Organelle-mimicking liposome dissociates G-quadruplexes and facilitates transcription

Nucleic Acids Research, Nov 2014

Important biological reactions involving nucleic acids occur near the surface of membranes such as the nuclear membrane (NM) and rough endoplasmic reticulum (ER); however, the interactions between biomembranes and nucleic acids are poorly understood. We report here that transcription was facilitated in solution with liposomes, which mimic a biomembrane surface, relative to the reaction in a homogeneous aqueous solution when the template was able to form a G-quadruplex. The G-quadruplex is known to be an inhibitor of transcription, but the stability of the G-quadruplex was decreased at the liposome surface because of unfavourable enthalpy. The destabilization of the G-quadruplex was greater at the surface of NM- and ER-mimicking liposomes than at the surfaces of liposomes designed to mimic other organelles. Thermodynamic analyses revealed that the G-rich oligonucleotides adopted an extended structure at the liposome surface, whereas in solution the compact G-quadruplex was formed. Our data suggest that changes in structure and stability of nucleic acids regulate biological reactions at membrane surfaces.

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Organelle-mimicking liposome dissociates G-quadruplexes and facilitates transcription

Abstract Important biological reactions involving nucleic acids occur near the surface of membranes such as the nuclear membrane (NM) and rough endoplasmic reticulum (ER); however, the interactions between biomembranes and nucleic acids are poorly understood. We report here that transcription was facilitated in solution with liposomes, which mimic a biomembrane surface, relative to the reaction in a homogeneous aqueous solution when the template was able to form a G-quadruplex. The G-quadruplex is known to be an inhibitor of transcription, but the stability of the G-quadruplex was decreased at the liposome surface because of unfavourable enthalpy. The destabilization of the G-quadruplex was greater at the surface of NM- and ER-mimicking liposomes than at the surfaces of liposomes designed to mimic other organelles. Thermodynamic analyses revealed that the G-rich oligonucleotides adopted an extended structure at the liposome surface, whereas in solution the compact G-quadruplex was formed. Our data suggest that changes in structure and stability of nucleic acids regulate biological reactions at membrane surfaces. INTRODUCTION Biomembranes play pivotal roles in not only the cell structure but also various intracellular functions. For example, the nuclear membrane (NM) in eukaryotic cells is a lipid bilayer that surrounds the genomic DNA and associated components. The NM serves as a physical boundary and may also be involved in chromatin function and gene expression (1). Liposomes, simple artificial systems that mimic biomembranes (2), have been used to study the dynamics and structural features of many cellular processes (3). For example, it was recently reported that DNA undergoes a conformational transition from a folded state in the aqueous phase to a coiled state on the phospholipid membrane in a cell-sized microdroplet and that the conformational transition regulated transcriptional activity (4). Self-replication of DNA is observed within a self-reproducible cationic giant vesicle that serves as a model protocell (5). Moreover, the efficiency of in vitro gene expression is enhanced in the presence of liposomes (6–8). It has been reported that the antimicrobial peptide mastoparan X undergoes a coil-to-helix transition upon binding to membranes (9). Liposomes have been used to reproduce membrane fusion (10) and ion channel formation (11) using purified proteins reconstituted in the liposomes. In living cells, biomembranes of organelles separate certain biomolecules from the rest of the cellular environment and create two kinds of environments (12). Inside organelles, such as nucleus, endoplasmic reticulum (ER) and mitochondria, high concentrations of biomolecules result in homogeneous crowding conditions (Figure 1). At the biomembrane surface, conditions are heterogeneous (Figure 1). Although the canonical structure of genomic DNA is a duplex, regions of DNA can undergo structural transitions from the duplex structure to non-canonical structures, such as G-quadruplexes, in response to environmental conditions (13–16). The formation of G-quadruplexes inhibits biological reactions, such as telomere elongation and transcription (17,18). To better predict whether G-quadruplexes form in cells, the structure and stability of the nucleic acids under conditions of molecular crowding induced by both non-interacting (19–22) and interacting (23) cosolutes have been studied. Formation of the G-quadruplexes is markedly facilitated by cosolutes (19). We have investigated the importance of heterogeneous confined media in the cell nucleus using reverse micelles and found that a significant fraction of the telomeric region of genomic DNA adopts non-canonical structures under these conditions (24). We have also recently shown that the formation of G-quadruplexes in open reading frames suppresses the translation of mRNA into protein (25). Although most proteins are translated on ribosomes that are free in the cytoplasm, translation of membrane proteins occurs on ribosomes bound to the membrane surface (12). The structures of mRNA on these membrane-bound ribosomes may be affected by the heterogeneous conditions at the membrane surface, in turn affecting translation efficiency. Figure 1. View largeDownload slide Schematic representation of intracellular crowding within organelles and heterogeneous conditions at the membrane surface. In this study, these intracellular conditions were mimicked using liposomes. Figure 1. View largeDownload slide Schematic representation of intracellular crowding within organelles and heterogeneous conditions at the membrane surface. In this study, these intracellular conditions were mimicked using liposomes. In the present study, we investigated the structure and stability of DNA hairpins and DNA and RNA G-quadruplexes in solutions containing liposomes to mimic the crowded condition present inside organelles and at liposome surfaces, which mimic the heterogeneous con (...truncated)


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Pramanik, Smritimoy, Tateishi-Karimata, Hisae, Sugimoto, Naoki. Organelle-mimicking liposome dissociates G-quadruplexes and facilitates transcription, Nucleic Acids Research, 2014, pp. 12949-12959, Volume 42, Issue 20, DOI: 10.1093/nar/gku998