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.

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.

Submolecular dissection reveals strong and specific binding of polyamide-pyridostatin conjugates to human telomere interface.

To modulate biological functions, G-quadruplexes in genome are often non-specifically targeted by small molecules. Here, specificity is increased by targeting both G-quadruplex and its flanking duplex DNA in a naturally occurring dsDNA-ssDNA telomere interface using polyamide (PA) and pyridostatin (PDS) conjugates (PA-PDS). We innovated a single-molecule assay in which dissociation constant (Kd) of the conjugate can be separately evaluated from the binding of either PA or PDS. We found Kd of 0.8 nM for PA-PDS, which is much lower than PDS (Kd ∼ 450 nM) or PA (Kd ∼ 35 nM). Functional assays further indicated that the PA-PDS conjugate stopped the replication of a DNA polymerase more efficiently than PA or PDS. Our results not only established a new method to dissect multivalent binding into actions of individual monovalent components, they also demonstrated a strong and specific G-quadruplex targeting strategy by conjugating highly specific duplex-binding molecules with potent quadruplex ligands.

Random Formation of G-Quadruplexes in the Full-Length Human Telomere Overhangs Leads to a Kinetic Folding Pattern with Targetable Vacant G-Tracts.

G-Quadruplexes formed in the 3' telomere overhang (∼200 nucleotides) have been shown to regulate biological functions of human telomeres. The mechanism governing the population pattern of multiple telomeric G-quadruplexes is yet to be elucidated inside the telomeric overhang in a time window shorter than thermodynamic equilibrium. Using a single-molecule force ramping assay, we quantified G-quadruplex populations in telomere overhangs over a full physiological range of 99-291 nucleotides. We found that G-quadruplexes randomly form in these overhangs within seconds, which leads to a population governed by a kinetic, rather than a thermodynamic, folding pattern. The kinetic folding gives rise to vacant G-tracts between G-quadruplexes. By targeting these vacant G-tracts using complementary DNA fragments, we demonstrated that binding to the telomeric G-quadruplexes becomes more efficient and specific for telomestatin derivatives.

Mechanical Cooperativity in DNA Cruciform Structures.

Unlike short-range chemical bonds that maintain chemical properties of a biological molecule, long-range mechanical interactions determine mechanochemical properties of molecules. Limited by experimental approaches, however, direct quantification of such mechanical interactions is challenging. Using magneto-optical tweezers, herein we found torque can change the topology and mechanochemical property of DNA cruciform, a naturally occurring structure consisting of two opposing hairpin arms. Both mechanical and thermodynamic stabilities of DNA cruciforms increase with positive torque, which have been attributed to the topological coupling between DNA template and the cruciform. The coupling exists simultaneously in both arms of a cruciform, which coordinates the folding and unfolding of the cruciform, leading to a mechanical cooperativity not observed previously. As DNA torque readily varies during transcriptions, our finding suggests that DNA cruciforms can modulate transcriptions by adjusting their properties according to the torque.

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.

Decay of Piwi-Interacting RNAs in Human Cells Is Primarily Mediated by 5' to 3' Exoribonucleases.

Piwi-interacting RNAs (piRNAs) are a group of small noncoding RNA molecules that regulate the activity of transposons and control gene expression. The cellular concentration of RNAs is generally maintained by their rates of biogenesis and degradation. Although the biogenesis pathways of piRNAs have been well defined, their degradation mechanism is still unknown. Here, we show that degradation of human piRNAs is mostly dependent on the 5'-3' exoribonuclease pathway. The presence of 3'-end 2'-O-methylation in piRNAs significantly reduced their degradation through the exosome-mediated decay pathway. The accumulation of piRNAs in XRN1 and XRN2 exoribonuclease-depleted cells further supports the 5'-3' exoribonuclease-mediated decay of piRNAs. Moreover, formation of stable secondary structures in piRNAs slows the rate of XRN1-mediated degradation. Our findings establish a framework for the piRNA degradation mechanism in cells and thus provide crucial information about how the basal level concentration of piRNAs is maintained in cells.

Reversal of G-Quadruplexes' Role in Translation Control When Present in the Context of an IRES.

G-quadruplexes (GQs) are secondary nucleic acid structures that play regulatory roles in various cellular processes. G-quadruplex-forming sequences present within the 5' UTR of mRNAs can function not only as repressors of translation but also as elements required for optimum function. Based upon previous reports, the majority of the 5' UTR GQ structures inhibit translation, presumably by blocking the ribosome scanning process that is essential for detection of the initiation codon. However, there are certain mRNAs containing GQs that have been identified as positive regulators of translation, as they are needed for translation initiation. While most cellular mRNAs utilize the 5' cap structure to undergo cap-dependent translation initiation, many rely on cap-independent translation under certain conditions in which the cap-dependent initiation mechanism is not viable or slowed down, for example, during development, under stress and in many diseases. Cap-independent translation mainly occurs via Internal Ribosomal Entry Sites (IRESs) that are located in the 5' UTR of mRNAs and are equipped with structural features that can recruit the ribosome or other factors to initiate translation without the need for a 5' cap. In this review, we will focus only on the role of RNA GQs present in the 5' UTR of mRNAs, where they play a critical role in translation initiation, and discuss the potential mechanism of this phenomenon, which is yet to be fully delineated.

Encounters between Cas9/dCas9 and G-Quadruplexes: Implications for Transcription Regulation and Cas9-Mediated DNA Cleavage.

Using the nuclease-dead Cas9 (dCas9), we targeted in cellulo a G-rich sequence, which contains multiple potentially G-quadruplex (GQ) forming sites, within the human tyrosine hydroxylase (TH) promoter. We demonstrate that transcription can be up or down regulated by targeting different parts of this G-rich sequence. Our results suggest that TH transcription levels correlate with stability of different GQs formed by this sequence and targeting them with dCas9 can modulate their stability. Unlike alternative approaches, regulating TH expression by targeting the promoter GQs with dCas9 enables a specific and potentially transient control and does not require mutations in the sequence. We also investigated whether the presence of GQs in target sequences impacts DNA cleavage activity of Cas9. We discovered significant reduction in cleavage activity when the vicinity of a high-stability GQ was targeted. Furthermore, this reduction is significantly more prominent for the G-rich strand compared to the complementary C-rich strand.

Single-Molecule Investigations of G-Quadruplex.

The genome-wide occurrence of G-quadruplexes and their demonstrated biological activities call for detailed understanding on the stability and transition kinetics of the structures. Although the core structural element in a G-quadruplex is simple and requires only four tandem repeats of Guanine rich sequences, there is rather rich conformational diversity in this structure. Corresponding to this structural diversity, it displays involved transition kinetics within individual G-quadruplexes and complicated interconversion among different G-quadruplex species. Due to the inherently high signal-to-noise ratio in the measurement, single-molecule tools offer a unique capability to investigate the thermodynamic, kinetic, and mechanical properties of G-quadruplexes with dynamic conformations. In this chapter, we describe different single molecule methods such as atomic-force microscopy (AFM), single-molecule fluorescence resonance energy transfer (smFRET), optical, magnetic, and magneto-optical tweezers to investigate G-quadruplex structures as well as their interactions with small-molecule ligands.

A Review on the Extraction of Quantitative Retinal Microvascular Image Feature.

Digital image processing is one of the most widely used computer vision technologies in biomedical engineering. In the present modern ophthalmological practice, biomarkers analysis through digital fundus image processing analysis greatly contributes to vision science. This further facilitates developments in medical imaging, enabling this robust technology to attain extensive scopes in biomedical engineering platform. Various diagnostic techniques are used to analyze retinal microvasculature image to enable geometric features measurements such as vessel tortuosity, branching angles, branching coefficient, vessel diameter, and fractal dimension. These extracted markers or characterized fundus digital image features provide insights and relates quantitative retinal vascular topography abnormalities to various pathologies such as diabetic retinopathy, macular degeneration, hypertensive retinopathy, transient ischemic attack, neovascular glaucoma, and cardiovascular diseases. Apart from that, this noninvasive research tool is automated, allowing it to be used in large-scale screening programs, and all are described in this present review paper. This paper will also review recent research on the image processing-based extraction techniques of the quantitative retinal microvascular feature. It mainly focuses on features associated with the early symptom of transient ischemic attack or sharp stroke.