ESI-MS Investigation of an Equilibrium between a Bimolecular Quadruplex DNA and a Duplex DNA/RNA Hybrid

Abstract

, J. L. (2015). ESI-MS investigation of an equilibrium between a bimolecular quadruplex DNA and a duplex DNA/RNA hybrid. Journal of the American Society for Mass Spectrometry, 26 (7), 1165-1173.ESI-MS investigation of an equilibrium between a bimolecular quadruplex DNA and a duplex DNA/RNA hybrid AbstractElectrospray ionization mass spectrometry (ESI-MS) conditions were optimized for simultaneous observation of a bimolecular qDNA and a Watson-Crick base-paired duplex DNA/RNA hybrid. The DNA sequence used was telomeric DNA, and the RNA contained the template for telomerase-mediated telomeric DNA synthesis. Addition of RNA to the quadruplex DNA (qDNA) resulted in formation of the duplex DNA/ RNA hybrid. Melting profiles obtained using circular dichroism spectroscopy confirmed that the DNA/RNA hybrid exhibited greater thermal stability than the bimolecular qDNA in solution. Binding of a 13-substituted berberine (1) derivative to the bimolecular qDNA stabilized its structure as evidenced by an increase in its stability in the mass spectrometer, and an increase in its circular dichroism (CD) melting temperature of 10°C. The DNA/RNA hybrid did not bind the ligand extensively and its thermal stability was unchanged in the presence of (1). The qDNA-ligand complex resisted unfolding in the presence of excess RNA, limiting the formation of the DNA/RNA hybrid. Previously, it has been proposed that DNA secondary structures, such as qDNA, may be involved in the telomerase mechanism. DNA/RNA hybrid structures occur at the active site of telomerase. The results presented in the current work show that if telomeric DNA was folded into a qDNA structure, it is possible for a DNA/RNA hybrid to form as is required during template alignment. The discrimination of ligand (1) AbstractElectrospray ionization mass spectrometry (ESI-MS) conditions were optimized for simultaneous observation of a bimolecular qDNA and a Watson-Crick base-paired duplex DNA/RNA hybrid. The DNA sequence used was telomeric DNA, and the RNA contained the template for telomerase-mediated telomeric DNA synthesis. Addition of RNA to the qDNA resulted in formation of the duplex DNA/RNA hybrid. Melting profiles obtained using circular dichroism spectroscopy confirmed that the DNA/RNA hybrid exhibited greater thermal stability than the bimolecular qDNA in solution. Binding of a 13-substituted berberine (1) derivative to the bimolecular qDNA stabilized its structure as evidenced by an increase in its stability in the mass spectrometer, and an increase in its (CD) melting temperature of 10 C. The DNA/RNA hybrid did not bind the ligand extensively and its thermal stability was unchanged in the presence of (1). The qDNA-ligand complex resisted unfolding in the presence of excess RNA, limiting the formation of the DNA/RNA hybrid.Previously, it has been proposed that DNA secondary structures, such as qDNA, may be involved in the telomerase mechanism. DNA/RNA hybrid structures occur at the active site of telomerase. The results presented in the current work show that if tel...

A mechanism for the extension and unfolding of parallel telomeric G-quadruplexes by human telomerase at single-molecule resolution

“…For telomerase, we favour a model in which the enzyme binds the G4 while it is still structured, followed by invasion by the RNA template; confirmation of this model will require further kinetic analyses. A similar template-mimicking RNA was unable to form a DNA-RNA hybrid with an antiparallel G4 of similar stability to the parallel G4 used in the present study ( Birrento et al, 2015 ), demonstrating that this is a specific interaction between this RNA and parallel G4 structures. Single-molecule FRET studies have illuminated the mechanisms used by other G4-unwinding proteins; for example, the helicase RHAU/DHX36 was shown to stack on the top of the G4 plane and bind to the exposed 3' single stranded DNA tail ( Chen et al, 2018 ), whereas the replication protein RPA has been proposed to bind to G4 structures via their exposed loops ( Ray et al, 2013 ).…”

May the Best Molecule Win: Competition ESI Mass Spectrometry

“…Valuable information is gathered quickly with regards to preferential binding of a ligand to DNA. Although several of the studies described later will focus primarily on the binding of small molecule ligands to DNA, this technique is not limited to these specific interactions and can be applicable to other biomolecules of interest, including proteins [ 2 , 22 , 35 , 36 , 37 , 38 ], carbohydrates [ 39 , 40 ], and other types of nucleic acids such as RNA [ 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 ] and peptide nucleic acids [ 52 , 53 , 54 ].…”

Enhancing Anticancer Potency of a 13-Substituted Berberine Derivative with Cationic Liposomes

“…It should be noted however that the situation with regard to telomerase inhibition by G-quadruplex DNA ligands has been re-evaluated and many may not be simple telomerase inhibitors. 10,11) A promising berberine-derived anticancer agent, 13-(2naphthylmethoxy) berberine bromide (1) (Fig. 1), was developed by us 12) and has been reported to have selective interaction with model G-quadruplex (G4) DNA structures over duplex DNA analogues, 11) as well as partially inhibiting telomerase unfolding and extension of an intramolecular parallel G4.…”