Peaceful coexistence: A double quadruplex composed of an i‐motif and a G‐quadruplex was constructed within one oligonucleotide strand (see picture). The defined double‐quadruplex structure can serve ...as a NOTIF logic gate on the basis of the fluorescence of crystal violet.
A label-free oligonucleotide-based luminescent switch-on assay has been developed for the selective detection of sub-nanomolar Pb2+ ions in aqueous solution and real water samples. An iridium(III) ...complex was employed as a G-quadruplex specific luminescent probe and a guanine rich DNA (PS2.M, 5′-GTG3TAG3CG3T2G2-3′) was employed as recognition unit for Pb2+ ions. The PS2.M exists in a single-stranded conformation in the absence of Pb2+ ions, and the weak binding of the iridium(III) probe to ssDNA results in a weak luminescence signal. Upon binding to Pb2+ ions, the single-stranded DNA sequence (PS2.M) is induced into a G-quadruplex conformation, which greatly enhances the luminescence emission of the iridium(III) probe. The assay can detect Pb2+ ions in aqueous media with a limit of detection of 600pM. It also exhibits good selectivity for Pb2+ ions over other heavy metal ions. Furthermore, the application of the assay for the detection of Pb2+ ions in spiked river water samples has been demonstrated.
► A label-free oligonucleotide-based luminescent switch-on assay for lead(II) ions has been constructed. ► A cyclometallated iridium(III) complex produces a switch-on luminescence response to the analyte. ► The system selectively detects lead(II) ions in aqueous solution with high sensitivity.
Recent studies have proven that the genetic landscape of pancreatic cancer is dominated by the KRAS oncogene. Its transcription is controlled by a G-rich motif (called 32R) located immediately ...upstream of the TSS. 32R may fold into a G-quadruplex (G4) in equilibrium between two G4 conformers: G9T (T M = 61.2 °C) and G25T (T M = 54.7 °C). We found that both G4s bind to hnRNPA1 and its proteolytic fragment UP1, promoting several contacts with the RRM protein domains. 1D NMR analysis of DNA imino protons shows that, upon binding to UP1, G25T is readily unfolded at both 5′ and 3′ tetrads, while G9T is only partially unfolded. The impact of hnRNPA1 on KRAS expression was determined by comparing Panc-1 cells with two Panc-1 knockout cell lines in which hnRNPA1 was deleted by the CRISPR/Cas9 technology. The results showed that the expression of KRAS is inhibited in the knockout cell lines, indicating that hnRNPA1 is essential for the transcription of KRAS. In addition, the knockout cell lines, compared to normal Panc-1 cells, show a dramatic decrease in cell growth and capacity of colony formation. Pull-down and Western blot experiments indicate that conformer G25T is a better platform than conformer G9T for the assembly of the transcription preinitiation complex with PARP1, Ku70, MAZ, and hnRNPA1. Together, our data prove that hnRNPA1, being a key transcription factor for the activation of KRAS, can be a new therapeutic target for the rational design of anticancer strategies.
The replication protein A (RPA) is a single-stranded DNA-binding protein that plays an essential role in DNA metabolism. RPA is able to unfold G-quadruplex (G4) structures formed by telomeric DNA ...sequences, a function important for telomere maintenance. To elucidate the mechanism through which RPA unfolds telomeric G4s, we studied its interaction with oligonucleotides that adopt a G4 structure extended with a single-stranded tail on either side of the G4. Binding and unfolding was characterized using several biochemical and biophysical approaches and in the presence of specific G4 ligands, such as telomestatin and 360A. Our data show that RPA can bind on each side of the G4 but it unwinds the G4 only from 5′ toward 3′. We explain the 5′ to 3′ unfolding directionality in terms of the 5′ to 3′ oriented laying out of hRPA subunits along single-stranded DNA. Furthermore, we demonstrate by kinetics experiments that RPA proceeds with the same directionality for duplex unfolding.
Guanine-rich DNA strands can fold in vitro into non-canonical DNA structures called G-quadruplexes. These structures may be very stable under physiological conditions. Evidence suggests that ...G-quadruplex structures may act as 'knots' within genomic DNA, and it has been hypothesized that proteins may have evolved to remove these structures. The first indication of how G-quadruplex structures could be unfolded enzymatically came in the late 1990s with reports that some well-known duplex DNA helicases resolved these structures in vitro. Since then, the number of studies reporting G-quadruplex DNA unfolding by helicase enzymes has rapidly increased. The present review aims to present a general overview of the helicase/G-quadruplex field.
KRAS is one of the most mutated oncogenes and still considered an undruggable target. An alternative strategy would consist in targeting its gene rather than the protein, specifically the formation ...of G-quadruplexes (G4) in its promoter. G4 are secondary structures implicated in biological processes, which can be formed among G-rich DNA (or RNA) sequences. Here we have studied the major conformations of the commonly known KRAS 32R, or simply 32R, a 32 residue sequence within the KRAS Nuclease Hypersensitive Element (NHE) region. We have determined the structure of the two major stable conformers that 32R can adopt and which display slow equilibrium (>ms) with each other. By using different biophysical methods, we found that the nucleotides G9, G25, G28 and G32 are particularly implicated in the exchange between these two conformations. We also showed that a triad at the 3' end further stabilizes one of the G4 conformations, while the second conformer remains more flexible and less stable.
Helicases, enzymes that unwind DNA or RNA structure, are present in the cell nucleus and in the mitochondrion. Although the majority of the helicases unwind DNA or RNA duplexes, some of these ...proteins are known to resolve unusual structures such as G-quadruplexes (G4) in vitro. G4 may form stable barrier to the progression of molecular motors tracking on DNA. Monitoring G4 unwinding by these enzymes may reveal the mechanisms of the enzymes and provides information about the stability of these structures. In the experiments presented herein, we developed a reliable, inexpensive and rapid fluorescence-based technique to monitor the activity of G4 helicases in real time in a 96-well plate format. This system was used to screen a series of G4 structures and G4 binders for their effect on the Pif1 enzyme, a 5' to 3' DNA helicase. This simple assay should be adaptable to analysis of other helicases and G4 structures.
G-quadruplexes unfolding by RHAU helicase Gueddouda, Nassima Meriem; Mendoza, Oscar; Gomez, Dennis ...
Biochimica et biophysica acta. General subjects,
20/May , Letnik:
1861, Številka:
5
Journal Article
Recenzirano
Odprti dostop
G-quadruplexes (G4) are RNA and DNA secondary structures formed by the stacking of guanine quartets in guanine rich sequences. Quadruplex-prone motifs may be found in key genomic regions such as ...telomeres, ribosomal DNA, transcriptional activators and regulators or oncogene promoters. A number of proteins involved in various biological processes are able to interact with G4s. Among them, proteins dedicated to nucleic acids unwinding such as WRN, BLM, FANCJ or PIF1, can unfold G4 structures. Mutations of these helicases are linked to genome instability and to increases in cancer risks. Here, we present a high-throughput fluorescence-based reliable, inexpensive and fast assay to study G4/RHAU interaction. RHAU is an RNA helicase known as the major source of G4 resolution in HeLa cells. Our assay allows to monitor the unfolding properties of RHAU towards DNA and RNA quadruplexes in parallel and to screen for the optimal conditions for its activity. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.
•Proteins dedicated to nucleic acids unwinding such as WRN, BLM, FANCJ or PIF1, can unfold G4 structures.•We present a high-throughput fluorescence-based reliable, inexpensive and fast assay to study G4/RHAU interaction.•Our assay allows to monitor the unfolding properties of RHAU towards DNA and RNA quadruplexes
The telomeric G-rich single-stranded DNA can adopt
in vitro an intramolecular quadruplex structure, which has been shown to directly inhibit telomerase activity. The reactivation of this enzyme in ...immortalized and most cancer cells suggests that telomeres and telomerase are relevant targets in oncology, and telomere ligands and telomerase inhibitors have been proposed as new potential anticancer agents. In this paper, we have analysed the FRET method used to measure the stabilization and selectivity of quadruplex ligands towards the human telomeric G-quadruplex. The stabilization value depends on the nature of the fluorescent tags, the incubation buffer, and the method chosen for
T
m calculation, complicating a direct comparison of the results obtained by different laboratories.
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