Characterization of Co-transcriptional Formation of G-Quadruplexes in Double-Stranded DNA
In vitro transcription of double-stranded DNA (dsDNA) induces a formation of two types of G-quadruplexes, intramolecular DNA G-quadruplexes or DNA:RNA hybrid G-quadruplexes, in clusters of guanine tracts. The formation of G-quadruplexes can be characteriz
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Introduction G-quadruplexes are nucleic acid secondary structures formed by four G-tracts via a stacking of G-quartet layers [1, 2]. Evidences show that G-quadruplex is stable both in vitro [3] and in vivo [4, 5]. In eukaryotic and prokaryotic cells, DNA exists in a double-stranded form where G-quadruplex can form when the base-pairing between two complementary strands is weakened or disrupted in processes, such as transcription, replication, and DNA repair. Transcription has been demonstrated to induce G-quadruplex formation in duplex DNA under both in vivo [6] and in vitro [7–11] conditions. Since the sequence of a RNA transcript is identical to the non-template DNA strand of a transcribed DNA, a distinctive form of G-quadruplex, i.e., DNA:RNA hybrid G-quadruplex which involve a participation of a non-template DNA strand and RNA transcript can also form in transcription [10–12]. Characterization of G-quadruplex structures in single-stranded oligonucleotides by biochemical techniques has been well described previously [13] with which the characterization of G-quadruplex formation in transcribed DNA shares similar
Danzhou Yang and Clement Lin (eds.), G-Quadruplex Nucleic Acids: Methods and Protocols, Methods in Molecular Biology, vol. 2035, https://doi.org/10.1007/978-1-4939-9666-7_14, © Springer Science+Business Media, LLC, part of Springer Nature 2019
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Fig. 1 Types and positions of G-quadruplex (GQ) formation in transcribed dsDNA. A motif of four or more G-tracts can form a canonical intramolecular DNA G-quadruplex (DQ) at both sides of a transcription start site (TSS). A motif of two or three G-tracts on the non-template strand in a transcribed region can form a DNA:RNA hybrid G-quadruplex (HQ) although it is unable to form a DQ
principles. In vitro transcription can be conveniently conducted using either phage RNA polymerases (RNAP) (T7, T3, SP6) or E. coli RNA polymerase in the presence of four nucleoside triphosphates (NTPs) with a dsDNA carrying a promoter at an appropriate position (Fig. 1). A dsDNA should be end-labeled with a fluorescent dye or radioisotope for visualization and can be prepared either from commercially synthesized oligonucleotides or by PCR amplification using pre-made plasmid or genomic DNA as template [8, 10]. This labeling not only indicate the position of dsDNA in electrophoresis, but also show the size of DNA fragments in DMS footprinting. In such an in vitro transcription model, maximal formation of G-quadruplexes is usually reached within 1 h at 37 C. We normally include PEG 200 in transcription and gel electrophoresis to stabilize G-quadruplexes to facilitate the detection [8]. Here, we present four methods used in our studies to detect G-quadruplexes formed in transcription. They are (1) native PEG polyacrylamide gel electrophoresis, (2) DMS footprinting, (3) DNA:RNA crosslinking, and (4) ligand-induced photocleavage footprinting.
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Materials
2.1 Preparing 50 -FAM-Labeled dsDNA Using PCR
1. Taq DNA Polymerase (Thermo Scientific). 2. DMSO
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