Biology subjects

Alfehaid, J.

Publications and source records attributed to Alfehaid, J..

4 recordsLinked to original sources

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

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↗