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. 2026 Mar 18;26(12):4143–4151. doi: 10.1021/acs.nanolett.5c06446

SM-NucSeq, Genetic and Epigenetic Sequencing of Nucleosomes with Single-Molecule Real-Time Sequencing

Pengyu Zheng †,‡, Noa Furth ‡, Seán E O’Leary §, Efrat Shema ‡,*, Meni Wanunu †,∥,⊥,*
PMCID: PMC13047728  PMID: 41848090

Abstract

Despite advances in deciphering chromatin structure and dynamics in recent years, mapping the sequence position and combinatorial modifications of individual nucleosomes, the building blocks of chromatin, has yet to be achieved. In this work, we develop SM-NucSeq: a technology that combines single-molecule immunoaffinity detection with single-molecule real-time (SMRT) sequencing in zero-mode waveguides (ZMWs), which are sub-wavelength well-like structures that confine the depth of the excitation beam to the nanometer scale. We show that SM-NucSeq can detect histone modifications on intact nucleosomes and identify their underlying DNA sequences. We leverage the ZMW chips to load nucleosome/polymerase complexes and detect co-occurring histone modifications with fluorescent antibodies, validating each step on a chromatically resolved micromirror TIRF microscope. Finally, the application of SM-NucSeq reveals that DNA synthesis rates are reduced in nucleosomes compared to the double-stranded DNA control sample and that DNA synthesis rate profiles are histone-modification-dependent.

Keywords: Nucleosomes, epigenetics, zero-mode waveguides, DNA sequencing, single molecule

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Nucleosomes, the basic units of chromatin, contain multiple layers of genetic and epigenetic information that are linked to gene activity in a cell. Chemical modifications on the core histone proteins, around which ∼150 bp DNA sequences are wound, can regulate the structural conformation and compactness of the chromatin, thus controlling the accessibility of the local gene. The large numbers of histone modifications, DNA modifications, and chromatin remodelers that interact with our genome suggest that multiple elements act combinatorially to direct specific outcomes. For example, Ruthenburg et al. discovered that the BPTF subunit of the nucleosome remodeling factor (NURF) selectively binds to nucleosomes with both H3 lysine 4 trimethylation (H3K4me3) and H4 lysine 16 acetylation (H4K16ac) modifications. Lee et al. found that the DNA methylation at CpG islands in the presence of trimethylation of H3 lysine 27 (H3K27me3) can attract transcriptional repressor MeCP2. Additionally, a recent study revealed that the biophysical information is electrically encoded in nucleosome core particles; thus, individual native mononucleosomes conserved sufficient information for chromatin organization. Detection of these structural features at the single-molecule level can expand our understanding of chromatin biology and our ability to understand the underlying processes that impact development as well as aging, cancer, and other diseases.

Current methodologies for studying chromatin modifications and their combinatorial patterns are limited, hindering an in-depth understanding of the epigenetic network and its regulation of genome function. Techniques such as chromatin immunoprecipitation followed by sequencing (ChIP-Seq, CUT&run, and CUT&tag) are widely used to investigate chromatin regulation. However, despite recent advancements, , these enrichment-based methods have notable limitations, particularly in their ability to simultaneously analyze multiple histone modifications. Importantly, the resulting signals are typically population-averaged, preventing the decoding of single-nucleosome-level information. In a previous report, Shema et al. presented a novel method based on single-molecule imaging to uncover combinatorial modifications on individual nucleosomes and found naturally bivalent nucleosomes with both repressive and activating marks. In this work, we introduce SM-NucSeq, where we combine single-molecule immunoaffinity detection with single-molecule real-time (SMRT) sequencing to decode histone modifications with their attached DNA sequences on intact nucleosomes (Figure A).

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(A) Schematic diagram of SM-NucSeq library preparation steps for converting nucleosomes to nuc-bells and loading DNA polymerase and schematic diagram of the main steps in SM-NucSeq: binding nuc-bells to ZMW surfaces, immunofluorescence-based histone modification detection, and nuc-bell SMRT sequencing. (B) Schematics of the custom micromirror TIRF microscope setup. PAGE gel electrophoresis result of SMRTbell ligation on the mononucleosome sample. (C) Lanes: 1, 50 bp DNA ladder; 3, intact nucleosomes, pre-ligation; 4, nucleosomal DNA, pre-ligation; 5, nuc-bells; and 6, DNA extracted from nuc-bells.

The key sensing element in SMRT sequencing is the zero-mode waveguide (ZMW), a nanosized well-like structure that transmits and confines excitation light in a thin layer of space near the bottom of the well. ZMWs and ZMW-based MEMS devices have also been used in biophysical investigations, such as direct observation of reverse transcription, observation of single-step protein translation by the ribosome, and mRNA 5′ cap recognition for translation, , among other studies. Due to its special ability to sequence native DNA molecules, SMRT sequencing can also detect some important epigenetic modifications in the DNA, such as methyl-cytosine and methyl-adenine, , by detecting anomalous kinetics in polymerase reactions.

Preparation of the SM-NucSeq Library and TIRF Setup for SM-NucSeq

We first built a custom micromirror TIRF microscope (Figure B), which features spatially separated excitation and emission paths and a set of double Amici prisms to spread and resolve the emitting fluorescence signal by wavelength, with adjustable dispersion. With this setup, we achieved a continuous control of the color dispersion (Figure S8A) for optimizing the spatial separation among the four nucleotide fluorescence labels on the emCCD sensor, essential for the differentiation of the DNA bases in prototypical ZMW platforms. , While both TIRF microscopy and ZMW restrict the excitation depth to minimize background noise, the combination of the two has shown a 2-fold further reduction in the background over widefield-illuminated ZMWs.

Single-pass base-calling has higher error rates − and allows only a short window of time to capture its signals. Pacific Biosciences overcame this by constructing SMRTbell libraries where circularized DNA can be replicated indefinitely by the DNA polymerase, which results in high-coverage readout with higher accuracy consensus sequences. ,

With the aim of sequencing native nucleosomal DNA using SMRT sequencing chemistry, we prepared nucleosome SMRTbells (nucleobells) by modifying the library preparation protocol so that nucleosome tails can be ligated to SMRTbell adapters while preserving intact nucleosomes. We made a few changes from the standard ligation protocol to maintain intact nucleosome samples: (1) The 65 °C incubation step for inactivating enzymes after end repair/A tailing was skipped to avoid disrupting the nucleosome structure. (2) In the post-ligation purification step, spin columns were used to purify the sample instead of the recommended AMPure PB beads, which can only retain DNA (see section 1 of the Supporting Information for details). SMRTbell ligation on nucleosomal DNA was verified by using gel electrophoresis. In the native polymacrylamide gel shown in Figure C, the ligated nucleosome (lane 5) showed a slightly higher (i.e., “slower”) band than the native nucleosome (lane 3), and no secondary band in the position corresponding to mononucleosomal DNA was observed, indicating that the histone/DNA complex survived the modified protocol for SMRTbell ligation. On the TapeStation, the DNA samples extracted from ligated nucleosomes showed a noticeable second peak at ∼230 bp (Figure S3), indicating the formation of nuc-bells. In the minus adapter control, we observed self-ligation among nucleosomes that formed duplexes, triplexes, etc. (Figure S3) but did not see the same peak at ∼230 bp. Analysis of the TapeStation band intensities revealed a ∼21% ligation yield for circularizing the tails of nucleosomal DNA to form nuc-bells.

Nucleosome Immobilization and Modification Detection on ZMWs

In SMRT sequencing, the library molecule is immobilized via a streptavidin–biotin interaction. Specifically, a streptavidin-conjugated DNA polymerase binds to the molecule, which is then anchored to the biotin-functionalized floor of a ZMW, within which the excitation beam is confined to a zeptoliter volume. To confirm the nucleosome and polymerase complex formation, we loaded the labeled nucleosome–polymerase complex, labeled with Cy3-dATP (section 1 of the Supporting Information), by incubating it on a PEG–biotin-coated coverslip within a flow cell for 10 min and then washed and imaged the glass coverslip using a TIRF microscope (Figure A). This yielded a large number of fluorescence spots from the nucleosome/polymerase complex binding to the biotinylated surface (Figure B). As controls, we also loaded and imaged three other samples, including a nucleosome library without polymerase, a complete DNA library, and a DNA library without polymerase (the DNA samples were extracted from the mononucleosomes in parallel lanes). There were relatively few fluorescence spots from the nucleosome sample without polymerase (Figure B) and even fewer spots from the DNA sample without polymerase, as counted (Figure C) vs non-specific binding (without polymerase) of the nucleosome sample and DNA sample. This experiment validated the SM-NucSeq library prep by confirming that the nucleosome sample can effectively bind to PacBio SMRT polymerase and subsequently be immobilized through streptavidin–biotin binding. Finally, the same nucleosome sample was added to an Astro chip, a prototypical ZMW chip that has arrays of 3000 100 nm size holes situated on a thin Al layer deposited on a fused silica sheet. This yielded immobilized fluorescence spots (Figure D, bottom panels), proving that the ZMW can anchor a nucleosome library similar to a standard DNA library. Noticeably, these two imaging surfaces have shown different signal spatial distributions (compare Figure B and D), as the ZMWs are arranged in a square array (Figure E).

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(A) Schematic diagram of preparing HEK293 extracted nucleosomes and Cy3 tagging of nucleosomal DNA, followed by nuc-bell generation, DNA polymerase–streptavidin fusion binding to the nuc-bells, and capture on biotinylated coverslips. (B) Fluorescence imaging of Cy3-nuc-bells bound to a biotinylated coverslip with (left) and without (right, negative control) polymerase–streptavidin fusion. (C) Captured nucleosome and DNA counts (±polymerase) on biotinylated coverslips (statistical data compiled from 96 FOVs in one experiment; error bars are SD). (D) Time-lapse images of H3K27me3 antibody binding on the nucleosome/DNA polymerase complex on a ZMW chip (showing the first four time points of the acquisition series). H3K27me3 antibody binding on the nucleosome/DNA polymerase complex on a ZMW chip (top) and the antibody binding signal co-localization with Cy3-tagged nucleosome (bottom; the nucleosome signals are compiled from all time points). The antibody signal is colored red to enhance visibility and does not reflect its actual fluorescence color. Scale bars = 5 μm. (E) Bright-field image of a top-illuminated ZMW array. (F) Illustration of histone modification detection on a biotinylated glass coverslip (left) or on a ZMW array (right). (G) Percentage of nucleosomes detected with H3K27me3 modification with biotinylated coverslips or ZMW arrays [statistical data compiled from 96 FOVs (for coverslip) or 15 FOVs (for ZMW) in one experiment; error bars are SD].

In the following histone modification detection test, the PEG–biotin-coated glass surface was also used in parallel as a control over the ZMW device surface. Following nuc-bell binding, the surfaces were incubated with fluorescently labeled histone modification antibodies (Figure F). Trimethylation of lysine 27 on the histone H3 protein subunit (H3K27me3, conjugated with Alexa 488), a modification associated with gene silencing, was selected for this experiment. The binding and dissociation events of the antibodies to their corresponding modified nucleosomes, immobilized on a glass surface vs ZMWs, were recorded on the TIRF microscope with images taken every 15 min for a total of 2 h. Figure D shows the fluorescence signals from the H3K27me3 antibody (blue) and their composite co-localizing images with nucleosomes (green). Binding and dissociating of the antibodies to the target nucleosomes on the ZMW chip showed similar dynamics to the patterns observed on the biotinylated glass surface and in agreement with our previous report ,, (Figure D and Figure S4). Image analysis with CellProfiler revealed that 6.8 and 6.4% of nucleosome signals were co-localized with H3K27me3 antibody signals on the coverslip surface and ZMW chip, respectively (Figure G). The percentages of H3K27me3-modified nucleosomes were in agreement with previously published results. Overall, the ZMW chip performed similarly to the more established single-molecule histone modification detection method, and the coexistence of DNA polymerase with nucleosome did not tamper with the binding of antibodies targeted to histone modifications.

Nucleosomal DNA Sequencing on ZMW on a TIRF Microscope

The key advantage of ZMWs over coverglass is that, in ZMWs, the sequencing of a single nucleosomal DNA molecule can be achieved in real time using SMRT sequencing (Figure A). We first tested nucleosomal DNA sequencing after histone modification detection on our custom-built TIRF microscope as a preliminary sequencing experiment (Figure B–D). The antibodies were removed by washing, and the sequencing buffer was added to the ZMW chip. During sequencing, the signals from each active ZMW were collected as a series of fluorescent bursts projected on four vertically spread locations on the EMCCD, representing four chromatically resolved bases (each tagged with a different fluorophore), and its chronological sequence represented the complementary sequence of the nucleosomal DNA template (Figure D). The dTTP and dGTP signals were easily identifiable; however, the dATP and dCTP signals had a significant overlap that required extra effort in data analysis to identify them (Figure D and Figure S8D). We have implemented a frame-by-frame approach to capture fast signals from enzymatic reactions that were subjected to higher random noise. To that end, we deployed a Fourier series fitting algorithm. First, the frames with fluorescence bursts in each ZMW region were isolated using blob detection. Then, the average signal intensities on the vertical axis of each frame were computed and fitted into a Fourier series model (section 2.5 of the Supporting Information) to obtain the fitting coefficient β1, an indicator sensitive to the location of the signal’s weight center. Each fluorescence burst from an incorporation event spanned from several to tens of frames and formed a cluster in the time trace (Figure E). Finally, we performed a density-based clustering method, the ordering points to identify the clustering structure (OPTICS) algorithm, to group and identify signals from each nucleotide, and the bases were called by evaluating the average location indicator β1 of such identified clusters (Figure E).

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(A) Schematic diagram of SM-NucSeq imaging processes. First, imaging antibody binding; second, the antibodies were washed away, and the spike in the labeled nucleotide was used to start the DNA polymerization and sequencing. (B) Fluorescence image of two histone modifications simultaneously on ZMW using the micromirror TIRF microscope. Red, H3K9ac antibody; blue, H3K27me3 antibody. (C) Average intensity image stacked from a sequencing video, highlighting an active ZMW from the ZMW array. (D) Reading the nucleotide sequence from a ZMW sequencing image stack. (E) Clustered fluorescence signals from a ZMW sequencing image stack. Histograms of the fitting coefficient β1 from each frame that has a fluorescence signal (has a blob detected) are shown on the side.

Next, we combined DNA sequencing with epigenetic information. Excitingly, we found that some of the sequencing signals co-localized with one or more histone modification antibody signal, providing a proof of concept for the sequencing of modified nucleosomes. Nevertheless, these experiments were limited by the throughput of the prototypical ZMW chip (fabricated with arrays of ∼3000 ZMWs) and the custom-built microscope (with the field of view cropped to satisfy the high frame rate demanded for monitoring the polymerization reaction in real time). With the concept proven viable, we turned to a commercially available PacBio RS II sequencer, which could simultaneously monitor and record ∼150 000 ZMWs on one SMRT cell, to magnify the data output.

High-Throughput Experiment on the RS II Sequencer

RS II sequencers with their commercially oriented software are dedicated to DNA sequencing and are unable to perform our SM-NucSeq workflow directly. Fortunately, a modified workflow and software for the RS sequencer were developed to allow a powerful, high-throughput platform for single-molecule fluorescence microscopy (SMFM), which provides great flexibility to the RS sequencer beyond standard DNA sequencing tasks. , We accessed a RS II sequencer equipped with this SMFM system at the University of California, Riverside, to perform SM-NucSeq experiments. In addition to the iconic well-like nanostructure, each ZMW unit in the SMRT cell used by the RS II features a cone-shaped micromirror structure attached to its bottom. It collects emission signals from the ZMW and directs them toward the imaging optics, greatly enhancing the signal collection.

The histone modification detection on the RS II sequencer was performed in a slightly modified method, as the RS can only run the continuous imaging mode. A PCA/PCD oxygen scavenging system and a built-in nitrogen gas flow were applied to reduce the oxidation and photobleaching of the antibody fluorophores. Thus, we were able to monitor the binding and dissociation of antibodies in real time with continuous imaging, which provided more information on antibody binding dynamics (Figure A). In this test, the H3K9ac antibody (Alexa 647 conjugated) and H3K4me3 antibody (trimethylation of lysine 4 on histone H3, Alexa 555 conjugated) were selected based on their compatibility with the RS II four-color fluorescence channels and their robust and consistent binding affinities observed from our previous experience. Both modifications are associated with active transcription and have been reported to mark the promoter regions of genes. ,

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(A) Fluorescence intensity traces of H3K9ac antibody detection (red) and H3K4me3 antibody detection (green) and co-localized detection events on SMRT cells, showing antibody binding and dissociation. We identified true binding events by detecting distinct single-molecule fluorescence features such as a single step rise from the baseline or a single drop down to the baseline, representing the arrival of a single molecule or the departure or photobleaching of the molecule. Small pictures inside the box show an image of a SMRT cell (Pacific Biosciences) and a reconstructed fluorescence image of the Cy3-nucleosome/polymerase complex immobilized on a SMRT cell. (B) Time trace of DNA sequencing on a SMRT cell. (C) Time trace of nucleosome sequencing on a SMRT cell. Four colored traces are from four nucleotide channels. (D) Histogram of polymerization rates from sequencing nucleosomes (gray) and ICC DNA (blue). (E) Histogram of polymerization rates from sequencing nucleosomes (gray), H3K9ac-detected nucleosomes (red), and H3K4me3-detected nucleosomes (green). For this kinetic comparison, we employed data from the same experiment on a single nucleosome library. (F) Polymerization rate profiles for several molecules, including DNA and nucleosome.

Because the SMFM program is independent of the standard RS sequencing software, it cannot directly execute the regular DNA sequencing protocol, which inevitably incapacitates the built-in data analysis and base-calling program. Therefore, we generated a self-made program to interpret and translate the raw intensity signals to DNA sequences. To validate this modified sequence workflow, we performed a sequencing experiment with the PacBio internal control complex (ICC, a commercially available pre-made DNA library). Figure B demonstrates an example of the ICC sequencing data showing a series of fluorescence bursts in the intensity traces of the four fluorescence channels. We used MATLAB scripts to detect the fluorescence burst events and decide the bases from the overlapping events (caused by channel bleed-through) by evaluating the relative intensity against the background noise (section 3 of the Supporting Information). We were able to successfully align the obtained sequences to the ICC reference sequence (Figure S11). Although our base-calling program is not as accurate (with around 70% accuracy) as PacBio’s software, as a result of lacking a sufficient training data set and circular consensus sequencing (CCS, a common tool to improve SMRT sequencing accuracy), it was sufficient for the primary goal of sequencing and alignment.

Subsequently, the nucleosome samples were successfully sequenced with this SMFM workflow (Figure C). Interestingly, the average nucleosome library polymerization rate was found to be slower than that of the ICC DNA library (Figure D). We fitted the average polymerization rate distribution of the nucleosome library to two Gaussian populations. The narrow peak around 0.5 base/s likely represented a population of misbehaving polymerases and ZMWs, including misaligned ZMWs with fluorescence lost in some channels, inactive ZMWs, and nucleosomes that rejected polymerase access. Another broadly distributed curve had a median polymerization rate of around 1 base/s. This can be explained by the additional time consumed by the process of unwinding DNA from the histones. We also discovered that the nucleosomes marked with H3K9ac modification or H3K4me3 modification had a slightly higher polymerization rate (at around 1.2 bases/s) (Figure E). This suggests that these two modifications can help DNA unwrap by altering the histone–DNA interaction. For example, acetylation can neutralize the positive charge on lysine, thus weakening histone’s attraction to DNA. Figure F shows examples of bases processed by the polymerase as a function of time. In some nucleosome traces, a distinct shift in the polymerization rate from a level similar to that of a DNA trace to a lower level can be observed.

Performing SM-NucSeq on Two Histone Modifications with the RS II Sequencer

With the mononucleosome extracted from the HEK293 cells, we found that 9.7% of nucleosomes carried H3K9ac modification (Figure A), higher than the 6.4% number obtained on Astro chips. A possible explanation was that the method to image every 15 min could miss some short-term binding and dissociation events. A total of 5.8% of nucleosomes were found carrying H3K4me3 modification (Figure A). Interestingly, 13.6% of H3K4me3-marked nucleosomes also had H3K9ac modification, and 8.3% of H3K9ac nucleosomes also had H3K4me3 modification (Figure A), confirming their association within some genomic regions.

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(A, Left) Percentages of K4me3 nucleosomes and K9ac nucleosomes. (Right) Percentage of H3K4me3 nucleosomes that were also detected with H3K9ac modification, and the percentage of H3K9ac nucleosomes that were also detected with H3K4me3 modification (statistical data compiled from three SMRT cells). (B) Distance of detected modified nucleosomes to the nearest TSS site. (C) Aligning detected bivalent H3K9ac and H3K4me3 modifications against the ChIP-seq data. Tracks from top to bottom: 1, detected H3K9ac nucleosomes; 2, detected H3K4me3 nucleosomes; 3, HEK293 cells H3K9ac ChIp-seq data from ENCODE; 4, HEK293 cells H3K4me3 ChIp-seq data from ENCODE; 5, HEK293 cells H3K9ac ChIp-seq peaks from ENCODE; 6, HEK293 cells H3K4me3 ChIp-seq peaks from ENCODE; and 7, corresponding genes from NCBI RefSeq.

Both H3K9ac and H3K4me3 modifications co-localize and are enriched near the transcription start sites (TSS). Using the Genomic Regions Enrichment of Annotations Tool (GREAT; , http://great.stanford.edu/public/html/), we calculated the distance of detected and aligned nucleosomes to the TSS and found that approximately 18% of the detected nucleosomes were associated with the TSS (Figure B). The modified nucleosome alignments were compared against two reference data sets from ENCODE: H3K9ac and H3K4me3 data were obtained from running ChIP-seq with HEK293 cells. Since only a fraction of the reads were from modified nucleosomes, they were sporadically scattered across all chromosomes at the current throughput. Nevertheless, we were able to locate some bivalently modified nucleosomes (Figure C).

In this work, we demonstrated the principal concept and feasibility of using zero-mode waveguides to detect and map histone modifications at a single-molecule level. Compared to established epigenetic techniques, such as ChIP-seq and CUT&run, SM-NucSeq utilizes single-molecule sequencing technology to provide a roadmap for full-scale sequencing that can simultaneously uncover genetic information and histone post-translational modifications. Although not demonstrated in this work, ZMW SMRT sequencing is also capable of detecting DNA methylation, providing a prospect of generating single-molecule data for both histone modifications and DNA methylation on individual nucleosomes. , As a single-molecule method, it also has the unique advantage of detecting bivalently and multivalently modified nucleosomes, critical for investigating combinatorial epigenetic effects.

Using a custom-built chromatically resolved TIRF microscope, we validated individual SM-NucSeq processes, including SM-NucSeq library prep, nucleosome library loading to the ZMW chip, imaging antibody binding, and SMRT sequencing of nucleosomal DNA. However, due to the limited optical field, this method offers intermediate throughput. On a commercial RS II sequencer with a specialized SMFM system, we successfully replicated the SM-NucSeq workflow and obtained a magnified set of data. With a much higher throughput, we were able to calculate the statistics of nucleosomes marked with both H3K9ac and H3K4me3 and map these nucleosomes to the genome. Interestingly, we also observed differences in replication rates between the nucleosome library and the DNA library and between the modified nucleosome and the unmodified nucleosome. This could open a new route for investigating the dynamics of unwrapping DNA around nucleosomes with distinct epigenetic features.

These proof-of-concept experiments harbor great potential for expanding ZMW sequencing technology beyond long-read DNA sequencing to become a multiplex genetic and epigenetic toolbox. Further development of this method requires a few key performance areas of improvement. First, a modified native RS II sequencing program could replace our custom-built data acquisition and analysis workflow and produce sequencing data of higher quality. Second, given the dumbbell structure of the nucleosomal DNA, CCS can be applied to produce additional information (e.g., detection of DNA modification sties). This platform can also be applied to investigate the effect of chromatin-modifying complexes, such as facilitates chromatin transcription (FACT), nucleosome remodeling factor (NURF), and nucleosome-destabilizing factor (NDF), on chromatin unwrapping and accessibility, as it can monitor the polymerase reactions in real time. Together, these advancements establish SM-NucSeq as a transformative single-molecule platform, offering unprecedented insights into chromatin dynamics and combinatorial modifications and paving the way for real-time, high-resolution studies of nucleosome biology and gene regulation.

Supplementary Material

nl5c06446_si_001.pdf (10.8MB, pdf)

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.nanolett.5c06446.

  • Materials and methods for sample preparation, chromatically resolved micromirror TIRF microscope setup, protocol for imaging antibody detection, method for DNA sequencing on the TIRF microscope, including data analysis, experimental details for running SM-NucSeq on the RS II sequencer, and Figures S1–S11 (PDF)

The authors declare no competing financial interest.

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