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G-quadruplex (G4) structures formed by guanine-rich nucleic acids are implicated in essential physiological and pathological processes and serve as important drug targets. The genome-wide detection of G4s in living cells is important for exploring the functional role of G4s but has not yet been achieved due to the lack of a suitable G4 probe. Here we report an artificial 6.7 kDa G4 probe (G4P) protein that binds G4s with high affinity and specificity. We used it to capture G4s in living human, mouse, and chicken cells with the ChIP-Seq technique, yielding genome-wide landscape as well as details on the positions, frequencies, and sequence identities of G4 formation in these cells. Our results indicate that transcription is accompanied by a robust formation of G4s in genes. In human cells, we detected up to >123 000 G4P peaks, of which >1/3 had a fold increase of ≥5 and were present in >60% promoters and ∼70% genes. Being much smaller than a scFv antibody (27 kDa) or even a nanobody (12–15 kDa), we expect that the G4P may find diverse applications in biology, medicine, and molecular devices as a G4 affinity agent.

Bioinformatic analysis reveals an enrichment of putative DNA:RNA hybrid G-quadruplex-forming sequences (PHQS) on both sides of the transcription start sites (TSSs) in the genome of warm-blooded animals, suggesting a positive selection of PHQSs in evolution and functional role of DNA:RNA hybrid G-quadruplexes (HQs) in transcription. The formation of HQs downstream of TSS in transcribed DNA has been documented under in vitro conditions; however, it is still not known if such HQs can form at the upstream side of TSSs. In this study, we report that such HQs can form in transcription in DNA with two to three guanine tracts if RNA carrying the required number of G-tracts is supplied. We also show that the formation of such HQs is dependent on the negative supercoiling generated by RNA polymerases. These results suggest that HQs may also form at the upstream side of TSSs in vivo and play a role in transcription since the two requirements are satisfied in cells.

A dual-functional peptide-PNA (peptide nucleic acid) conjugate consisting of a PNA G3-tract and an RHAU23 peptide is devised to target nucleic acids bearing three tandem guanine tracts (G-tracts). The PNA G3-tract joins the three G-tracts to form a stable bimolecular G-quadruplex (G4) and the resulting G4 is then bound by the RHAU23 moiety to form an extra stable G4-peptide complex. Owing to this synergistic dual structural enforcement, the conjugate accomplished extremely high selectivity and nM to sub-nM affinities towards its targets that are up to 1000 times greater than the small molecule G4 ligands. As a result, the conjugate impacts the tracking activity of motor proteins on DNA with superior selectivity and potency that are rarely seen in other G4-targeting approaches.

Stabilization of G-quadruplexes (G4s) formed in guanine-rich (G-rich) nucleic acids by small-molecule ligands has been extensively explored as a therapeutic approach for diseases such as cancer. Finding ligands with sufficient affinity and specificity toward G4s remains a challenge, and many ligands reported seemed to compromise between the two features. To cope with this challenge, we focused on targeting a particular type of G4s, i.e., the G-vacancy-bearing G-quadruplexes (GVBQs), by taking a structure complementation strategy to enhance both affinity and selectivity. In this approach, a G-quadruplex-binding peptide RHAU23 is guided toward a GVBQ by a guanine moiety covalently linked to the peptide. The filling-in of the vacancy in a GVBQ by the guanine ensures an exclusive recognition of GVBQ. Moreover, the synergy between the RHAU23 and the guanine dramatically improves both the affinity toward and stabilization of the GVBQ. Targeting a GVBQ in DNA by this bifunctional peptide strongly suppresses in vitro replication. This study demonstrates a novel and promising alternative targeting strategy to a distinctive panel of G4s that are as abundant as the canonical ones in the human genome.

The particular enrichment of G-quadruplex-forming sequences near transcription start sites signifies the involvement of G-quadruplexes in the regulation of transcription. The characterization of G-quadruplex formation, which holds the key to understand the function it plays in physiological and pathological processes, is mostly performed under simplified in vitro experimental conditions. Formation of G-quadruplexes in cells, however, occurs in an environment far different from the ones in which the in vitro studies on G-quadruplexes are normally carried out. Therefore, the characteristics of G-quadruplex structures obtained under the in vitro conditions may not faithfully reveal how the G-quadruplexes would behave in a physiologically relevant situation. In this mini-review, we attempt to briefly summarize the differences in a few important characteristics, including kinetics, conformation, and stability of G-quadruplex formation observed under the two conditions to illustrate how the intracellular environment might affect the behavior of G-quadruplexes largely based on the previous work carried out in the authors’ laboratory. We also propose that unstable G-quadruplex variants may be better drug target candidates to improve selectivity and potency.

DNA supercoiling is an important regulator of gene activity. The transmission of transcription-generated supercoiling wave along a DNA helix provides a way for a gene being transcribed to communicate with and regulate its neighboring genes. Currently, the dynamic behavior of supercoiling transmission remains unclear owing to the lack of a suitable tool for detecting the dynamics of supercoiling transmission. In this work, we established a torsion sensor that quantitatively monitors supercoiling transmission in real time in DNA. Using this sensor, we studied the transmission of transcriptionally generated negative supercoiling in linear and multi-way DNA duplexes. We found that transcription-generated dynamic supercoiling not only transmits along linear DNA duplex but also equally diverges at and proceeds through multi-way DNA junctions. We also show that such a process is regulated by DNA–protein interactions and non-canonical DNA structures in the path of supercoiling transmission. These results imply a transcription-coupled mechanism of dynamic supercoiling-mediated intra- and inter-chromosomal signal transduction pathway and their regulation in DNA.

Transcription induces formation of intramolecular G-quadruplex structures at the upstream region of a DNA duplex by an upward transmission of negative supercoiling through the DNA. Currently the regulation of such G-quadruplex formation remains unclear. Using plasmid as a model, we demonstrate that while it is the dynamic negative supercoiling generated by a moving RNA polymerase that triggers a formation of a G-quadruplex, the constitutional superhelicity determines the potential and range of the formation of a G-quadruplex by constraining the propagation of the negative supercoiling. G-quadruplex formation is maximal in negatively supercoiled and nearly abolished in relaxed plasmids while being moderate in nicked and linear ones. The formation of a G-quadruplex strongly correlates with the presence of an R-loop. Preventing R-loop formation virtually abolished G-quadruplex formation even in the negatively supercoiled plasmid. Enzymatic action and protein binding that manipulate supercoiling or its propagation all impact the formation of G-quadruplexes. Because chromosomes and plasmids in cells in their natural form are maintained in a supercoiled state, our findings reveal a physical basis that justifies the formation and regulation of G-quadruplexes in vivo. The structural features involved in G-quadruplex formation may all serve as potential targets in clinical and therapeutic applications.

G-quadruplex (GQ) structures are implicated in important physiological and pathological processes. Millions of GQ-forming motifs are enriched near transcription start sites (TSSs) of animal genes. Transcription can induce the formation of GQs, which in turn regulate transcription. The kinetics of the formation and persistence of GQs in transcription is crucial for the role they play but has not yet been explored. We established a method based on the fluorescence resonance energy transfer (FRET) technique to monitor in real-time the cotranscriptional formation and post-transcriptional persistence of GQs in DNA. Using a T7 transcription model, we demonstrate that a representative intramolecular DNA GQ and DNA:RNA hybrid GQ promptly form in proportion to transcription activity and, once formed, are maintained for hours or longer at physiological temperature even after transcription is stopped. Both their formation and persistence strongly depend on R-loop, a DNA:RNA hybrid duplex formed during transcription. Enzymatic removal of R-loop dramatically slows their formation and accelerates their unfolding. These results suggest that a transcription event is promptly read-out by GQ-forming motifs and the GQ formed can either perform regulation in fast response to transcription and/or memorized in DNA to mediate time-delayed regulation under the control of RNA metabolism and GQ-resolving activity. Alternatively, GQs need to be timely resolved to warrant success of translocating activities such as replication. The kinetic characteristics of GQs and its connection with the R-loop may have implications in transcription regulation, signal transduction, G-quadruplex processing, and genome stability.

RNA G-quadruplex formation in a defined sequence in living bacterial cells is detected by bimolecular fluorescence complementation with sequence and structure specificity., G-quadruplexes are implicated in many essential cellular processes and sequences with potential to form a G-quadruplex are widely present in DNA and RNA. However, it is difficult to know whether a sequence of interest naturally forms a G-quadruplex in living cells. Here we report the detection of a G-quadruplex in defined RNA sequences in living cells in a natural intracellular environment. A G-quadruplex forming sequence in a RNA transcript is tagged at proximity with an aptamer. The two structures are recognized respectively by two probe proteins each of which is fused with a split half of enhanced green fluorescent protein (eGFP). Simultaneous binding of the two proteins to RNA brings the two halves of eGFP into proximity, permitting them to reconstitute into a functional eGFP that emits fluorescence to signal the formation of a G-quadruplex in RNA. We show that a G-quadruplex can form in RNA and can be detected with sequence and structure specificity under both in vitro and in vivo conditions. The results, therefore, provide direct evidence for the formation of RNA G-quadruplexes in live cells and the method provides a useful tool to validate G-quadruplex formation in a specific sequence under a natural cellular condition.

Chromosomes in human cells are protected by telomeres. Telomere shortens during each round of cell division because of the DNA end-replication problem. Cancer cells maintain telomere length homeostasis by either telomerase or/and the alternative lengthening of telomere (ALT) mechanism to sustain their division potential. Telomeric DNA tends to form G-quadruplex preferentially at the extreme 3' end. This unique feature prevents the 3' end from being used as a substrate of telomerase and as a primer in the ALT. Therefore, stabilizing telomere G-quadruplex is expected to inhibit both pathways and limit the proliferation of cancer cells. Based on a mathematical modeling and experimental results, this mini-review proposes a hypothesis that the formation of G-quadruplex in telomere may constitute a significant contribution to the incomplete end-replication of telomere DNA by preventing the priming of DNA synthesis near the 3' end during telomere replication. According to this, stabilization of telomere G-quadruplex by chemical ligand may promise to accelerate telomere shortening in proliferating cells.

G-quadruplex-forming sequences are enriched near transcription start sites (TSSs) in animal genes. They readily form G-quadruplexes in transcription, which in turn regulate transcription. Therefore, the control of G-quadruplex formation is important for their functionality. It is now shown that G-quadruplexes form efficiently on the non-template, but hardly on the template DNA strand in the downstream vicinity of TSSs in DNA duplexes when they are transcribed by the T7 RNA polymerase (RNAP). Structural analysis reveals that the T7 RNAP causes distortion in a DNA duplex both inside and in front of the enzyme. This structural distortion leads to strand-biased G-quadruplex formation when a G-quadruplex-forming sequence is partially fed into the T7 RNAP to a position about seven nucleotides away from the front of RNA synthesis. Based on these facts, we propose a model for the strand-biased formation of G-quadruplexes in transcribed DNA duplexes.

G-quadruplexes are implicated in important cellular processes. Previous studies mostly focused on intramolecular G-quadruplexes of three or more G-quartets. Those composed of two G-quartets were only shown to form in single-stranded oligonucleotides. On the basis of electrophoresis, DMS footprinting, fluorescence labeling, and photo-cross-linking, we detected the formation of DNA:RNA hybrid G-quadruplexes (HQs) of two G-quartets during the transcription of DNA duplexes. These HQs have a lifetime on the minute scale and are stabilized by a stabilizing ligand. They are far shorter-lived than the HQs of three G-quartets, which last for hours. The occurrence of putative formation motifs of such HQs shows a transcription-dependent strand-biased selection, thus supporting their formation and function in genomes. They are present in almost all human genes in large amounts. We speculate that the two-G-quartet HQs may be a distinct type of G-quadruplexes that may play a role in timely responsive processes and for purposes of fine-tuning.

Recently, we reported the co-transcriptional formation of DNA:RNA hybrid G-quadruplex (HQ) structure by the non-template DNA strand and nascent RNA transcript, which in turn modulates transcription under both in vitro and in vivo conditions. Here we present bioinformatic analysis on putative HQ-forming sequences (PHQS) in the genomes of eukaryotic organisms. Starting from amphibian, PHQS motifs are concentrated in the immediate 1000-nt region downstream of transcription start sites, implying their potential role in transcription regulation. Moreover, their occurrence shows a strong bias toward the non-template versus the template strand. PHQS has become constitutional in genes in warm-blooded animals, and the magnitude of the strand bias correlates with the ability of PHQS to form HQ, suggesting a selection based on HQ formation. This strand bias is reversed in lower species, implying that the selection of PHQS/HQ depended on the living temperature of the organisms. In comparison with the putative intramolecular G-quadruplex-forming sequences (PQS), PHQS motifs are far more prevalent and abundant in the transcribed regions, making them the dominant candidates in the formation of G-quadruplexes in transcription. Collectively, these results suggest that the HQ structures are evolutionally selected to function in transcription and other transcription-mediated processes that involve guanine-rich non-template strand.

G-quadruplexes, four-stranded structures formed by Guanine-rich nucleic acids, are implicated in many physiological and pathological processes. G-quadruplex-forming sequences are abundant in genomic DNA, and G-quadruplexes have recently been shown to exist in the genome of mammalian cells. However, how G-quadruplexes are formed in the genomes remains largely unclear. Here, we show that G-quadruplex formation can be remotely induced by downstream transcription events that are thousands of base pairs away. The induced G-quadruplexes alter protein recognition and cause transcription termination at the local region. These results suggest that a G-quadruplex-forming sequence can serve as a sensor or receiver to sense remote DNA tracking activity in response to the propagation of mechanical torsion in a DNA double helix. We propose that the G-quadruplex formation may provide a mean for long-range sensing and communication between distal genomic locations to coordinate regulatory transactions in genomic DNA.

G-quadruplex formation in genomic DNA is considered to regulate transcription. Previous investigations almost exclusively focused on intramolecular G-quadruplexes formed by DNA carrying four or more G-tracts, and structure formation has rarely been studied in physiologically relevant processes. Here, we report an almost entirely neglected, but actually much more prevalent form of G-quadruplexes, DNA:RNA hybrid G-quadruplexes (HQ) that forms in transcription. HQ formation requires as few as two G-tracts instead of four on a non-template DNA strand. Potential HQ sequences (PHQS) are present in >97% of human genes, with an average of 73 PHQSs per gene. HQ modulates transcription under both in vitro and in vivo conditions. Transcriptomal analysis of human tissues implies that maximal gene expression may be limited by the number of PHQS in genes. These features suggest that HQs may play fundamental roles in transcription regulation and other transcription-mediated processes.

A guanine-vacancy-bearing G-quadruplex (GVBQ) interacts with guanine and derivatives by a structural complementation to form a more stable and intact G-quadruplex. Sensors using GVBQs are devised to detect guanine and other nucleobases, and their derivatives derived from structurally similar compounds. A strict requirement of Hoogsteen hydrogen bonds between the GVBQ and analyte in the structural complementation confers exceptional selectivity on the analyte. As such, subtle modifications on analytes affecting even a single hydrogen bond can preclude the recognition. In principle, the strategy may also be expanded to detect many planar cyclic compounds. Because nucleobases and derivatives/metabolites are involved in many physiological and pathological processes, this type of sensor may find applications in risk assessment of pathogenesis and therapeutics related to nucleic acid metabolism.

Ionizing radiation has the potential to cause extensive damage to living organisms. It can directly act on DNA, proteins and lipids, resulting in ionizing excitation and chemical bond cleavage, which can lead to molecular and cellular damage. Ionizing radiation can hydrolyze water molecules in the body, resulting in increased numbers of free radicals with strong oxidation ability. This process indirectly leads to tissue degeneration and necrosis, which can possibly result in cancer. In this paper, the intervention mechanism of flavonoids on ionizing radiation was analyzed. It has been revealed that the intervention mechanism associated with flavonoids may offer protective properties for DNA, prevent scavenging free radicals, and protect against auto-immune damage. In addition, this invention mechanism can protect the hematopoietic system and reduce inflammation Keywords: Ionizing radiation, Flavonoids, Radioprotective mechanisms, Molecular and cellular damage, DNA, Hematopoietic system, Inflammation

G-quadruplex structures formed by guanine-rich nucleic acids are implicated in essential physiological and pathological processes and nanodevices. G-quadruplexes are normally composed of four Gn (n ≥ 3) tracts assembled into a core of multiple stacked G-quartet layers. By dimethyl sulfate footprinting, circular dichroism spectroscopy, thermal melting, and photo-cross-linking, here we describe a unique type of intramolecular G-quadruplex that forms with one G2 and three G3 tracts and bears a guanine vacancy (G-vacancy) in one of the G-quartet layers. The G-vacancy can be filled up by a guanine base from GTP or GMP to complete an intact G-quartet by Hoogsteen hydrogen bonding, resulting in significant G-quadruplex stabilization that can effectively alter DNA replication in vitro at physiological concentration of GTP and Mg(2+). A bioinformatic survey shows motifs of such G-quadruplexes are evolutionally selected in genes with unique distribution pattern in both eukaryotic and prokaryotic organisms, implying such G-vacancy-bearing G-quadruplexes are present and play a role in gene regulation. Because guanine derivatives are natural metabolites in cells, the formation of such G-quadruplexes and guanine fill-in (G-fill-in) may grant an environment-responsive regulation in cellular processes. Our findings thus not only expand the sequence definition of G-quadruplex formation, but more importantly, reveal a structural and functional property not seen in the standard canonical G-quadruplexes.

A matter of speed: when allowed to fold in a K(+)/poly(ethylene glycol) solution, the guanine (G)-rich strand of vertebrate telomere DNA forms a parallel/antiparallel G-quadruplex, which is a (3+1) hybrid, within microseconds before slowly transforming into the parallel one within hours. Thus, the conformation that a G-quadruplex initially adopts under physiological conditions may not be the one it adopts at the equilibrium state.

Guanine rich (G-rich) nucleic acids form G-quadruplex structures that are implicated in many biological processes, pharmaceutical applications, and molecular machinery. The folding equilibrium constant (K(F)) of the G-quadruplex not only determines its stability and competition against duplex formation in genomic DNA but also defines its recognition by proteins and drugs and technical specifications. The K(F) is most conveniently derived from thermal melting analysis that has so far yielded extremely diversified results for the human telomere G-quadruplex. Melting analysis cannot be used for nucleic acids associated with proteins, thus has difficulty to study how protein association affects the folding equilibrium of G-quadruplex structure. In this work, we established an isothermal differential hybridization (IDH) method that is able to determine the K(F) of G-quadruplex, either alone or associated with proteins. Using this method, we studied the folding equilibrium of the core sequence G(3)(T(2)AG(3))(3) from vertebrate telomere in K(+) and Na(+) solutions and how it is affected by proteins associated at its adjacent regions. Our results show that the K(F) obtained for the free G-quadruplex is within 1 order of magnitude of most of those obtained by melting analysis and protein binding beside a G-quadruplex can dramatically destabilize the G-quadruplex.

Large numbers of guanine-rich sequences with potential to form G-quadruplexes have been identified in genomes of various organisms. Such sequences are constrained at both ends by long DNA duplex with a complementary strand in close proximity to compete for duplex formation. G-quadruplex/duplex competition in long double-stranded DNA has rarely been studied. In this work, we used DMS footprinting and gel electrophoresis to study G-quadruplex formation in long double-stranded DNA derived from human genome under both dilute and molecular crowding condition created by PEG. G-quadruplex formation was observed in the process of RNA transcription and after heat denaturation/renaturation under molecular crowding condition. Our results showed that the heat denaturation/renaturation treatment followed by gel electrophoresis could provide a simple method to quantitatively access the ability of G-quadruplex formation in long double-stranded DNA. The effect of K(+) and PEG concentration was investigated and we found that stable G-quadruplexes could only form under the crowding condition with PEG at concentrations near the physiological concentration of biomass in living cells. This observation reveals a physical basis for the formation of stable G-quadruplexes in genome and supports its presence under the in vivo molecular crowding condition.

Telomere DNA in human cells shortens during each round of DNA replication. In cancer cells, telomere shortening is compensated by telomerase or the alternative lengthening of telomere (ALT) mechanism to maintain cell division potential. The G-rich strand of telomere DNA can fold into a G-quadruplex structure and disrupt these two processes. Therefore, stabilization of the G-quadruplex by chemical ligands is emerging as a promising anticancer strategy. So far, in vitro studies on such ligands are exclusively carried out in dilute solutions. However, the intracellular environment is highly crowded with biomolecules. How G-quadruplex ligands behave under molecular crowding condition is critical for their in vivo anticancer effect. In this work, we studied several ligands for their ability to stabilize the telomere G-quadruplex and inhibit telomerase under both dilute and crowding conditions. Surprisingly, the ligands became significantly less effective or even lost the ability to stabilize the G-quadruplex and inhibit telomerase under crowding conditions. Our data attributed this consequence to the decreased binding affinity of ligands to the G-quadruplex as a result of reduced water activity and increased viscosity of the medium associated with molecular crowding. This effect is irrelevant to and overweighs the influences from other factors such as the G-quadruplex structure, cation, and ligand species. Our work illustrates a possibility that molecular crowding inside cells may reduce or limit the potency of ligands although they may be effective in dilute solution, thus strongly arguing for the necessity of evaluating ligands under more physiologically relevant conditions and designing drugs with this concern in mind.

Background PHIS transposon superfamily belongs to DNA transposons and includes PIF/Harbinger, ISL2EU, and Spy transposon groups. These three groups have similar DDE domain-containing transposases; however, their coding capacity, species distribution, and target site duplications (TSDs) are significantly different. Results In this study, we systematically identified and analyzed PHIS transposons in 836 sequenced eukaryotic genomes using transposase homology search and structure approach. In total, 380 PHIS families were identified in 112 genomes and 168 of 380 families were firstly reported in this study. Besides previous identified PIF/Harbinger, ISL2EU, and Spy groups, three new types (called Pangu, NuwaI, and NuwaII) of PHIS superfamily were identified; each has its own distinctive characteristics, especially in TSDs. Pangu and NuwaII transposons are characterized by 5′-ANT-3′ and 5′-C|TNA|G-3′ TSDs, respectively. Both transposons are widely distributed in plants, fungi, and animals; the NuwaI transposons are characterized by 5′-CWG-3′ TSDs and mainly distributed in animals. Conclusions Here, in total, 380 PHIS families were identified in eukaryotes. Among these 380 families, 168 were firstly reported in this study. Furthermore, three new types of PHIS superfamily were identified. Our results not only enrich the transposon diversity but also have extensive significance for improving genome sequence assembly and annotation of higher organisms. Electronic supplementary material The online version of this article (doi:10.1186/s13100-015-0043-7) contains supplementary material, which is available to authorized users.

DNA with four guanine tracts can fold into G-quadruplexes that are targets of transcription regulation. We recently found that hybrid DNA:RNA G-quadruplexes (HQs) can form during in vitro transcription. However, it is unclear whether they can form in cells. Evidence is presented supporting their formation in plasmids in bacterial cells. The formation of the HQs is indicated by a unique pattern of prematurely terminated transcripts under two conditions where the RNA transcripts do or do not participate in G-quadruplex assembly and further supported by a number of chemical and biochemical analysis. HQs dominate over the intramolecular DNA G-quadruplexes (DQ) in mediating the transcription termination when both structures are able to form. These findings provide the first evidence of HQ formation in cells and suggest that the competition/conversion between HQ and DQ may regulate transcription and serve as drug target in pharmaceutical applications.

Many sequences in genomic DNA are able to form unique tetraplex structures. Such structures are involved in a variety of important cellular processes and are emerging as a new class of therapeutic targets for cancers and other diseases. Screening for molecules targeting the tetraplex structure has been explored using such sequences immobilized on solid surfaces. Immobilized nucleic acids, in certain situations, may better resemble the molecules under in vivo conditions. In this report, we studied the formation of tetraplex structure of both the G-rich and C-rich strands of surface-immobilized human telomere sequence by surface plasmon resonance using the single-stranded DNA binding protein from Escherichia coli as probe. We demonstrate how the formation of G-quadruplex and i-motif could be probed under various conditions by this sequence-universal method. Our results also show that immobilization destabilized the tetraplex structure.

Nucleic acid molecules may fold into secondary structures, and the formation of such structures is involved in many biological processes and technical applications. The folding and unfolding rate constants define the kinetics of conformation interconversion and the stability of these structures and is important in realizing their functions. We developed a method to determine these kinetic parameters using an optical biosensor based on surface plasmon resonance. The folding and unfolding of a nucleic acid is coupled with a hybridization reaction by immobilization of the target nucleic acid on a sensor chip surface and injection of a complementary probe nucleic acid over the sensor chip surface. By monitoring the time course of duplex formation, both the folding and unfolding rate constants for the target nucleic acid and the association and dissociation rate constants for the target-probe duplex can all be derived from the same measurement. We applied this method to determine the folding and unfolding rate constants of the G-quadruplex of human telomere sequence (TTAGGG)(4) and its association and dissociation rate constants with the complementary strand (CCCTAA)(4). The results show that both the folding and unfolding occur on the time scale of minutes at physiological concentration of K(+). We speculate that this property might be important for telomere elongation. A complete set of the kinetic parameters for both of the structures allows us to study the competition between the formation of the quadruplex and the duplex. Calculations indicate that the formation of both the quadruplex and the duplex is strand concentration-dependent, and the quadruplex can be efficiently formed at low strand concentration. This property may provide the basis for the formation of the quadruplex in vivo in the presence of a complementary strand.

Motivation: Linear chromosomes carry on both ends repetitive DNA sequences called telomere. In the conventional model of semi-conservative DNA replication, the 3′-end of a linear DNA strand cannot be fully replicated, resulting in a single-stranded 3′ overhang at one end of the double-stranded DNA product. In this model, the length of the overhang is expected to be about the size of an RNA primer (about nine nucleotides for human cells). However, it has been found that the telomere overhangs in human cells can be as long as several hundred nucleotides. At present, the opinion regarding how such long overhangs are produced is controversial. Results: In order to gain insight into the mechanism by which long telomere overhangs are produced, we derived a mathematical model that can perfectly describe the length distribution of telomere overhangs in several human cell strains. The model suggests that the production of telomere overhangs can be explained by three contributions corresponding to three regions on the G-rich telomere template strand, namely, the region occupied by the last primer, that missed out by this primer at its 5′-side and the 3′-terminus of the template strand that is inaccessible to primase. The model can also be used to simulate incomplete telomere replication. Contact: tanclswu@public.wh.hb.cn * To whom correspondence should be addressed.

We recently reported that a DNA:RNA hybrid G-quadruplex (HQ) forms during transcription of DNA that bears two or more tandem guanine tracts (G-tract) on the nontemplate strand. Putative HQ-forming sequences are enriched in the nearby 1000 nt region right downstream of transcription start sites in the nontemplate strand of warm-blooded animals, and HQ regulates transcription under both in vitro and in vivo conditions. Therefore, knowledge of the mechanism of HQ formation is important for understanding the biological function of HQ as well as for manipulating gene expression by targeting HQ. In this work, we studied the mechanism of HQ formation using an in vitro T7 transcription model. We show that RNA synthesis initially produces an R-loop, a DNA:RNA heteroduplex formed by a nascent RNA transcript and the template DNA strand. In the following round of transcription, the RNA in the R-loop is displaced, releasing the RNA in single-stranded form (ssRNA). Then the G-tracts in the RNA can jointly form HQ with those in the nontemplate DNA strand. We demonstrate that the structural cascade R-loop → ssRNA → HQ offers opportunities to intercept HQ formation, which may provide a potential method to manipulate gene expression.

Telomere G-quadruplex is emerging as a promising anti-cancer target due to its inhibition to telomerase, an enzyme expressed in more than 85% tumors. Telomerase-mediated telomere extension and some other reactions require a free 3' telomere end in single-stranded form. G-quadruplex formation near the 3' end of telomere DNA can leave a 3' single-stranded tail of various sizes. How these terminal structures affect reactions at telomere end is not clear. In this work, we studied the 3' tail size-dependence of telomere extension by either telomerase or the alternative lengthening of telomere (ALT) mechanism as well as telomere G-quadruplex unwinding. We show that these reactions require a minimal tail of 8, 12 and 6 nt, respectively. Since we have shown that G-quadruplex tends to form at the farthest 3' distal end of telomere DNA leaving a tail of no more than 5 nt, these results imply that G-quadruplex formation may play a role in regulating reactions at the telomere ends and, as a result, serve as effective drug target for intervening telomere function.

Sequences with the potential to form G-quadruplex structures are spread throughout genomic DNA. G-quadruplexes in promoter regions can play regulatory roles in gene expression. Expression of protein-encoding genes involves processing of DNA and RNA molecules at the level of transcription and translation, respectively. In order to examine how the G-quadruplex affects processing of nucleic acids, we established a real-time fluorescent assay and studied the unwinding of intramolecular G-quadruplex formed by the human telomere, ILPR and PSMA4 sequences by the BLM helicase. Through comparison with their corresponding duplex substrates, we found that the unwinding of intramolecular G-quadruplex structures was much less efficient than that of the duplexes. This result is in contrast to previous reports that multistranded intermolecular G-quadruplexes are far better substrates for the BLM and other RecQ family helicases. In addition, the unwinding efficiency varied significantly among the G-quadruplex structures, which correlated with the stability of the structures. These facts suggest that G-quadruplex has the capability to modulate the processing of DNA and RNA molecules in a stability-dependent manner and, as a consequence, may provide a mechanism to play regulatory roles in events such as gene expression.

The association and dissociation rate constants (k(a) and k(d)) of DNA hybridizations involving dual, single or no stacking with different base-pairing sizes were measured, which reveals the advantage of stacking hybridization in both the kinetic and steady state.

Human chromosome ends are protected with kilobases repeats of TTAGGG. Telomere DNA shortens at replication. This shortening in most tumor cells is compensated by telomerase that adds telomere repeats to the 3′ end of the G-rich telomere strand. Four TTAGGG repeats can fold into G-quadruplex that is a poor substrate for telomerase. This property has been suggested to regulate telomerase activity in vivo and telomerase inhibition via G-quadruplex stabilization is considered a therapeutic strategy against cancer. Theoretically G-quadruplex can form anywhere along the long G-rich strand. Where G-quadruplex forms determines whether the 3′ telomere end is accessible to telomerase and may have implications in other functions telomere plays. We investigated G-quadruplex formation at different positions by DMS footprinting and exonuclease hydrolysis. We show that G-quadruplex preferentially forms at the very 3′ end than at internal positions. This property provides a molecular basis for telomerase inhibition by G-quadruplex formation. Moreover, it may also regulate those processes that depend on the structure of the very 3′ telomere end, for instance, the alternative lengthening of telomere mechanism, telomere T-loop formation, telomere end protection and the replication of bulky telomere DNA. Therefore, targeting telomere G-quadruplex may influence more telomere functions than simply inhibiting telomerase.

Single-stranded guanine-rich (G-rich) DNA can fold into a four-stranded G-quadruplex structure and such structures are implicated in important biological processes and therapeutic applications. So far, bioinformatic analysis has identified up to several hundred thousand of putative quadruplex sequences in the genome of human and other animal. Given such a large number of sequences, a fast assay would be desired to experimentally verify the structure of these sequences. Here we describe a method that identifies the quadruplex structure by a single-stranded DNA binding protein from a thermoautotrophic archaeon. This protein binds single-stranded DNA in the unfolded, but not in the folded form. Upon binding to DNA, its fluorescence can be quenched by up to 70%. Formation of quadruplex greatly reduces fluorescence quenching in a K+-dependent manner. This structure-dependent quenching provides simple and fast detection of quadruplex in DNA at low concentration without DNA labelling. Copyright © 2007 John Wiley & Sons, Ltd.

The G-rich strand of human telomeric DNA can fold into a four-stranded structure called G-quadruplex and inhibit telomerase activity that is expressed in 85-90% tumor cells. For this reason, telomere quadruplex is emerging as a potential therapeutic target for cancer. Information on the structure of the quadruplex in the physiological environment is important for structure-based drug design targeting the quadruplex. Recent studies have raised significant controversy regarding the exact structure of the quadruplex formed by human telomeric DNA in a physiological relevant environment. Studies on the crystal prepared in K+ solution revealed a distinct propeller-shaped parallel-stranded conformation. However, many later works failed to confirm such structure in physiological K+ solution but rather led to the identification of a different hybrid-type mixed parallel/antiparallel quadruplex. Here we demonstrate that human telomere DNA adopts a parallel-stranded conformation in physiological K+ solution under molecular crowding conditions created by PEG. At the concentration of 40% (w/v), PEG induced complete structural conversion to a parallel-stranded G-quadruplex. We also show that the quadruplex formed under such a condition has unusual stability and significant negative impact on telomerase processivity. Since the environment inside cells is molecularly crowded, our results obtained under the cell mimicking condition suggest that the parallel-stranded quadruplex may be the more favored structure under physiological conditions, and drug design targeting the human telomeric quadruplex should take this into consideration.

Chromosomes in vertebrates are protected at both ends by telomere DNA composed of tandem (TTAGGG)n repeats. DNA replication produces a blunt-ended leading strand telomere and a lagging strand telomere carrying a single-stranded G-rich overhang at its end. The G-rich strand can form G-quadruplex structure in the presence of K+ or Na+. At present, it is not clear whether quadruplex can form in the double-stranded telomere region where the two complementary strands are constrained in close vicinity and quadruplex formation, if possible, has to compete with the formation of the conventional Watson–Crick duplex. In this work, we studied quadruplex formation in oligonucleotides and double-stranded DNA containing both the G- and C-rich sequences to better mimic the in vivo situation. Under such competitive condition only duplex was observed in dilute solution containing physiological concentration of K+. However, quadruplex could preferentially form and dominate over duplex structure under molecular crowding condition created by PEG as a result of significant quadruplex stabilization and duplex destabilization. This observation suggests quadruplex may potentially form or be induced at the blunt end of a telomere, which may present a possible alternative form of structures at telomere ends.

Telomere length homeostasis is a prerequisite for the generation and growth of cancer. In >85% cancer cells, telomere length is maintained by telomerase that add telomere repeats to the end of telomere DNA. Because the G-rich strand of telomere DNA can fold into G-quadruplex that inhibits telomerase activity, stabilizing telomere quadruplex by small molecules is emerging as a potential therapeutic strategy against cancer. In these applications, the specificity of small molecules toward quadruplex over other forms of DNA is an important property to ensure no processes other than telomere elongation are interrupted. The evaluating assays currently available more or less have difficulty identifying or distinguishing quadruplex-irrelevant effect from quadruplex stabilization. Here, we describe an exonuclease I hydrolysis assay that evaluates quadruplex stabilization by DNA-interacting compounds, discriminates inhibitory effect from different sources and helps determine the optimal compound concentration.

Background Horizontal transfer (HT) of transposable elements (TEs) into a new genome is considered as an important force to drive genome variation and biological innovation. However, most of the HT of DNA transposons previously described occurred between closely related species or insects. Results In this study, we carried out a detailed analysis of four DNA transposons, which were found in the first sequenced twisted-wing parasite, Mengenilla moldrzyki. Through the homology-based strategy, these transposons were also identified in other insects, freshwater planarian, hydrozoans, and bats. The phylogenetic distribution of these transposons was discontinuous, and they showed extremely high sequence identities (>87%) over their entire length in spite of their hosts diverging more than 300 million years ago (Mya). Additionally, phylogenies and comparisons of transposons versus orthologous gene identities demonstrated that these transposons have transferred into their hosts by independent HTs. Conclusions Here, we provided the first documented example of HT of CACTA transposons, which have been so far extensively studied in plants. Our results demonstrated that bats had continuously acquired new DNA elements via HT. This implies that predation on a large quantity of insects might increase bat exposure to HT. In addition, parasite-host interaction might facilitate exchanging of their genetic materials. Electronic supplementary material The online version of this article (doi:10.1186/s13100-014-0033-1) contains supplementary material, which is available to authorized users.