Biology subjects

Balci, H.

Publications and source records attributed to Balci, H..

17 recordsLinked to original sources

bioRxiv2026

Cap-Independent Translation Regulation of VEGF Using CRISPR-dCas13d

Vascular endothelial growth factor (VEGF) is a central driver of pathological angiogenesis in cancer and retinal disease. A G-quadruplex (GQ) structure that forms in an Internal Ribosome Entry Site (IRES) of VEGF was proposed to recruit the 40S ribosome and initiate cap-independent translation under hypoxia. We demonstrate that blocking this GQ with dCas13d represses VEGF translation by up to 5-fold, without impacting mRNA abundance, under hypoxia. Similar levels of repression were attained in HeLa and human umbilical vein endothelial cells (HUVECs) using immunofluorescence and western blot assays, as well as a bicistronic dual-luciferase experiment. Single molecule Forster resonance energy transfer (smFRET) and fluorescence enhancement assays demonstrated destabilization of the GQ by CRISPR-dCas13d. We also tested the functional consequences of this regulation using tube formation assay in bovine aortic endothelial cells (BAOECs), which demonstrated consistent results with VEGF expression levels. Our study demonstrates the feasibility of using CRISPR-dCas13d as a sequence-specific and transient translation regulator for a large group of genes that contain translationally-active secondary structures.

biophysics↗

bioRxiv2025

Accessibility of Telomeric Overhangs to Stabilizing Small-Molecule Ligands

Human chromosomes terminate in 50-300 nucleotide (nt) long single-stranded telomeric overhangs composed of repeating d(TTAGGG) sequences, which can fold into tandem G-quadruplex (GQ) structures that protect chromosome ends. Stabilization of GQs by small-molecule ligands inhibits telomerase activity, motivating extensive efforts to develop GQ-targeting anti-cancer therapeutics. However, how interactions between successive GQs and bound ligands influence small-molecule accessibility remains poorly understood. Here, we employ single-molecule fluorescence microscopy and stepwise photobleaching analysis to quantify the binding stoichiometry of a fluorescently-labeled oxazole telomestatin derivative (L1Cy5-7OTD) to telomeric overhangs capable of forming 1-6 GQs (30-162 nt long), spanning much of the physiologically relevant range. We find that longer overhangs accommodate more ligands on average but exhibit consistently lower binding stoichiometry than the theoretical maximum, saturating at six molecules even in constructs with twelve binding sites. This trend was further supported by experiments showing increased L1Cy5-7OTD binding when the inter-GQ spacer was extended from 3-nt to 9-nt. This effect was independently confirmed by ensemble fluorescence enhancement experiments utilizing N-methyl mesoporphyrin IX (NMM) as a ligand. Complementary modeling with a one-dimensional lattice model describing equilibrium ligand binding to partially ordered telomeric overhangs revealed positive cooperativity between folding of successive GQs, negative cooperativity between ligands bound opposing (top and bottom) faces of successive GQs, and reduced binding affinity to GQs located at the junction of double stranded and single stranded telomeres. Together, these findings demonstrate how telomeric overhang architecture governs ligand accessibility and provide mechanistic insight to guide the rational design of GQ-targeting anticancer agents.

biophysics↗

bioRxiv2025

Liquid Crystalline Ordering of Banana-Shaped Gapped DNA Duplexes

In-phase adenine-tracts (A-tracts) introduce intrinsic bending to double stranded DNA resulting in banana-shaped macromolecules. In this study, we investigate how such sequence-dependent bending influences DNA-based liquid crystalline (LC) phases formed by gapped DNA (GDNA) constructs. By incorporating three in-phase A-tracts into each duplex arm, we created a GDNA construct with bent duplexes and examined the LC phases they form using temperature-resolved synchrotron small-angle X-ray scattering and polarizing optical microscopy. Like the analogous constructs containing straight (rod-like) duplexes, the bent constructs exhibit a transition from bi-layer smectic-B phase to a monolayer smectic-A phase, although at ~30 {degrees}C lower temperatures. By comparing the monolayer spacing between the bent and straight constructs, we estimate a bending angle of ~11{degrees} per A-tract at room temperature and at physiologically relevant salt and cDNA. The bending angle decreases with increasing DNA concentration and temperature. Moreover, we demonstrate that divalent cations enhance the stability of the smectic-B phase up to ~30 mM Mg2+ but reduce it beyond that. The reduced thermal stabilities of the bilayer and in-layer ordering of bent duplexes imply reduced propensity for DNA condensation and heterochromatin formation under physiological conditions.

biophysics↗

bioRxiv2025

Locked Nucleic Acid Stabilized Liquid Crystalline Phases

Liquid crystalline (LC) phases formed by gapped DNA (GDNA) constructs, where two rigid duplexes are connected with a flexible single stranded linker, offer a versatile platform to investigate interactions between DNA molecules. Base pairs containing a locked nucleic acid (LNA-DNA or LNA-LNA pairs) are generally more stable compared to DNA-DNA pairs due to enhanced hydrogen bonding and/or attractive base stacking interactions. In concentrated solutions of GDNA constructs, the stability of terminal base pairs and the base stacking interactions between neighboring duplexes are critical for the formation of a bilayer smectic phase. By using temperature-resolved synchrotron small-angle X-ray scattering (SAXS) measurements, we quantified the impact of single LNA modification of terminal base pairs on the thermal stability of smectic LC phases. We observe that LNA-DNA terminal AT base pairing (A+T) increases the stability of the bilayer smectic phase by [~]9-18 {degrees}C relative to DNA-DNA pairing at the same duplex concentrations. While relatively large, this increase is still significantly less than the up to [~]30 {degrees}C increase observed when AT DNA-DNA pairs are replaced by GC pairs, suggesting the stacking interactions between A+T LNA-DNA base pairs are significantly weaker than those between unmodified GC base pairs. Our study illustrates the sensitivity of LC ordering in dense DNA solutions to a single nucleotide modification and demonstrates that LNA modifications can provide a new mechanism for tuning the stability of nucleic acid-based materials.

biophysics↗

bioRxiv2025

Impact of Molecular Crowding on Accessibility of Telomeric Overhangs Forming Multiple G-quadruplexes

Telomeric overhangs, composed of repeating GGGTTA sequences, can fold into multiple G-quadruplex (GQ) structures that are essential for maintaining genomic stability and regulating telomere length. Molecular crowding--a defining feature of the cellular environment--affects folding kinetics, conformation, and stability of individual GQs. However, its influence on the overall architecture and accessibility of telomeric overhangs containing multiple GQs remains largely unexplored. In this study, we employed single-molecule Forster Resonance Energy Transfer (smFRET) and FRET-Point Accumulation for Imaging in Nanoscale Topography (FRET-PAINT) to address this question. We examined the accessibility of telomeric overhangs capable of forming one to six GQs to a short complementary peptide nucleic acid (PNA) imager probe under molecular crowding conditions. These conditions were simulated by polyethylene glycol (PEG) molecules of two different molecular weights: 200 Da (PEG-200) and 6000 Da (PEG-6000). Our results reveal a systematic reduction in the overhang accessibility with increasing PEG concentration--showing approximately a 3-fold reduction at 30% (v/v) PEG-200 and an 8-fold reduction at 30% PEG-6000. We also observed a progressive compaction of the overhang as PEG concentration increased, suggesting molecular crowding promotes architectural condensation, thereby reducing probe accessibility. These findings offer new insights into how the crowded cellular environment may compact telomeric overhangs and modulate their structural and functional properties.

biophysics↗

bioRxiv2024

Sequence-Dependent Liquid Crystalline Ordering of Gapped DNA

We investigate the impact of poly-adenine (poly-A) sequences on the type and stability of liquid crystalline (LC) phases formed by concentrated solutions of gapped DNA (two duplex arms bridged by a flexible single strand) using synchrotron small angle X-ray scattering and polarizing optical microscopy. While samples with mixed sequence form layered (smectic) phases, poly-A samples demonstrate a columnar phase at lower temperatures (5-35 {degrees}C), not previously observed in GDNA samples, and a smectic-B phase of exceptional stability at higher temperatures (35-65 {degrees}C). We present a model that connects formation of these LC phases with the unique characteristics of poly-A sequences, which manifest in various biological contexts, including DNA condensation and nucleosome formation.

biophysics↗

bioRxiv2024

Combining the CRISPR Activation and Interference Capabilities Using dCas9 and G-Quadruplex Structures

We demonstrate that both CRISPR interference and CRISPR activation can be achieved at RNA and protein levels by targeting the vicinity of a putative G-quadruplex forming sequence (PQS) in the c-Myc promoter with nuclease-dead Cas9 (dCas9). The achieved suppression and activation in Burkitts Lymphoma cell line and in in vitro studies are at or beyond those reported with alternative approaches. When the template strand (contains the PQS) was targeted with CRISPR-dCas9, the G-quadruplex was destabilized and c-Myc mRNA and protein levels increased by 2.1-fold and 1.6-fold, respectively, compared to controls in the absence of CRISPR-dCas9. Targeting individual sites in the non-template strand with CRISPR-dCas9 reduced both the c-Myc mRNA and protein levels (by 1.8-fold and 2.5-fold, respectively), while targeting two sites simultaneously further suppressed both the mRNA (by 3.6-fold) and protein (by 9.8-fold) levels. These were consistent with cell viability assays when single or dual sites in the non-template strand were targeted (1.7-fold and 4.7-fold reduction in viability, respectively). We also report extensive in vitro biophysical studies which are in quantitative agreement with these cellular studies and provide important mechanistic details about how the transcription is modulated via the interactions of RNA polymerase, CRISPR-dCas9, and the G-quadruplex.

molecular biology↗

bioRxiv2024

Reusable Microfluidic Chambers for Single-Molecule Microscopy

Maintaining a consistent environment in single molecule microfluidic chambers containing surface-bound molecules requires laborious cleaning and surface passivation procedures. Despite such efforts, variations in non-specific binding and background signals commonly occur across different chambers. Being able to reuse the chambers without degrading the surface promises significant practical and fundamental advantages; however, this necessitates removing the molecules attached to the surface, such as DNA, proteins, lipids or nanoparticles. Biotin-streptavidin attachment is widely used for such attachments as biotin can be readily incorporated to these molecules. In this study, we present single-molecule fluorescence experiments that demonstrate effective resetting and recycling of the chambers at least 10 times using photocleavable biotin (PC-biotin) and UV light exposure. This method differs from alternatives as it does not utilize any harsh chemical treatment of the surface. We show that all bound molecules (utilizing various PC-biotin attachment chemistries) can be removed from the surface by a 5-min UV exposure of a specific wavelength. Non-optimal wavelengths and light sources showed varying degrees of effectiveness. Our approach does not result in any detectable degradation of surface quality, as assessed by non-specific binding of fluorescently labeled DNA and protein samples, and recovery of DNA secondary structure and protein activity. The speed and efficiency of the resetting process, the cost-effectiveness of the procedure, and the widespread use of biotin-streptavidin attachment make this approach adaptable for a wide range of single-molecule applications.

biophysics↗

bioRxiv2024

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 between 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 to 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 to POT1 alone by bridging single- and double-stranded telomeric tracts. Table of Content Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/608453v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@a58e79org.highwire.dtl.DTLVardef@12cb172org.highwire.dtl.DTLVardef@135bf40org.highwire.dtl.DTLVardef@19f3fe4_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2024

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.

molecular biology↗

bioRxiv2023

Liquid Crystalline Layering and Divalent Cations Cooperatively Enhance DNA Condensation

The layered liquid crystalline (LC) 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 lateral interactions that drive the condensation of DNA molecules. The resulting layer structure exhibits long-range inter-layer and intra-layer positional correlations. Using synchrotron small-angle x-ray scattering (SAXS) measurements, we investigate the impact of divalent Mg2+ cations on the stability of the inter- and intra-layer DNA ordering as a function of temperature between 5-65 {degrees}C and for different terminal base pairings at the blunt ends of the gapped DNA constructs, which mediate the strength of the attractive end-to-end interaction. We demonstrate that the stabilities at a fixed DNA concentration of both inter-layer and intra-layer order are significantly enhanced even at a few mM Mg2+ concentration. The stability continues to increase up to [~]30 mM Mg2+ concentration, but at higher ([~]100 mM) Mg2+ content repulsion between positive ions counteracts and reverses the increase. On the other hand, sufficiently strong base-stacking interactions promote intra-layer order even in the absence of multivalent cations, which demonstrates the impact of liquid crystal layering on the DNA condensation process. We discuss the implications of these results in terms cation-mediated DNA-DNA attraction.

biophysics↗

bioRxiv2023

Effect of selective end-to-end base stacking interactions on the stability of smectic liquid crystal ordering in concentrated gapped 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 {degrees}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 {degrees}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. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/525591v2_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@1ba488borg.highwire.dtl.DTLVardef@f0cbb2org.highwire.dtl.DTLVardef@cd3eaforg.highwire.dtl.DTLVardef@ce59b9_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2022

Impact of Shelterin Complex on Telomere Accessibility

Shelterin plays critical roles in maintaining and protecting telomeres by regulating access of various physiological agents to telomeric DNA. We present single molecule measurements investigating the impact of the POT1 and a four-component shelterin complex on the accessibility of human telomeric DNA overhangs with physiologically relevant lengths (28-150 nt), which to our knowledge is the first direct approach to measure this effect on such telomeric constructs. To quantify telomere accessibility, we monitored transient binding events of a short peptide nucleic acid (PNA) probe that is complementary to telomeric overhangs using FRET-PAINT. Although POT1 has a mild G-quadruplex unfolding activity, it reduced 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 reduced the accessibility of telomeric overhangs by [~]5-fold. This enhanced protection suggests shelterin restructures the region between single and double stranded telomere, which is otherwise the most accessible part of the overhang, by a synergistic cooperation of shelterin components located on single and double stranded telomere.

biophysics↗

bioRxiv2022

Rtt105 configurationally staples RPA and blocks facilitated exchange and interactions with RPA-interacting proteins

Replication Protein A (RPA) binds to single-stranded DNA (ssDNA) and recruits over three dozen RPA-interacting proteins (RIPs) to coordinate multiple aspects of DNA metabolism including DNA replication, repair, and recombination. Rtt105 is a molecular chaperone that regulates nuclear localization of RPA. Whether and how Rtt105 regulates the activities of RPA is poorly understood. Here, we show that Rtt105 binds to multiple DNA binding and protein-interaction domains of RPA and configurationally staples the complex. In the absence of ssDNA, Rtt105 inhibits RPA binding to Rad52, thus preventing spurious binding to RPA-interacting proteins (RIPs). When ssDNA is available, Rtt105 promotes formation of high-density RPA nucleoprotein filaments and dissociates during this process. Free Rtt105 further stabilizes the RPA-ssDNA filaments by inhibiting RPA facilitated exchange. Collectively, our data suggest that Rtt105 sequesters free RPA in the nucleus to prevent untimely RIP interaction, while stabilizing RPA-ssDNA filaments at DNA lesion sites.

biochemistry↗

bioRxiv2021

A Single Molecule Investigation of I-Motif: Stability, Folding Kinetics, and Potential as an In-situ pH Sensor

We present a collection of single molecule work on the i-motif structure formed by the human telomeric sequence. Even though it was largely ignored in earlier years of its discovery due to its modest stability and requirement for physiologically low pH levels (pH<6.5), the i-motif has been attracting more attention recently as both a physiologically relevant structure and as a potent pH sensor. In this manuscript, we establish single molecule Forster resonance energy transfer (smFRET) as a tool to study the i-motif over a broad pH and ionic conditions. We demonstrate pH and salt dependence of i-motif formation under steady state conditions and illustrate the kinetics of i-motif folding in real time at the single molecule level. We also show the prominence of intermediate folding states and reversible folding/unfolding transitions. We present an example of using the i-motif as an in-situ pH sensor and use this sensor establish the time scale for the pH drop in a commonly used oxygen scavenging system.

biophysics↗

bioRxiv2021

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 1-7 tandem G-quadruplex (GQ) structures. Using 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 PNA 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 telomere 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 telomere 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. Significance StatementThe ends of eukaryotic linear chromosomes are capped by telomeres which terminate with a single-stranded overhang. Telomeric overhangs fold into compact structures, called G-quadruplex, that inhibit access to these critical genomic sites. We report single molecule measurements and computational modeling studies probing the accessibility of a set of human telomeric overhangs that covers a significant portion of the physiologically relevant length scale. We observe novel accessibility patterns which have a well-defined periodicity and show that certain regions are significantly more accessible than others. These accessibility patterns also suggest the underlying folding frustration of G-quadruplexes depends on telomere length. These patterns have significant implications for regulating the access of DNA processing enzymes and DNA binding proteins that can target telomeric overhangs.

biophysics↗

bioRxiv2020

Targeting G-quadruplex Forming Sequences with Cas9

Clustered Regularly Interspaced Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) proteins, particularly Cas9, have provided unprecedented control on targeting and editing specific DNA sequences. If the target sequences are prone to folding into non-canonical secondary structures, such as G-quadruplex (GQ), the conformational states and activity of CRISPR-Cas9 complex would be influenced, but the impact has not been assessed. Using single molecule FRET, we investigated structural characteristics of the complex formed by CRISPR-Cas9 and target DNA, which contains a potentially GQ forming sequence (PQS) in either the target or the non-target strand (TS or NTS). We observed different conformational states and dynamics depending on the stability of the GQ and the position of PQS. When PQS was in NTS, we observed evidence for GQ formation for both weak and stable GQs. This is consistent with R-loop formation between TS and crRNA releasing NTS from Watson-Crick pairing and facilitating secondary structure formation in it. When PQS was in TS, R-loop formation was adequate to maintain a weak GQ in the unfolded state but not a GQ with moderate or high stability. The observed structural heterogeneity within the target dsDNA and the R-loop strongly depended on whether the PQS was in TS or NTS. We propose these variations in the complex structures to have functional implications for Cas9 activity.

biophysics↗