Plasmonic polarization conversion offers significant advantages over conventional methods, including a smaller device footprint and easier integration into photonic circuits. In this work, we numerically and experimentally investigate the polarization conversion properties of a plasmonic double-hole structure surrounded by circular nanograting, i.e., a bull's eye antenna. Using a combination of polarimetric imaging via back focal plane (BFP) microscopy and Stokes parameter analysis, we demonstrate the functionality of our structure as a miniature on-chip polarization converter. Our results show that this nanostructure enables complex polarization transformations, including converting linear to circular polarization and vice versa. Polarization conversion efficiency is found to be dependent on the periodicity of the circular gratings and is particularly pronounced in the central region of Fourier space. Moreover, strong asymmetric scattering leads to distinctive patterns in the Stokes parameters across various incident polarization states. This work provides insights into the plasmonic manipulation of light polarization at the nanoscale with potential applications in miniature on-chip polarization convertors, polarization-controlled emitters, and advanced sensing technologies.

We numerically study a double-slit interferometer that detects the modification of local phase and amplitude signal due to a refractive index contrast in a nanoplasmonic sensor after the plasmonic wave has been coupled to the farfield. Specifically, the sensor consists of two elongated zero-mode waveguides (ZMW) which function to launch surface plasmons as well as transmit light directly into the farfield. The surface plasmon waves are coupled to the farfield by surface gratings which cover one quarter of a bull-eye antenna, with different transmission directions. In the farfield, the coupled plasmon waves interfere with the directly transmitted reference wave, which reveals a combination of the phase and amplitude modification by the asymmetry of refractive index in the lumen of the ZMW. We report a drastic increase in sensitivity over interferometers that only utilize the directly transmitted fraction.

DNA confined to nanofluidic channels with a cross-section from tens to hundreds of nm wide and hundreds of microns long stretches in an equilibrium process free of flow or end tethering. Because DNA is free to move along the channel axis, its extension is exquisitely sensitive to DNA–DNA interactions and the DNA persistence length, as well as the contour length. We discuss how this sensitivity has been used to probe DNA-protein interactions at physiological concentrations of both DNA and proteins.

Superfluorescence (SF) is a unique optical phenomenon that consists of an ensemble of emitters coupling collectively to produce a short but extremely intense burst of light. Despite our recently published works showing that room temperature anti-Stokes shifted SF were achieved in a few randomly assembled or even single lanthanide-doped upconversion nanoparticle (UCNP), the coupling required to produce and optimize Burnham-Chiao ringing (echoing of pulses) is not understood. Such ringing could be particularly useful to provide timing and multiplexing in potential applications as an alternative light source device. We previously found a lack of Burnham Chiao ringing in single nanocrystals, but strong ringing in a random cluster. The ordered assembly of these crystals will not only create a SF superburst, but also enable understanding of the periodicity of the Burnham Chiao ringing. This work explores SF microrod (MR), with enhanced SF performance and the closely spaced assembly of MR result in a greater active volume, which gives rise to greater reabsorption of the initial emission, which is then re-emitted, leading to greater oscillatory fluorescence or Burnham Chiao ringing. We also correlate the MR dimension and orientation with the corresponding SF spectral properties.

Telomeres, complexes of DNA and proteins, protect ends of linear chromosomes. In humans, the two shelterin proteins TRF1 and TIN2, along with cohesin subunit SA1, were proposed to mediate telomere cohesion. Although the ability of the TRF1-TIN2 and TRF1-SA1 systems to compact telomeric DNA by DNA-DNA bridging has been reported, the function of the full ternary TRF1-TIN2-SA1 system has not been explored in detail. Here, we quantify the compaction of nanochannel-stretched DNA by the ternary system, as well as its constituents, and obtain estimates of the relative impact of its constituents and their interactions. We find that TRF1, TIN2, and SA1 work synergistically to cause a compaction of the DNA substrate, and that maximal compaction occurs if all three proteins are present. By altering the sequence with which DNA substrates are exposed to proteins, we establish that compaction by TRF1 and TIN2 can proceed through binding of TRF1 to DNA, followed by compaction as TIN2 recognizes the previously bound TRF1. We further establish that SA1 alone can also lead to a compaction, and that compaction in a combined system of all three proteins can be understood as an additive effect of TRF1-TIN2 and SA1-based compaction. Atomic force microscopy of intermolecular aggregation confirms that a combination of TRF1, TIN2, and SA1 together drive strong intermolecular aggregation as it would be required during chromosome cohesion.

The transmission of light through sub-wavelength apertures (zero-mode waveguides, ZMW) in metal films is well-explored. It introduces both an amplitude modulation as well as a phase shift to the oscillating electromagnetic field. We propose a nanophotonic interferometer by bringing two ZMW (∼100 nm diameter) in proximity and monitoring the distribution of transmitted light in the back-focal plane of collecting microscope objective (1.3 N.A.). We demonstrate that both an asymmetry induced by the binding of a quantum dot in one of the two ZMW, as well as an asymmetry in ZMW diameter yield qualitatively similar transmission patterns. We find that the complex pattern can be quantified through a scalar measure of asymmetry along the symmetry axis of the aperture pair. In a combined experimental and computational exploration of detectors with differing ZMW diameters, we find that the scalar asymmetry is a monotonous function of the diameter difference of the two apertures, and that the scalar asymmetry measure is higher if the sample is slightly displaced from the focal plane of the collecting microscope objective. An optimization of the detector geometry determined that the maximum response is achieved at an aperture separation that is comparable to the wavelength on the exit side of the sensor. For small separations of apertures, on the order of a quarter of the wavelength and less, the signal is strongly polarization dependent, while for larger separations, on the order of the wavelength or larger, the signal becomes essentially polarization-independent.

Telomeres, complexes of DNA and proteins, protect ends of linear chromosomes. In humans, the two shelterin proteins TRF1 and TIN2, along with cohesin subunit SA1, were proposed to mediate telomere cohesion. While the ability of the TRF1-TIN2 and TRF1-SA1 systems to compact telomeric DNA by DNA-DNA bridging has been reported, the function of the full ternary TRF1-TIN2-SA1 system has not been explored in detail.

The methyl-CpG-binding domain 2 and 3 proteins (MBD2 and MBD3) provide structural and DNA-binding function for the Nucleosome Remodeling and Deacetylase (NuRD) complex. The two proteins form distinct NuRD complexes and show different binding affinity and selectivity for methylated DNA. Previous studies have shown that MBD2 binds with high affinity and selectivity for a single methylated CpG dinucleotide while MBD3 does not. However, the NuRD complex functions in regions of the genome that contain many CpG dinucleotides (CpG islands). Therefore, in this work, we investigate the binding and diffusion of MBD2 and MBD3 on more biologically relevant DNA templates that contain a large CpG island or limited CpG sites. Using a combination of single-molecule and biophysical analyses, we show that both MBD2 and MBD3 diffuse freely and rapidly across unmethylated CpG-rich DNA. In contrast, we found methylation of large CpG islands traps MBD2 leading to stable and apparently static binding on the CpG island while MBD3 continues to diffuse freely. In addition, we demonstrate both proteins bend DNA, which is augmented by methylation. Together, these studies support a model in which MBD2-NuRD strongly localizes to and compacts methylated CpG islands while MBD3-NuRD can freely mobilize nucleosomes independent of methylation status.

Single-molecule techniques provide important details supplementing the framework obtained from traditional bulk experiments. Many cellular processes such as DNA replication, DNA repair, and telomere maintenance involve interaction among multiple proteins, their co-factors, and DNA. To investigate such interactions and to differentiate the function of each component necessitate a technique that allows the combinatorial exposure of DNA to multiple proteins and co-factors as well as manipulation of the DNA configuration. We present a nanofluidic device with the capability of active combinatorial exchange of up to three buffers in real-time and dynamic manipulation of DNA under physiologically relevant conditions. We demonstrate its utility in monitoring compaction of DNA by telomeric proteins, DNA modification by an endonuclease, and DNA loop extrusion by cohesin.

Previous single-molecule studies of dynamics of shelterin assembles on DNA focused on short telomeric repeats (32 TTAGGG) in the context of λ DNA sequences. Studies based on the short telomeric sequences showed that shelterin complexes form individual complexes on DNA. However, previous electron microscopy, atomic force microscopy and bulk biochemical assays also demonstrated that TRF2 forms multi-protein complexes and compacts DNA. There are discrepancies from these previous studies whether TIN2 regulates DNA compaction by TRF2 on physiologically long telomeric DNA. To directly address these long-standing questions, we have studied the TIN2 regulated telomeric DNA compaction on long telomeric substrates purified from mouse liver tissue. We also studied how TIN2 helps TRF2 in protecting the telomeres by forming T-loops. Given that in human and mouse cells, TRF1, TRF2, TIN2, and RapI are approximately 10 times more abundant than POT1 and TPP1, majority of the TRF1 and TRF2 proteins exist in complexes without POT1 and TPP1 at telomeres. Therefore, in this study, we focused on how TIN2 (TIN2S and TIN2L) regulates DNA binding by TRF2 on telomeric DNA purified from mouse livers.

ABSTRACT The shelterin complex consisting of TRF1, TRF2, RAP1, TIN2, TPP1, and POT1, functions to prevent false recognition of telomeres as double-strand DNA breaks, and to regulate telomerase and DNA repair protein access. TIN2 is a core component linking double-stranded telomeric DNA binding proteins (TRF1 and TRF2) and proteins at the 3’ overhang (TPP1-POT1). Since knockdown of TIN2 also removes TRF1 and TRF2 from telomeres, determining TIN2’s unique mechanistic function has been elusive. Here, we investigated DNA molecular structures promoted by TRF1-TIN2 using complementary single-molecule imaging platforms, including atomic force microscopy (AFM), total internal reflection fluorescence microscopy (TIRFM), and the DNA tightrope assay. We demonstrate that TIN2S and TIN2L isoforms facilitate TRF1-mediated DNA compaction ( cis -interactions) and DNA-DNA bridging ( trans -interactions) in a telomeric sequence- and length-dependent manner. On the short telomeric DNA substrate (6 TTAGGG repeats), the majority of TRF1 mediated telomeric DNA-DNA bridging events are transient with a lifetime of ~1.95 s. On longer DNA substrates (270 TTAGGG), TIN2 forms multi-protein complexes with TRF1 and stabilizes TRF1-mediated DNA-DNA bridging events that last for at least minutes. Preincubation of TRF1 with its regulator protein Tankyrase 1 significantly reduces TRF1-TIN2 mediated DNA-DNA bridging, whereas TIN2 protects the disassembly of TRF1-TIN2 mediated DNA-DNA bridging upon Tankyrase 1 addition. Our study provides evidence that TIN2 functions to promote TRF1 mediated trans -interactions of telomeric DNA, leading to new mechanistic insight into sister telomere cohesion.

The telomere specific shelterin complex, which includes TRF1, TRF2, RAP1, TIN2, TPP1 and POT1, prevents spurious recognition of telomeres as double-strand DNA breaks and regulates telomerase and DNA repair activities at telomeres. TIN2 is a key component of the shelterin complex that directly interacts with TRF1, TRF2 and TPP1. In vivo, the large majority of TRF1 and TRF2 are in complex with TIN2 but without TPP1 and POT1. Since knockdown of TIN2 also removes TRF1 and TRF2 from telomeres, previous cell-based assays only provide information on downstream effects after the loss of TRF1/TRF2 and TIN2. Here, we investigated DNA structures promoted by TRF2-TIN2 using single-molecule imaging platforms, including tracking of compaction of long mouse telomeric DNA using fluorescence imaging, atomic force microscopy (AFM) imaging of protein-DNA structures, and monitoring of DNA-DNA and DNA-RNA bridging using the DNA tightrope assay. These techniques enabled us to uncover previously unknown unique activities of TIN2. TIN2S and TIN2L isoforms facilitate TRF2-mediated telomeric DNA compaction (cis-interactions), dsDNA-dsDNA, dsDNA-ssDNA and dsDNA-ssRNA bridging (trans-interactions). Furthermore, TIN2 facilitates TRF2-mediated T-loop formation. We propose a molecular model in which TIN2 functions as an architectural protein to promote TRF2-mediated trans and cis higher-order nucleic acid structures at telomeres.

TIN2 is a core component of the shelterin complex linking double-stranded telomeric DNA-binding proteins (TRF1 and TRF2) and single-strand overhang-binding proteins (TPP1-POT1). In vivo, the large majority of TRF1 and TRF2 exist in complexes containing TIN2 but lacking TPP1/POT1; however, the role of TRF1-TIN2 interactions in mediating interactions with telomeric DNA is unclear. Here, we investigated DNA molecular structures promoted by TRF1-TIN2 interaction using atomic force microscopy (AFM), total internal reflection fluo-rescence microscopy (TIRFM), and the DNA tightrope assay. We demonstrate that the short (TIN2S) and long (TIN2L) isoforms of TIN2 facilitate TRF1-mediated DNA compaction (cis-in-teractions) and DNA-DNA bridging (trans-interactions) in a telomeric sequence-and length-dependent manner. On the short telomeric DNA substrate (six TTAGGG repeats), the ma-jority of TRF1-mediated telomeric DNA-DNA bridging events are transient with a lifetime of similar to 1.95 s. On longer DNA substrates (270 TTAGGG repeats), TIN2 forms multiprotein complexes with TRF1 and stabilizes TRF1-mediated DNA-DNA bridging events that last on the order of minutes. Preincubation of TRF1 with its regulator protein Tankyrase 1 and the cofactor NAD+ significantly reduced TRF1-TIN2 mediated DNA-DNA bridging, whereas TIN2 protected the disassembly of TRF1-TIN2 mediated DNA-DNA bridging upon Tankyrase 1 addition. Furthermore, we showed that TPP1 inhibits TRF1-TIN2L-mediated DNA-DNA bridging. Our study, together with previous findings, supports a molecular model in which protein assemblies at telomeres are heterogeneous with distinct subcomplexes and full shelterin complexes playing distinct roles in telomere protection and elongation.

Cohesin SA1 (STAG1) and SA2 (STAG2) are key components of the cohesin complex. Previous studies have highlighted the unique contributions by SA1 and SA2 to 3D chromatin organization, DNA replication fork progression, and DNA double-strand break (DSB) repair. Recently, we discovered that cohesin SA1 and SA2 are DNA binding proteins. Given the recently discovered link between SA2 and RNA-mediated biological pathways, we investigated whether or not SA1 and SA2 directly bind to RNA using a combination of bulk biochemical assays and single-molecule techniques, including atomic force microscopy (AFM) and the DNA tightrope assay. We discovered that both SA1 and SA2 bind to various RNA containing substrates, including ssRNA, dsRNA, RNA:DNA hybrids, and R-loops. Importantly, both SA1 and SA2 localize to regions on dsDNA that contain RNA. We directly compared the SA1/SA2 binding and R-loops sites extracted from Chromatin Immunoprecipitation sequencing (ChIP-seq) and DNA-RNA Immunoprecipitation sequencing (DRIP-Seq) data sets, respectively. This analysis revealed that SA1 and SA2 binding sites overlap significantly with R-loops. The majority of R-loop-localized SA1 and SA2 are also sites where other subunits of the cohesin complex bind. These results provide a new direction for future investigation of the diverse biological functions of SA1 and SA2.

BACKGROUND:MeCP2 and MBD2 are members of a family of proteins that possess a domain that selectively binds 5-methylcytosine in a CpG context. Members of the family interact with other proteins to modulate DNA packing. Stretching of DNA-protein complexes in nanofluidic channels with a cross-section of a few persistence lengths allows us to probe the degree of compaction by proteins. RESULTS:We demonstrate DNA compaction by MeCP2 while MBD2 does not affect DNA configuration. By using atomic force microscopy (AFM), we determined that the mechanism for compaction by MeCP2 is the formation of bridges between distant DNA stretches and the formation of loops. CONCLUSIONS:Despite sharing a similar specific DNA-binding domain, the impact of full-length 5-methylcytosine-binding proteins can vary drastically between strong compaction of DNA and no discernable large-scale impact of protein binding. We demonstrate that ATTO 565-labeled MBD2 is a good candidate as a staining agent for epigenetic mapping.

Telomeres, consisting of duplex TTAGGG repeats and associating with protein complexes at chromosome ends, play a crucial role in maintaining the stability of chromosomes. The protein complex - shelterin contains six subunits (TRF1, TRF2, RAP1, TIN2, TPP1 and POT1), which bind to telomeres and protect the chromosome ends from DNA repairing, and recruit telomerase when chromosome gets shortened abnormally. Among these six subunits, TIN2 has no affinity to either double-strand or single-strand DNA. However, it is a core component bridging the double-strand DNA binding proteins (TRF1 and TRF2) to single-strand DNA binding protein complex (TPP1-POT1). Loss of TRF1 or TRF2 binding domain in TIN2 can trigger DNA damage response. Moreover, TIN2 without the TPP1 binding domain is capable of fully supporting the stabilization of TRF1 and TRF2/RAP1. Despite the significance of TIN2 in telomere maintenance, the mechanism underlying TIN2 remains elusive. To understand how TIN2 affects TRF1 binding dynamics and how TIN2 influences with TRF1-mediated telomeric DNA pairing, we conducted Atomic Force Microscopy (AFM) and used nanochannels confined DNA to study the telomeric DNA conformation upon TRF1 and TIN2 binding. We observed high-order protein-DNA complexes formation indicating TIN2 facilitates TRF1 accumulation on telomeric DNA. We also carried out single molecule fluorescence technique to investigate how TIN2 affects TRF1 binding dynamics on telomeric DNA sequences. Our results show that TIN2 can accelerate DNA-DNA pairing and stabilize TRF1 on telomeric DNA.

R-loops are three-stranded nucleic acid structures consisting of an RNA-DNA hybrid and a displaced single-stranded DNA (ssDNA) loop. R-loops collectively occupy up to 5% of the mammalian genome, which occur at conserved hotspot including promoter and terminator regions of poly(A) dependent genes. R-loops are proposed to be the "double-edged sword" that functions as powerful regulators of gene expression and induces genome instability if it is un-regulated. Despite the importance of R-loops in a wide range of biological pathways, proteins that mediate R-loop dependent cellular function are not fully understood. Recently The Bishop group showed that Ewing sarcoma cells displayed a higher level of R-loops compared to control cell lines. EWSR1 depletion induces R-loop accumulation. In addition, the second most common mutation in Ewing sarcoma cells was found in cohesin SA2. These observations raise the important question of whether EWSR1 as well as cohesin SA1 and SA2 subunits are sensors of R-loops that regulate R-loop processing. Using atomic force microscopy (AFM) and bulk fluorescence anisotropy, we tested the hypothesis that cohesin SA1, SA2, and EWSR1 directly bind to R-loops. We observed that cohesin SA1/SA2, and EWSR1 specifically recognize R-loops. These observations suggest direct roles of cohesin SA1/SA2 and EWSR1 in R-loop regulation and how the loss of SA2 in Ewing sarcoma may provide some benefit to these cancers.

Telomeres are nucleoprotein structures that prevent the degradation or fusion of the ends of linear chromosomes by shielding them from activating DNA damage response (DDR) and DNA double-strand break (DSB) repair. Human telomeres contain ∼2 to 20 kb of TTAGGG repeats and a G-rich 3’ overhang. A specialized six-protein shelterin complex, including TRF1, TRF2, RAP1, TIN2, TPP1 and POT1, binds and prevents telomeres from being falsely recognized as double strand breaks, and regulates telomerase and DNA repair protein access. Extensive telomere shortening or dramatic telomere loss due to DNA damage causes chromosome ends to be recognized as DNA breaks, which triggers cell senescence and aging-related pathologies. Human telomeric DNA is arranged into T-loops, in which the 3’ single-stranded overhang invades the upstream double-stranded region. A recent study suggests that T-loops at telomeres function as conformational switches that regulate ATM activation [1]. Further, it was also shown that TRF2 mediated DNA compaction drives T-loop formation [2]. While it has been shown that TRF2 protein is required for the T-loop formation, essential information regarding the dynamics of its formation still remains unknown. Moreover, most of the previous studies were done using short telomeric DNA sequences ∼ 20 kb. To elucidate the dynamics of TRF2 mediated DNA compaction and T-loop formation, we used single molecule methods which frequently require long DNA substrates. We established a new method for extracting and purifying long telomeric DNA from mouse liver cells using Region specific Extraction (RSE) [3].