Characterization of small interfering RNA by non-denaturing ion-pair reversed-phase liquid chromatography

Noll, Bernhard; Seiffert, Stephan; Vornlocher, Hans‐Peter; Roehl, Ingo

doi:10.1016/j.chroma.2011.06.057

Cited by 37 publications

(16 citation statements)

References 48 publications

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“…MS has become a method of choice to study structural properties [19][20][21], as well as for quantitative analyses of ODNs. Other chromatographic strategies (utilizing capillary electrophoresis or ion exchange in combination with fluorescence or UV detection [22][23][24][25]) are also reported, although these methods are often incompatible with MS. There are several technical issues associated with LC-MS analysis of ODNs as they are large acidic molecules with limited ionization efficiency in ESI-MS. High polarity of ODNs leads to limited chromatographic retention.…”

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confidence: 99%

Quantitative Analysis of Imetelstat in Plasma with LC–MS/MS Using Solid-Phase or Hybridization Extraction

et al. 2017

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Aim: Imetelstat, a 13-mer oligonucleotide with a lipid tail is being evaluated for treating hematologic myeloid malignancies. This report describes the development of extraction and quantification methods for imetelstat. Methodology & results: Imetelstat was extracted using SPE (rat plasma) or by hybridization using a biotinylated capture probe (human plasma) and was quantified by LC-MS/MS. Calibration curves were established (0.1-50 μg/ml). Stability of imetelstat in plasma was demonstrated. Concentrations of imetelstat extracted using either of the methods and quantified with LC-MS/MS were comparable with a validated ELISA. Conclusion: Two extraction methods (solid phase and hybridization) were developed for quantifying imetelstat in plasma using LC-MS/MS. The hybridization extraction in combination with LC-MS/MS is a novel extraction approach. In recent years, oligonucleotide drugs (ODNs) have gained popularity owing to their ability to target specific genes and provide specificity. ODNs comprise an oligonucleotide sequence, containing 10-50 nucleotides [1,2]. Numerous ODNs have been investigated so far [1][2][3][4]. But only six ODNs (fomivirsen, pegaptanib, mipomersen, eteplirsen, nusinersen and defibrotide sodium) have been approved by the US FDA since 1998 [5].Pharmaceutical development of ODNs has several technical challenges such as maintaining in vivo stability, ensuring delivery and bioavailability, and minimizing off-target effects. These challenges can be minimized by chemical modifications of ODNs to enhance their pharmacokinetic and pharmacodynamic properties [4,6]. These chemical modifications include: sulfurization of the phosphodiester bond to avert degradation by enlindogenous exonucleases, addition of methoxy or methoxyethyl groups to sugar moieties [7,8], locked and unlocked nucleic acids, and alterations in the internucleotide linkage (e.g., amide linkage) resulting in peptide nucleic acids [1]. Sulfurization results in phosphorothioate analogs that are more hydrophobic, exhibit more complex secondary structures with higher protein binding and greater accumulation in organs than phosphodiester analogs [9][10][11].Imetelstat is a novel, first-in-class telomerase inhibitor ODN [12], being investigated for the treatment of hematologic myeloid malignancies [13,14] and certain solid tumors [12,15,16]. Imetelstat is a 13-mer oligonucleotide N3 -P5 thio-phosphoramidate (NPS) with a covalently linked C16 (palmitoyl) lipid moiety at the 5 -end (Figure 1; Supplementary Table 1). Addition of a lipid chain and the modified oligonucleotide backbone enables imetelstat to penetrate cells and tissues, with high biodistribution into normal and malignant cells [12].Imetelstat differs from antisense oligonucleotides in its mechanism of action. Imetelstat is complementary to the template region of the RNA component of telomerase, to which it binds with high affinity and specificity, and directly competes with telomere binding, thereby inhibiting telomerase activity [17]. Thus, imetelstat acts as a classical ac...

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Quantitative Analysis of Imetelstat in Plasma with LC–MS/MS Using Solid-Phase or Hybridization Extraction

et al. 2017

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“…To date, no method has been reported that uses HPLC to resolve any RNAs of the same length, but longer than 25 nt, solely based on structure [24]. In attempts to separate AN59-M1 from AN59-M2 on HPLC, we first tested the effect of magnesium concentration during loading of the sample.…”

Section: Resultsmentioning

confidence: 99%

“…It should be also noted that the buffer we use contains TEA/HFIP (TEA is also an ion-pair agent). TEA/HFIP is a widely used buffer in the analysis of oligonucleotides using HPLC [24; 43; 44]. The use of HFIP is particularly desirable because it is compatible with post-HPLC analysis of RNA species using ESI-MS [24; 43; 44].…”

Section: Discussionmentioning

confidence: 99%

“…TEA/HFIP is a widely used buffer in the analysis of oligonucleotides using HPLC [24; 43; 44]. The use of HFIP is particularly desirable because it is compatible with post-HPLC analysis of RNA species using ESI-MS [24; 43; 44]. Furthermore, HFIP is known to reduce the impact of oligonucleotide hydrophobicity upon retention [33].…”

Section: Discussionmentioning

confidence: 99%

“…The latter technique is less time consuming, and is more suitable for high throughput analysis [20]. To date, ion-pair, reverse-phase HPLC is a standard platform for separation and quantification of a wide range of labeled/unlabeled, regular/modified RNAs [23], such as siRNAs [24; 25; 26], mRNA [27], and large ribosomal RNA (rRNA) [28]. However, HPLC can be used to separate short RNAs (~20 nt) by single-nucleotide resolution [29; 30; 31].…”

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Single-nucleotide resolution of RNAs up to 59 nucleotides by high-performance liquid chromatography

Huang

Jayaseelan

Hebert

et al. 2013

Analytical Biochemistry

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Ion-pair, reverse-phase high performance liquid chromatography (HPLC) is a standard analytical platform for separating, purifying and analyzing RNAs. However, a single-nucleotide resolution by using HPLC is currently limited to RNAs shorter than 25 nucleotides (nt). Here we describe a method of separating three RNA aptamers with 57, 58 and 59 nt on an XBridge ion-pair, reverse-phase HPLC column by a single-nucleotide resolution. Under a similar condition, we also show the capability of our method to resolve two structurally different, yet sequence or mass identical 59-nt aptamers. We establish that the optimal condition to achieve a single-nucleotide resolution correlates to 50 °C and zero magnesium concentration in mobile phases. The ion-pairing agent, the buffer and the solvent we use are also compatible for post-HPLC analysis such as mass spectrometry. Therefore, our method provides a new way of detecting, analyzing and separating RNAs by conformation or structure, and extends the ability of separating RNAs that are longer than 25-nt in length by single-nucleotide resolution.

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Evidence for Rod‐Shaped DNA‐Stabilized Silver Nanocluster Emitters

et al. 2013

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Characterization of small interfering RNA by non-denaturing ion-pair reversed-phase liquid chromatography

Cited by 37 publications

References 48 publications

Quantitative Analysis of Imetelstat in Plasma with LC–MS/MS Using Solid-Phase or Hybridization Extraction

Quantitative Analysis of Imetelstat in Plasma with LC–MS/MS Using Solid-Phase or Hybridization Extraction

Single-nucleotide resolution of RNAs up to 59 nucleotides by high-performance liquid chromatography

Evidence for Rod‐Shaped DNA‐Stabilized Silver Nanocluster Emitters

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