The use of strong anion-exchange (SAX) magnetic particles for the extraction of therapeutic siRNA and their analysis by liquid chromatography/mass spectrometry

被引:13
|
作者
Ye, Guofeng [1 ]
Beverly, Michael [1 ]
机构
[1] NIBR Inc, RNAi Therapeut, NIBR Biol Ctr, Cambridge, MA 02139 USA
关键词
SINGLE-STEP METHOD; PLASMID DNA; RNA ISOLATION; PHOSPHOROTHIOATE OLIGONUCLEOTIDES; MASS-SPECTROMETRY; BIOLOGICAL-FLUIDS; CHLOROFORM; PURIFICATION; SEPARATION; ANTISENSE;
D O I
10.1002/rcm.5221
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Traditional methods for extracting oligonucleotides from serum and other biological fluids are often time-consuming and require multiple steps. Magnetic particle based separation of oligonucleotides has gained importance recently due to the advantages of simplicity and high efficiency. Here we report the development and optimization of commercially available strong anion-exchange (SAX) magnetic beads for the extraction of siRNA from human serum. The beads allowed for rapid extraction of siRNA from human serum in 100-200 mu L of liquid chromatography/mass spectrometry (LC/MS)-compatible buffer in less than 1 h for a 96-well plate with no further drying steps. Due to the strong cation-binding properties of oligonucleotides, volatile ammonium salts such as triethylammonium bicarbonate (TEAB), ammonium bicarbonate, and NH4Cl were used to elute the siRNA from the beads. For more hydrophobic siRNA sequences, the addition of 5-10% organic solvent was required for elution. The recovery of chemically modified siRNA from human serum was around 80% for two types of beads examined; however, the recovery for highly modified sequences differed greatly between the two types of beads. In addition to extracting highly modified oligonucleotides, the SAX beads were also able to extract liposomal formulated siRNAs from serum with no interference from the lipid formulation. The extraction of siRNA from human serum was linear over the tested range of 50 ng/mL to 10 mu g/mL. Using this extraction methodology, we have created a workflow to monitor siRNA serum stability by LC/MS. Initial observations confirm that RNase A type degradation with strand cleavage on the 3' side of uridine or cytosine is the dominant cleavage pattern in serum. This finding has implications for the selection and modification of therapeutic siRNAs and demonstrates the utility of magnetic beads as a simple and rapid extraction technique for siRNA. Copyright (C) 2011 John Wiley & Sons, Ltd.
引用
收藏
页码:3207 / 3215
页数:9
相关论文
共 50 条
  • [1] Strong anion-exchange liquid chromatography coupled with isotope ratio mass spectrometry using a Liquiface interface
    Morrison, Douglas J.
    Taylor, Karen
    Preston, Tom
    RAPID COMMUNICATIONS IN MASS SPECTROMETRY, 2010, 24 (12) : 1755 - 1762
  • [2] ADAPTATION OF A THERMOSPRAY LIQUID-CHROMATOGRAPHY MASS-SPECTROMETRY INTERFACE FOR USE WITH ALKALINE ANION-EXCHANGE LIQUID-CHROMATOGRAPHY OF CARBOHYDRATES
    SIMPSON, RC
    FENSELAU, CC
    HARDY, MR
    TOWNSEND, RR
    LEE, YC
    COTTER, RJ
    ANALYTICAL CHEMISTRY, 1990, 62 (03) : 248 - 252
  • [4] A Hydrophilic Strong Anion-Exchange Hybrid Monolith for Capillary Liquid Chromatography
    Liu, Jun
    Weng, Qianfeng
    Li, Jinxiang
    LCGC EUROPE, 2022, 35 (08) : 319 - 324
  • [5] Integration of microfiltration and anion-exchange nano particles-based magnetic separation with MALDI mass spectrometry for bacterial analysis
    Li, Shuping
    Guo, Zhongxian
    Liu, Ying
    Yang, Zhaoguang
    Hui, Hui Kim
    TALANTA, 2009, 80 (01) : 313 - 320
  • [6] Determination of strong binding chelators and their metal complexes by anion-exchange chromatography and inductively coupled plasma mass spectrometry
    Ammann, AA
    JOURNAL OF CHROMATOGRAPHY A, 2002, 947 (02) : 205 - 216
  • [7] ANALYSIS FOR DAMINOZIDE IN APPLE JUICE BY ANION-EXCHANGE CHROMATOGRAPHY PARTICLE BEAM MASS-SPECTROMETRY
    KIM, IS
    SASINOS, FI
    STEPHENS, RD
    BROWN, MA
    JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, 1990, 38 (05) : 1223 - 1226
  • [8] HIGH-PERFORMANCE ANION-EXCHANGE CHROMATOGRAPHY THERMOSPRAY MASS-SPECTROMETRY IN THE ANALYSIS OF OLIGOSACCHARIDES
    NIESSEN, WMA
    VANDERHOEVEN, RAM
    VANDERGREEF, J
    SCHOLS, HA
    LUCASLOKHORST, G
    VORAGEN, AGJ
    BRUGGINK, C
    RAPID COMMUNICATIONS IN MASS SPECTROMETRY, 1992, 6 (07) : 474 - 478
  • [9] Analysis of carbohydrates by anion exchange chromatography and mass spectrometry
    Bruggink, C
    Maurer, R
    Herrmann, H
    Cavalli, S
    Hoefler, F
    JOURNAL OF CHROMATOGRAPHY A, 2005, 1085 (01) : 104 - 109
  • [10] Improvement of sugar analysis sensitivity using anion-exchange chromatography-electrospray ionization mass spectrometry with sheath liquid interface
    Xu, Xian-Bing
    Liu, Ding-Bo
    Guo, Xiao Ming
    Yu, Shu-Juan
    Yu, Pei
    JOURNAL OF CHROMATOGRAPHY A, 2014, 1366 : 65 - 72