Resolution of Quadruplex Polymorphism by Size‐Exclusion Chromatography
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Abstract
This unit describes a method for separation of quadruplex species formed from the same sequence via size?exclusion chromatography (SEC). Polymorphism is inherent to quadruplex formation, and even relatively simple quadruplex?forming sequences, such as the human telomere sequence d(GGG(TTAGGG)3 ), can form a myriad of possible configurations. HPLC, especially using reversed?phase and anion?exchange methods, has been a mainstay of nucleic acids research and purification for many decades. These methods have been applied for separation of individual quadruplex species formed in a mixture from the same parent sequence. Curr. Protoc. Nucleic Acid Chem. 45:17.3.1?17.3.18. © 2011 by John Wiley & Sons, Inc.
Keywords: SEC; quadruplex; size?exclusion chromatography; telomere
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PDF or HTML at Wiley Online LibraryTable of Contents
- Introduction
- Basic Protocol 1: Size‐Exclusion Chromatography of G‐Quadruplexes
- Support Protocol 1: Preparation of Quadruplex Sample
- Commentary
- Literature Cited
- Figures
- Tables
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Basic Protocol 1: Size‐Exclusion Chromatography of G‐Quadruplexes
Materials
Support Protocol 1: Preparation of Quadruplex Sample
Materials
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Figure 17.3.1 Calibration of the Superdex 75 10/300 column. Top: A mixture of protein standards resolved on the column. 1: conalbumin (75 kDa, V R = 9.36); 2: ovalbumin (43 kDa, V R = 10.19); 3: carbonic anhydrase (29 kDa, V R = 11.32); 4: ribonuclease A (13.7 kDa; V R = 12.84); 5: bovine lung aprotonin (6.5 kDa, V R = 14.66). Bottom: Linear calibration curve developed by graphing V R / V 0 vs. logMW. V 0 (7.61 mL) was determined by injection of blue dextran as described. View Image -
Figure 17.3.2 Typical elution profile of AS1411. At a glance, there are at least eight isolatable species with varying levels of separation. View Image -
Figure 17.3.3 Possible topologies of the human telomere sequence include (A) basket (Protein Database code: 143D), (B) hybrid1 (2HY9), (C) hybrid2 (2JPZ), and (D) propeller (1KF1). View Image -
Figure 17.3.4 (A) 1 H NMR of imino regions of the human telomere sequence AG3 (T2 AG3 )3 and of the AS1411 sequence (G2 T)4 TGT(G2 T)3 G2 . The human telomere exhibits 23‐24 GN1H signals, indicative of the presence of two quadruplex species. AS1411 yields an irresolvable mixture of GN1H resonances known to be the result of more than eight quadruplex species. (B) CD of the same two sequences. The CD data provide no indication of the polymorphism evident from the NMR data. Conventional wisdom would associate the data for AS1411 with a single, parallel‐stranded species in solution. View Image -
Figure 17.3.5 HPLC results for the following sequences: (A) AS1411, (B) Her2, (C) c‐myc, (D) G4 T4 G4 T4 G4 T4 , (E) HuTel 22, (F) HuTel Hybrid 1, (G) HuTel Hybrid 2, (H) TG4 T. View Image -
Figure 17.3.6 Resolution of AS1411 polymorphism. Top: Typical chromatographic separation for AS1411 demonstrating multiple isolatable species with apparent molecular weights ranging from 45,000 g/mol to less than 6,000 g/mol. Bottom: 1 H NMR of the fraction of AS1411 corresponding to roughly 14,000 g/mol (marked with * in A). The spectrum shows roughly 16 GN1H signals corresponding to a single, monomeric species. View Image
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Literature Cited
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