performance using different input descriptors resulting from different step lengths in the ACC transformation, indicating the neighboring effect between bases should not be neglected in the features related to the chromatographic retention of oligonucleotides. As a whole, the GA‐SVM predictors obtained from more than 20 training samples can produce satisfying performance in predicting the chromatographic retention of oligonucleotides at a range of temperatures (30 °C, 40 °C, 50 °C, 60 °C and 80 °C), respectively. The present approach based on the SGBP‐ACC‐GA‐SVM combination shows great application prospect in the field of separation and analysis science, bioinformatics and proteomics.

Histidine affinity chromatography of homo‐oligonucleotides. Role of multiple interactions on retention

The recent application of histidine-agarose affinity supports in plasmid purification takes advantage of the biorecognition of nucleic acid bases by the histidine ligand. This consideration prompted the need for better understanding the interactions involved in affinity chromatography of plasmid DNA with the histidine-agarose support. In this work, we used synthetic homo-deoxyoligonucleotides with different sizes (1-30 nucleotides long), to explore the effect of several conditions like hydrophobic character of the individual bases, presence of secondary structures, temperature, pH and salt concentration on the mechanism of retention of nucleic acids to histidine-agarose support. One of the most striking results shows that histidine interacts preferentially with guanine, and the presence of secondary structures on polyA and polyG oligonucleotides has a significant influence on retention. Otherwise, the temperature manipulation has not shown a direct influence on oligonucleotide retention, only inducing conformational changes on secondary structures. Overall, the results obtained provide valuable information for the future development and implementation of histidine and other amino acids as ligands in chromatography for the purification of plasmid DNA and other nucleic acids, by improving the knowledge of the interactions involved as well as of the parameters influencing the retention.

A statistical learning approach to the modeling of chromatographic retention of oligonucleotides incorporating sequence and secondary structure data

We propose a new model for predicting the retention time of oligonucleotides. The model is based on ν support vector regression using features derived from base sequence and predicted secondary structure of oligonucleotides. Because of the secondary structure information, the model is applicable even at relatively low temperatures where the secondary structure is not suppressed by thermal denaturing. This makes the prediction of oligonucleotide retention time for arbitrary temperatures possible, provided that the target temperature lies within the temperature range of the training data.We describe different possibilities of feature calculation from base sequence and secondary structure, present the results and compare our model to existing models.

Simultaneous analysis of four stereoisomers of anti-benzo[ a]pyrene diol epoxide–deoxyguanosine adducts in short oligodeoxynucleotides using reversed-phase high-performance liquid chromatography

anti-Benzo[ a]pyrene-7,8-dihydrodiol-9,10-epoxide ( anti-BPDE), a reactive metabolite of the environmental carcinogen benzo[ a]pyrene, predominantly binds to deoxyguanine in DNA and forms four stereoisomeric adducts. Here we developed an improved method for simultaneous analysis and purification of four stereoisomeric adducts in short oligonucleotides using reversed-phase high-performance liquid chromatography, providing a selection strategy of stationary phase for analysis and separation of polyaromatic hydrocarbon–DNA adducts. This work demonstrates that secondary retention of oligonucleotides on C18 stationary phases induced by exposed silanol heavily affects the separation of four stereosiomeric adducts on C18 stationary phases, and the silicone polymer monolayer coating for completely capping exposed silica or silanol greatly reduces such secondary retention, thereby displaying a much better resolution of the four stereoisomeric adducts. We further demonstrate that aromatic group (phenyl)-based stationary phase can significantly improve stereoisomeric separation of four anti-BPDE–deoxyguanosine (dG) adducts in short oligonucleotides over nonaromatic C18 stationary phase due to enhancement of the selective interaction with aromatic anti-BPDE moiety in oligonucleotides. The developed method was also used for purification and preparation of anti-BPDE-oligoncleotide adducts.

Structure–Activity Relationships in Chromatography: Retention Prediction of Oligonucleotides with Support Vector Regression

The prediction of molecular properties for a given molecular structure is of considerable interest for many physicochemical and biochemical processes. Efforts are particularly being made to theoretically predict retention times in the area of chromatographic separations that are based on molecular interactions between the molecules partitioning in separating systems comprised of two phases. Linear free-enthalpy relationships model chromatographic retention as a sum of individual energy contributions (dispersion, dipole–dipole, p–p, proton donor–acceptor interactions, etc.). However, owing to their complex molecular structures, as in the case of biopolymers such as peptides or nucleic acids, use is frequently made of the addition of empirically calculated retention contributions of the individual amino acids or nucleotides that are then corrected by terms that take account of the total structure of the molecule. However, for more complex molecules, this prediction model is imprecise and the relevant descriptors are very complex to determine. Sequence information (apart from the total composition) and information on secondary structures are not considered in these models. Models have been developed for peptides that do not derive retention from the properties of the molecular building blocks, but learn them from data sets obtained with test analyses of known structures. The retention data of approximately 7000 peptides have been used, for example, to train an artificial neural network (ANN) for the prediction of peptide retention times from peptide sequences with an accuracy of 3–10%. Other methods from the field of statistical learning, for example, support vector machines (SVMs), may be used for regression problems. In addition to the advantage of leading exactly to a globally optimal solution (unlike ANNs), support vector approaches have also proved themselves for practical use with chemical problems. A model based on the determination and addition of the retention contributions of the nucleotides has been developed to predict the retention of oligonucleotides in ion-pair reversed-phase chromatography (IP-RPC). This model provided satisfactory results at relatively high separating temperatures (60 8C) for cases in which secondary structures are less pronounced, whereas at lower temperatures, the influence of hairpin or partial double strands led to a poor correlation between the prediction and the experiment (own measurement results). Our model for the retention of oligonucleotides in IPRPC, even at low temperatures, is based on n-support vector regression (SVR) as proposed by Sch:lkopf et al. This method determines a model for a given data set that at the same time minimizes the model error and the model complexity. The training of this model is accomplished with a low number of 50–100 oligonucleotides. A test data set was created by the measurement of the retention times of 72 oligonucleotides. To record the influence of the sequence on the retention, 41 of the oligonucleotide sequences were generated by variation of a sequence of a 24mer (GTA CTC AGT GTA GCC CAG GAT GCC). To take into account other possible secondary structures, four further sequences were selected that form stable hairpin structures even at higher temperatures. The remaining sequences were finally selected so that they covered a length range of 15–48 nucleotides. Quantitative structure–property relationships (QSPR) code the input structures in the form of characteristic vectors. Relevant characteristics for the property to be predicted are, [*] Dipl.-Chem. S. Quinten, Dr. B. M. Mayr, Prof. Dr. C. G. Huber Fachbereich Chemie Instrumentelle Analytik und Bioanalytik Universit0t des Saarlandes Geb0ude B2.2, 66123 Saarbr2cken (Germany) Fax: (+49)681-302-2433 E-mail: christian.huber@mx.uni-saarland.de

Unusual chromatographic behavior of oligonucleotide sequence isomers on two different anion exchange HPLC columns

The retention behavior of the unmodified phosphodiester oligonucleotide sequence isomers was investigated on two different anion exchange columns: Biospher GMB 1000Q (based on DEAE-modified glycidyl methacrylate) and PolyWAX LP (based on silica with a crosslinked coating of linear polyethyleneimine). There was a notable difference in retention of oligonucleotides of the same composition but differing in the position of a single base. The most pronounced difference was observed between the oligonucleotides with the variable base in the end and in the center of the sequence. The use of either acetonitrile or 2-propanol as a mobile phase organic modifier did not markedly affect the retention time patterns. Prediction of the retention times of oligonucleotides must take into account the base position as well as identity. This is the first report of such a “same composition different sequence” effect, described for the short peptides, for synthetic oligonucleotides.

Separation of modified 2′-deoxyoligonucleotides using ion-pairing reversed-phase HPLC

A group of 18-mers of the same base sequence, but with differing alkyl modifications is used to investigate effects of these modifications on retention of oligonucleotides using ion-pairing reversed-phase liquid chromatography (IP-RPLC). It is shown that IP-RPLC is able to distinguish between oligonucleotides differing only by a single alkyl group. The identity of the nucleobase and position and length of the alkyl adduct affect retention of the oligonucleotide. These separation phenomena result from changes in charge and hydrophobicity upon alkylation. As demonstrated in this paper; chromatographic selectivity, unique to IP-RPLC, greatly facilitates the purification process of modified oligonucleotides.