Emerging accessibility patterns in long telomeric overhangs.

We present single-molecule experimental and computational modeling studies investigating the accessibility of human telomeric overhangs of physiologically relevant lengths. We studied 25 different overhangs that contain 4-28 repeats of GGGTTA (G-Tract) sequence and accommodate one to seven tandem G-quadruplex (GQ) structures. Using the FRET-PAINT method, we probed the distribution of accessible sites via a short imager strand, which is complementary to a G-Tract and transiently binds to available sites. We report accessibility patterns that periodically change with overhang length and interpret these patterns in terms of the underlying folding landscape and folding frustration. Overhangs that have [4n]G-Tracts, (12, 16, 20…) demonstrate the broadest accessibility patterns where the peptide nucleic acid probe accesses G-Tracts throughout the overhang. On the other hand, constructs with [4n+2]G-Tracts, (14, 18, 22…) have narrower patterns where the neighborhood of the junction between single- and double-stranded telomeres is most accessible. We interpret these results as the folding frustration being higher in [4n]G-Tract constructs compared to [4n+2]G-Tract constructs. We also developed a computational model that tests the consistency of different folding stabilities and cooperativities between neighboring GQs with the observed accessibility patterns. Our experimental and computational studies suggest the neighborhood of the junction between single- and double-stranded telomeres is least stable and most accessible, which is significant as this is a potential site where the connection between POT1/TPP1 (bound to single-stranded telomere) and other shelterin proteins (localized on double-stranded telomere) is established.

Protection of the Telomeric Junction by the Shelterin Complex.

Shelterin serves critical roles in suppressing superfluous DNA damage repair pathways on telomeres. The junction between double-stranded telomeric tracts (dsTEL) and single-stranded telomeric overhang (ssTEL) is the most accessible region of the telomeric DNA. The shelterin complex contains dsTEL and ssTEL binding proteins and can protect this junction by bridging the ssTEL and dsTEL tracts. To test this possibility, we monitored shelterin binding to telomeric DNA substrates with varying ssTEL and dsTEL lengths and quantified its impact on telomere accessibility using single-molecule fluorescence microscopy methods in vitro. We identified the first dsTEL repeat nearest the junction as the preferred binding site for creating the shelterin bridge. Shelterin requires at least two ssTEL repeats, while the POT1 subunit of shelterin that binds to ssTEL requires longer ssTEL tracts for stable binding to telomeres and effective protection of the junction region. The ability of POT1 to protect the junction is significantly enhanced by the 5'-phosphate at the junction. Collectively, our results show that shelterin enhances the binding stability of POT1 to ssTEL and provides more effective protection compared with POT1 alone by bridging single- and double-stranded telomeric tracts.

Impact of Divalent Cations on In-Layer Positional Order of DNA-Based Liquid Crystals: Implications for DNA Condensation.

The layered liquid crystalline phases formed by DNA molecules, which include rigid and flexible segments ("gapped DNA"), enable the study of both end-to-end stacking and side-to-side (helix-to-helix) lateral interactions, forming a model system to study such interactions at physiologically relevant DNA and ion concentrations. The observed layer structure exhibits long-range interlayer and in-layer positional correlations. In particular, the in-layer order has implications for DNA condensation, as it reflects whether these normally repulsive interactions become attractive under certain ionic conditions. Using synchrotron small-angle X-ray scattering measurements, we investigate the impact of divalent Mg2+ cations (in addition to a constant 150 mM Na+) on the stability of the inter- and in-layer DNA ordering as a function of temperature between 5 and 65 °C. DNA constructs with different terminal base pairings were created to mediate the strength of the attractive end-to-end stacking interactions between the blunt ends of the gapped DNA constructs. We demonstrate that the stabilities at a fixed DNA concentration of both interlayer and in-layer order are significantly enhanced even at a few mM Mg2+ concentration. The stabilities are even higher at 30 mM Mg2+; however, a marked decrease is observed at 100 mM Mg2+, suggesting a change in the nature of side-by-side interactions within this Mg2+ concentration range. We discuss the implications of these results in terms of counterion-mediated DNA-DNA attraction and DNA condensation.

Shelterin reduces the accessibility of telomeric overhangs.

Telomeres terminate with a 50-300 bases long single-stranded G-rich overhang, which can be misrecognized as a DNA damage repair site. Shelterin plays critical roles in maintaining and protecting telomere ends by regulating access of various physiological agents to telomeric DNA, but the underlying mechanism is not well understood. Here, we measure how shelterin affects the accessibility of long telomeric overhangs by monitoring transient binding events of a short complementary peptide nucleic acid (PNA) probe using FRET-PAINT in vitro. We observed that the POT1 subunit of shelterin reduces the accessibility of the PNA probe by ∼2.5-fold, indicating that POT1 effectively binds to and protects otherwise exposed telomeric sequences. In comparison, a four-component shelterin stabilizes POT1 binding to the overhang by tethering POT1 to the double-stranded telomeric DNA and reduces the accessibility of telomeric overhangs by ∼5-fold. This enhanced protection suggests shelterin restructures the junction between single and double-stranded telomere, which is otherwise the most accessible part of the telomeric overhang.

Mono- and bilayer smectic liquid crystal ordering in dense solutions of "gapped" DNA duplexes.

Although its mesomorphic properties have been studied for many years, only recently has the molecule of life begun to reveal the true range of its rich liquid crystalline behavior. End-to-end interactions between concentrated, ultrashort DNA duplexes-driving the self-assembly of aggregates that organize into liquid crystal phases-and the incorporation of flexible single-stranded "gaps" in otherwise fully paired duplexes-producing clear evidence of an elementary lamellar (smectic-A) phase in DNA solutions-are two exciting developments that have opened avenues for discovery. Here, we report on a wider investigation of the nature and temperature dependence of smectic ordering in concentrated solutions of various "gapped" DNA (GDNA) constructs. We examine symmetric GDNA constructs consisting of two 48-base pair duplex segments bridged by a single-stranded sequence of 2 to 20 thymine bases. Two distinct smectic layer structures are observed for DNA concentration in the range [Formula: see text] mg/mL. One exhibits an interlayer periodicity comparable with two-duplex lengths ("bilayer" structure), and the other has a period similar to a single-duplex length ("monolayer" structure). The bilayer structure is observed for gap length ≳10 bases and melts into the cholesteric phase at a temperature between 30 °C and 35 °C. The monolayer structure predominates for gap length ≲10 bases and persists to [Formula: see text]C. We discuss models for the two layer structures and mechanisms for their stability. We also report results for asymmetric gapped constructs and for constructs with terminal overhangs, which further support the model layer structures.

Stability of End-to-End Base Stacking Interactions in Highly Concentrated DNA Solutions.

Positionally ordered bilayer liquid crystalline nanostructures formed by gapped DNA (GDNA) constructs provide a practical window into DNA-DNA interactions at physiologically relevant DNA concentrations; concentrations several orders of magnitude greater than those in commonly used biophysical assays. The bilayer structure of these states of matter is stabilized by end-to-end base stacking interactions; moreover, such interactions also promote in-plane positional ordering of duplexes that are separated from each other by less than twice the duplex diameter. The end-to-end stacked as well as in-plane ordered duplexes exhibit distinct signatures when studied via small-angle X-ray scattering (SAXS). This enables analysis of the thermal stability of both the end-to-end and side-by-side interactions. We performed synchrotron SAXS experiments over a temperature range of 5-65 °C on GDNA constructs that differ only by the terminal base-pairs at the blunt duplex ends, resulting in identical side-by-side interactions, while end-to-end base stacking interactions are varied. Our key finding is that bilayers formed by constructs with GC termination transition into the monolayer state at temperatures as much as 30 °C higher than for those with AT termination, while mixed (AT/GC) terminations have intermediate stability. By modeling the bilayer melting in terms of a temperature-dependent reduction in the average fraction of end-to-end paired duplexes, we estimate the stacking free energies in DNA solutions of physiologically relevant concentrations. The free-energies thereby determined are generally smaller than those reported in single-molecule studies, which might reflect the elevated DNA concentrations in our studies.

Detecting secondary structure formation with FRET-PAINT.

We present single molecule studies demonstrating the capabilities of the FRET-PAINT method to detect secondary structures that would be challenging to detect with alternative methods, particularly single molecule FRET (smFRET). Instead of relying on the change in end-to-end separation as in smFRET, we use the change in accessibility to a small probe as the criterion for secondary structure formation and relative stability. As a model system, we study G-triplex formation by human telomeric repeat sequences in different structural contexts.

Interrogating accessibility of telomeric sequences with FRET-PAINT: evidence for length-dependent telomere compaction.

Single-stranded telomeric overhangs are ∼200 nucleotides long and can form tandem G-quadruplex (GQ) structures, which reduce their accessibility to nucleases and proteins that activate DNA damage response. Whether these tandem GQs further stack to form compact superstructures, which may provide better protection for longer telomeres, is not known. We report single-molecule measurements where the accessibility of 24-144 nucleotide long human telomeric DNA molecules is interrogated by a short PNA molecule that is complementary to a single GGGTTA repeat, as implemented in the FRET-PAINT method. Binding of the PNA strand to available GGGTTA sequences results in discrete FRET bursts which were analyzed in terms of their dwell times, binding frequencies, and topographic distributions. The binding frequencies were greater for binding to intermediate regions of telomeric DNA compared to 3'- or 5'-ends, suggesting these regions are more accessible. Significantly, the binding frequency per telomeric repeat monotonically decreased with increasing telomere length. These results are consistent with telomeres forming more compact structures at longer lengths, reducing accessibility of these critical genomic sites.

Quantifying the impact of small molecule ligands on G-quadruplex stability against Bloom helicase.

G-quadruplex (GQ) stabilizing small molecule (SM) ligands have been used to stabilize human telomeric GQ (hGQ) to inhibit telomerase activity, or non-telomeric GQs to manipulate gene expression at transcription or translation level. GQs are known to inhibit DNA replication unless destabilized by helicases, such as Bloom helicase (BLM). Even though the impact of SM ligands on thermal stability of GQs is commonly used to characterize their efficacy, how these ligands influence helicase-mediated GQ unfolding is not well understood. Three prominent SM ligands (an oxazole telomestatin derivative, pyridostatin, and PhenDC3), which thermally stabilize hGQ at different levels, were utilized in this study. How these ligands influence BLM-mediated hGQ unfolding was investigated using two independent single-molecule approaches. While the frequency of dynamic hGQ unfolding events was used as the metric in the first approach, the second approach was based on quantifying the cumulative unfolding activity as a function of time. All three SM ligands inhibited BLM activity at similar levels, 2-3 fold, in both approaches. Our observations suggest that the impact of SM ligands on GQ thermal stability is not an ideal predictor for their inhibition of helicase-mediated unfolding, which is physiologically more relevant.

Ribosomal RNA Methyltransferase RsmC Moonlights as an RNA Chaperone.

Ribosomes are ribonucleoprotein particles that are essential for protein biosynthesis in all forms of life. During ribosome biogenesis, transcription, folding, modification, and processing of rRNA are coupled to the assembly of proteins. Various assembly factors are required to synchronize all different processes that occur during ribosome biogenesis. Herein, the RNA chaperone and RNA strand annealing activity of rRNA modification enzyme ribosome small subunit methyltransferase C (RsmC), which modifies guanine to 2-methylguanosine (m2 G) at position 1207 of 16S rRNA (Escherichia coli nucleotide numbering) located at helix 34 (h34), are reported. A 25-fold increase in the h34 RNA strand annealing rates is observed in the presence of RsmC. Single-molecule FRET experiments confirmed the ability of protein RsmC to denature a non-native structure formed by one of the two h34 strands and to form a native-like duplex. This observed RNA chaperone activity of protein RsmC might play a vital role in the rapid generation of functional ribosomes.

A single molecule study of a fluorescently labeled telomestatin derivative and G-quadruplex interactions.

The potential use of G-quadruplex (GQ) stabilizing small molecules as anti-cancer drugs has created a flurry of activity on various aspects of these molecules. Telomestatin and oxazole telomestatin derivatives (OTD) are some of the most prominent of such molecules, yet the underlying dynamics of their interactions with GQ and the extent of heterogeneities in these interactions are not known. We performed single molecule measurements to study binding kinetics, rotational freedom, and dwell time distributions of a Cy5-labeled OTD (L1Cy5-7OTD) as it interacted with several different GQ structures. Our measurements show that L1Cy5-7OTD dwells on more stable GQ for longer times and binds to such GQ with higher frequency. The dwell times showed a broad distribution, but were longer than a minute for a significant fraction of molecules (characteristic dwell time τ = 192 ± 15 s and τ = 98 ± 15 s for the more and less stable GQ, respectively). In addition, L1Cy5-7OTD might be able to bind to GQ in at least two different primary orientations and occasionally transition between these orientations. The dwell time in one of these orientations was significantly longer than that in the other one, suggesting different stabilities for different binding orientations.

Impact of Small Molecules on Intermolecular G-Quadruplex Formation.

We performed single molecule studies to investigate the impact of several prominent small molecules (the oxazole telomestatin derivative L2H2-6OTD, pyridostatin, and Phen-DC3) on intermolecular G-quadruplex (i-GQ) formation between two guanine-rich DNA strands that had 3-GGG repeats in one strand and 1-GGG repeat in the other (3+1 GGG), or 2-GGG repeats in each strand (2+2 GGG). Such structures are not only physiologically significant but have recently found use in various biotechnology applications, ranging from DNA-based wires to chemical sensors. Understanding the extent of stability imparted by small molecules on i-GQ structures, has implications for these applications. The small molecules resulted in different levels of enhancement in i-GQ formation, depending on the small molecule and arrangement of GGG repeats. The largest enhancement we observed was in the 3+1 GGG arrangement, where i-GQ formation increased by an order of magnitude, in the presence of L2H2-6OTD. On the other hand, the enhancement was limited to three-fold with Pyridostatin (PDS) or less for the other small molecules in the 2+2 GGG repeat case. By demonstrating detection of i-GQ formation at the single molecule level, our studies illustrate the feasibility to develop more sensitive sensors that could operate with limited quantities of materials.

A practical guide to studying G-quadruplex structures using single-molecule FRET.

In this article, we summarize the knowledge and best practices learned from bulk and single-molecule measurements to address some of the frequently experienced difficulties in single-molecule Förster resonance energy transfer (smFRET) measurements on G-quadruplex (GQ) structures. The number of studies that use smFRET to investigate the structure, function, dynamics, and interactions of GQ structures has grown significantly in the last few years, with new applications already in sight. However, a number of challenges need to be overcome before reliable and reproducible smFRET data can be obtained in measurements that include GQ. The annealing and storage conditions, the location of fluorophores on the DNA construct, and the ionic conditions of the experiment are some of the factors that are of critical importance for the outcome of measurements, and many of these manifest themselves in unique ways in smFRET assays. By reviewing these aspects and providing a summary of best practices, we aim to provide a practical guide that will help in successfully designing and performing smFRET studies on GQ structures.

ATP-dependent G-quadruplex unfolding by Bloom helicase exhibits low processivity.

Various helicases and single stranded DNA (ssDNA) binding proteins unfold G-quadruplex (GQ) structures. However, the underlying mechanisms of this activity have only recently come to focus. We report kinetic studies on Bloom (BLM) helicase and human telomeric GQ interactions using single-molecule Förster resonance energy transfer (smFRET). Using partial duplex DNA (pdDNA) constructs with different 5' ssDNA overhangs, we show that BLM localizes in the vicinity of ssDNA/double-stranded DNA (dsDNA) junction and reels in the ssDNA overhang in an ATP-dependent manner. A comparison of DNA constructs with or without GQ in the overhang shows that GQ unfolding is achieved in 50-70% of reeling attempts under physiological salt and pH conditions. The unsuccessful attempts often result in dissociation of BLM from DNA which slows down the overall BLM activity. BLM-mediated GQ unfolding is typically followed by refolding of the GQ, a pattern that is repeated several times before BLM dissociates from DNA. BLM is significantly less processive compared to the highly efficient GQ destabilizer Pif1 that can repeat GQ unfolding activity hundreds of times before dissociating from DNA. Despite the variations in processivity, our studies point to possible common patterns used by different helicases in minimizing the duration of stable GQ formation.

G-quadruplex formation in telomeres enhances POT1/TPP1 protection against RPA binding.

Human telomeres terminate with a single-stranded 3' G overhang, which can be recognized as a DNA damage site by replication protein A (RPA). The protection of telomeres (POT1)/POT1-interacting protein 1 (TPP1) heterodimer binds specifically to single-stranded telomeric DNA (ssTEL) and protects G overhangs against RPA binding. The G overhang spontaneously folds into various G-quadruplex (GQ) conformations. It remains unclear whether GQ formation affects the ability of POT1/TPP1 to compete against RPA to access ssTEL. Using single-molecule Förster resonance energy transfer, we showed that POT1 stably loads to a minimal DNA sequence adjacent to a folded GQ. At 150 mM K(+), POT1 loading unfolds the antiparallel GQ, as the parallel conformation remains folded. POT1/TPP1 loading blocks RPA's access to both folded and unfolded telomeres by two orders of magnitude. This protection is not observed at 150 mM Na(+), in which ssTEL forms only a less-stable antiparallel GQ. These results suggest that GQ formation of telomeric overhangs may contribute to suppression of DNA damage signals.

A Comparative Study of G-Quadruplex Unfolding and DNA Reeling Activities of Human RECQ5 Helicase.

RECQ5 is one of five members of the RecQ family of helicases in humans, which include RECQ1, Bloom (BLM), Werner (WRN), RECQ4, and RECQ5. Both WRN and BLM have been shown to resolve G-quadruplex (GQ) structures. Deficiencies in unfolding GQ are known to result in DNA breaks and genomic instability, which are prominent in Werner and Bloom syndromes. RECQ5 is significant in suppressing sister chromatid exchanges during homologous recombination but its GQ unfolding activity are not known. We performed single-molecule studies under different salt (50-150 mM KCl or NaCl) and ATP concentrations on different GQ constructs including human telomeric GQ (with different overhangs and polarities) and GQ formed by thrombin-binding aptamer to investigate this activity. These studies demonstrated a RECQ5-mediated GQ unfolding activity that was an order of magnitude weaker than BLM and WRN. On the other hand, BLM and RECQ5 demonstrated similar single-stranded DNA (ssDNA) reeling activities that were not coupled to GQ unfolding. These results demonstrate overlap in function between these RecQ helicases; however, the relatively weak GQ destabilization activity of RECQ5 compared to BLM and WRN suggests that RECQ5 is not primarily associated with GQ destabilization, but could substitute for the more efficient helicases under conditions where their activity is compromised. In addition, these results implicate a more general role for helicase-promoted ssDNA reeling activity such as removal of proteins at the replication fork, whereas the association of ssDNA reeling with GQ destabilization is more helicase-specific.

RecQ-core of BLM unfolds telomeric G-quadruplex in the absence of ATP.

Various helicases and single-stranded DNA (ssDNA) binding proteins are known to destabilize G-quadruplex (GQ) structures, which otherwise result in genomic instability. Bulk biochemical studies have shown that Bloom helicase (BLM) unfolds both intermolecular and intramolecular GQ in the presence of ATP. Using single molecule FRET, we show that binding of RecQ-core of BLM (will be referred to as BLM) to ssDNA in the vicinity of an intramolecular GQ leads to destabilization and unfolding of the GQ in the absence of ATP. We show that the efficiency of BLM-mediated GQ unfolding correlates with the binding stability of BLM to ssDNA overhang, as modulated by the nucleotide state, ionic conditions, overhang length and overhang directionality. In particular, we observed enhanced GQ unfolding by BLM in the presence of non-hydrolysable ATP analogs, which has implications for the underlying mechanism. We also show that increasing GQ stability, via shorter loops or higher ionic strength, reduces BLM-mediated GQ unfolding. Finally, we show that while WRN has similar activity as BLM, RecQ and RECQ5 helicases do not unfold GQ in the absence of ATP at physiological ionic strength. In summary, our study points to a novel and potentially very common mechanism of GQ destabilization mediated by proteins binding to the vicinity of these structures.

G-Quadruplex-Enabling Sequence within the Human Tyrosine Hydroxylase Promoter Differentially Regulates Transcription.

G-Quadruplexes (GQs) found within the promoter regions of genes are known to mostly act as repressors of transcription. Here we report a guanosine (G)-rich segment in the 3'-proximal promoter region of human tyrosine hydroxylase (TH), which acts as a necessary element for transcription. Tyrosine hydroxylase catalyzes the rate-limiting step in the catecholamine biosynthesis and is linked to several common neurological disorders such as Parkinson's and schizophrenia. A 45 nucleotide (nt) sequence (wtTH49) within the human TH promoter contains multiple G-stretches that are extremely well conserved among the primates but deviate in rodents, which raises the possibility of variation in the GQ structures formed in the two orders with the potential for a distinctive functional outcome. Biochemical and biophysical studies, including single-molecule Förster resonance energy transfer, indicate that the wtTH49 sequence can adopt multiple GQ structures by using different combinations of G-stretches. A functional assay performed with 2.8 kb of the 3'-proximal end of the TH promoter and a mutated version (TH49fm; mutated wtTH49) that is unable to form any GQ structure indicates that overall the GQ-enabling wtTH49 sequence is functionally necessary and enhances human TH promoter activity by 5-fold compared to that of the mutant. Two additional mutants, each of which was designed to form distinct GQs, differentially affected reporter gene transcription. A cationic porphyrin TMPyP4 destabilizes the wtTH49 GQ and lowers the level of reporter gene expression, although its analogue, TMPyP2, fails to elicit any response. The 45 nt G-rich sequence within the human TH promoter can form multiple GQ structures, is a necessary element in transcription, and depending on the utilized combination of G-stretches affects transcription in different ways.

Structure, Topology, and Stability of Multiple G-quadruplexes in Long Telomeric Overhangs.

Telomeres and their single stranded overhangs gradually shorten with successive cell divisions, as part of the natural aging process, but can be elongated by telomerase, a nucleoprotein complex which is activated in the majority of cancers. This prominent implication in cancer and aging has made the repetitive telomeric sequences (TTAGGG repeats) and the G-quadruplex structures that form in their overhangs the focus of intense research in the past several decades. However, until recently most in vitro efforts to understand the structure, stability, dynamics, and interactions of telomeric overhangs had been focused on short sequences that are not representative of longer sequences encountered in a physiological setting. In this review, we will provide a broad perspective about telomeres and associated factors, and introduce the agents and structural characteristics involved in organizing, maintaining, and protecting telomeric DNA. We will also present a summary of recent research performed on long telomeric sequences, nominally defined as those that can form two or more tandem G-quadruplexes, i.e., which contain eight or more TTAGGG repeats. Results of experimental studies using a broad array of experimental tools, in addition to recent computational efforts will be discussed, particularly in terms of their implications for the stability, folding topology, and compactness of the tandem G-quadruplexes that form in long telomeric overhangs.

Stalling of Transcription by Putative G-quadruplex Sequences and CRISPR-dCas9.

Putative G-quadruplex forming sequences (PQS) have been identified in promoter sequences of prominent genes that are implicated among others in cancer and neurological disorders. We explored mechanistic aspects of CRISPR-dCas9-mediated gene expression regulation, which is transient and sequence specific unlike alternative approaches that lack such specificity or create permanent mutations, using the PQS in tyrosine hydroxylase (TH) and c-Myc promoters as model systems. We performed in vitro ensemble and single molecule investigations to study whether G-quadruplex (GQ) structures or dCas9 impede T7 RNA polymerase (RNAP) elongation process and whether orientation of these factors is significant. Our results demonstrate that dCas9 is more likely to block RNAP progression when the non-template strand is targeted. While the GQ in TH promoter was effectively destabilized when the dCas9 target site partially overlapped with the PQS, the c-Myc GQ remained folded and stalled RNAP elongation. We also determined that a minimum separation between the transcription start site and the dCas9 target site is required for effective stalling of RNAP by dCas9. Our study provides significant insights about the factors that impact dCas9-mediated transcription regulation when dCas9 targets the vicinity of sequences that form secondary structures and provides practical guidelines for designing guide RNA sequences.