Adams, Asher_Senior Thesis 2026

Transcription Factor Regulation, Cofactor Regulation, the Development of a Liquid-Based ProteinBinding Microarray Assay and its Applications to Biomedical Research Asher Adams Senior Thesis | 2026

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TRANSCRIPTION FACTOR REGULATION, COFACTOR REGULATION, THE DEVELOPMENT OF A LIQUID-BASED PROTEIN-BINDING MICROARRAY ASSAY AND ITS APPLICATIONS TO BIOMEDICAL RESEARCH By Asher Weston Adams

Senior Thesis Boston University | Boston University Academy March 2026 Thesis Advisor: Olivia Waring

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1. Introduction 1a. TF-COF Significance and the Methodological Gap Transcription factors (TFs) and cofactors (COFs) form the core regulatory machinery that governs gene expression. TFs recognize specific DNA sequences and recruit COFs, which in turn modulate chromatin structure, stabilize protein-DNA interactions, and recruit enzymatic complexes that activate or repress transcription. These interactions operate within a multilayered regulatory environment, where DNA accessibility, histone modifications, and cooperative binding networks collectively determine when and where genes are expressed.

Understanding TF-COF interactions is therefore essential for understanding transcriptional regulation itself, as these partnerships shape the specificity, strength, and context dependence of gene expression across cell types and developmental states. [1]

Despite their importance, TF-COF interactions remain difficult to study quantitatively. Many existing methods capture DNA binding profiles or protein-protein interactions, but few can measure how TFs and COFs cooperate on DNA under controlled conditions. Traditional biochemical assays often lack scalability or require purification strategies that remove essential COFs. Genomic assays performed in cells offer a rich biological context but make it challenging to isolate direct interactions or indirect, secondary effects. These limitations create a methodological gap, and

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researchers need tools that can more deeply analyze TF-COF binding in a systematic, high-throughput, and controlled environment. [2] 1b. PBMs as a Platform and their Core Limitations Protein Binding Microarrays (PBMs) have emerged as a major platform for mapping TF-DNA binding specificity, the degree to which a transcription factor preferentially recognizes and binds one DNA sequence over all others, with high resolution by immobilizing a comprehensive library of DNA sequences on a microarray and measuring TF binding through fluorescence. PBMs allow for rapid discovery of binding motifs, the short, characteristic DNA sequences that a given TF recognizes and binds, as well as quantitative comparison across large TF families. However, PBMs also imposed various constraints. They require custom array fabrication and large amounts of purified protein, and their short, surface-bound DNA probes limit the range of interactions that can be observed. Most importantly, PBMs are fundamentally solid-phase assays, which means they cannot fully capture the dynamics, flexibility, and multicomponent assembly that characterize TF-COF interactions in solution. [3] 1c. Project Purpose and Primary Research Question These limitations motivate the central problem addressed in my thesis.

While PBMs are powerful, they are also costly, material-intensive, and constrained by probe length and immobilization, restricting their adaptability and making them poorly suited for studying TF-COF complexes. [3] Critically, the field currently lacks high-throughput tools capable of measuring TF-COF binding interactions systematically and at scale, a gap that limits both basic research into transcriptional regulation and applied efforts in drug discovery. The goal of this study is therefore to develop and optimize a liquid-based PBM, a solution-phase assay design that preserves the quantitative strengths of PBMs while affording a more realistic biochemical environment for interaction, and reducing the resource burden associated with the traditional platform.

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The purpose of this project is to determine whether a liquid PBM can serve as a flexible, scalable, and cost-effective complement to solid-phase PBMs. Specifically, this work seeks to determine whether the liquid assay can generate reproducible and biologically meaningful readouts of TF-COF interactions while requiring less material and avoiding the structural constraints imposed by immobilized DNA. The primary research question guiding this study is: Can a liquid-based PBM achieve consistent and specific detection of TF-COF interactions while reducing the material, cost, and technical barriers associated with traditional PBMs? 1d. Project Goals The first goal is to define reaction conditions under which the liquid PBM can reliably detect true, antibody-mediated, proximity ligation events, rather than background or non-specific ligation. This includes identifying the buffer conditions, incubation parameters, and reaction temperatures that produce stable, interpretable bands across repeated experiments, ensuring that the assay behaves consistently. Because the liquid PBM is highly sensitive to component concentrations, the project systematically varies each reagent to find conditions that maximize signal while minimizing nonspecific background. The result is a set of working concentrations that can act as a standardized baseline on which future experiments can be based. This work examines potential sources of experimental noise, including PCR contamination, endogenous ligase activity in nuclear extracts, pipetting error, and enzymatic (DNase) activity. Through targeted controls and troubleshooting, the project aims to determine which factors most strongly disrupt the readout and how to reduce or eliminate these sources of error. Beyond optimization, the project assesses whether the liquid PBM can function as a scalable, lower-cost complement to traditional slide-based PBMs. This includes comparing the requirements, workflow complexity, and data quality across both formats and determining whether the assay can, in principle, be expanded to profile multiple TF-COF pairs systematically.

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2. Background 2a. TF-COF Regulatory Biology and Chromatin Context To understand the motivation and design of the liquid PBM, it is necessary to first examine the biology of TF-COF interactions and the landscape of existing tools used to study them. The human genome encodes over 1600 TFs with diverse binding domains and functions, each with characteristic binding conditions and roles, illustrating the scale of transcriptional control. [4] These proteins function within larger complexes that coordinate transcription initiation, elongation, and chromatin accessibility. [5] Cofactors (COFs) are generally non-DNA-binding partners that interact with TFs to stabilize complexes, remodel chromatin, or allow for enzymatic activities that modify histones or the transcriptional machinery. Experiments such as those by Siggers et al. showed that COFs can enhance the DNA-binding specificity of a TF. [6] Work by Wang and coauthors demonstrated that particular combinations of TFs and COFs determine which types of blood cells develop from stem cells through lineage-specific gene regulation, a process called hematopoiesis. [7] COFs often bridge TFs to the enzymes that place or remove histone modifications, coupling sequence-specific recognition with broader chromatin remodeling. [8] This layered structure means that transcriptional output depends not exclusively on DNA binding affinity but also on the chromatin state surrounding the binding site, making epigenetic regulation inseparable from TF function.

2b. Quantification Challenges and CoRec Despite progress in identifying transcriptional regulators, it remains challenging to quantify TF-COF networks. Their interactions are brief, context-dependent, and influenced by the surrounding environment, leading to variability that traditional biochemical or genomic assays cannot fully capture. A recently developed high-throughput system to profile these networks, called CoRec, revealed organizing principles of epigenetic regulation through its protein-binding microarray (PBM) approach. It demonstrated strong reproducibility across replicates and

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successfully quantified the efficiency of cofactor recruitment, DNA binding affinity, and TF-COF interaction affinity. However, like other microarray-based methods, CoRec uses short, unchromatinized DNA probes, meaning naked DNA not wrapped around histone proteins and therefore lacking the nucleosome structure and epigenetic modifications present in living cells. As a result, these probes cannot fully simulate the binding dynamics that occur on timescales of seconds in vivo. [2] These limitations expose the central methodological gap in the field: existing approaches provide static snapshots of aggregate binding under fixed conditions, but cannot resolve the kinetic parameters, including association and dissociation rates, residence times, and dynamic cofactor exchange, that govern transcriptional output in physiologically complex environments.

2c. PBM History, Design, and Workflow The PBM emerged in the mid-2000s as a systematic high-throughput platform for measuring TF-DNA binding specificity. [9] The “universal” PBM, first introduced in 2006, is a microarray design incorporating synthetic double-stranded DNA probes that collectively represent all possible 10-base pair sequences. [10] This allows simultaneous measurement of a TF's binding to every potential short motif on a single array. This approach provides an alternative to the serial low-throughput methods that were then dominant, such as electrophoretic mobility shift assays, by quantifying TF binding across thousands of unique sequences in parallel. [11] The follow-up protocol paper standardized the workflow, detailing each step from TF purification and fluorescent labeling to hybridization, washing, and scanning. [3] The protocol also formalized computational processing, including the calculation of enrichment scores, which rank each DNA sequence by how strongly a TF binds relative to background, and the derivation of position weight matrices, quantitative models that capture the probability of each nucleotide appearing at each position within a binding site and together summarize a TF's sequence preference across the full probe set. Together, these works established PBMs as the reference framework for in vitro quantitative assessment of TF-DNA interaction and provided the basis for subsequent large-scale

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binding databases. Because the probe set is exhaustive, PBMs can also reveal subtle variations in binding specificity arising from amino acid substitutions or post-translational modifications, giving them an advantage over purely binary binding assays. [3]

A typical PBM experiment uses a glass microarray slide containing tens of thousands of double-stranded DNA probes, 60 nucleotides long, with sequences chosen so that every possible k-mer site (a short DNA sequence of length k) is represented multiple times. Purified TFs, often expressed with an affinity tag (a guide for an antibody), are incubated with the array under controlled conditions. After binding and washing, the protein is detected with a fluorescently labeled antibody specific to the tag. The fluorescence intensity at each spot corresponds to the relative affinity of the TF for that probe sequence, allowing quantitative comparison across the entire sequence space. Following data acquisition, enrichment scores are computed for each binding site and aggregated to generate sequence logos—tools to determine how often each nucleotide appears at each position in a binding site, summarizing a transcription factor’s DNA preference. The key strengths of the PBM design are reproducibility, throughput, and universality: a single array can capture both known and previously uncharacterized motifs, and parallel assays allow direct comparison of TF families. Because the probe set is

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exhaustive, PBMs can reveal subtle variations in specificity arising from amino acid substitutions, post-translational modifications, or the presence of cofactors when these are included experimentally. The fluorescence-based quantification also enables a semi-quantitative estimation of relative affinity, giving PBMs an advantage over purely binary binding assays. [3] 2d. PBM Structural Constraints Protein-DNA interactions on a rigid substrate may display altered affinities or reduced flexibility compared to those in a three-dimensional nuclear environment. Each experiment demands purified protein at relatively high concentration, specialized microarray fabrication, and fluorescence scanning equipment. These demands limit scalability outside dedicated facilities, especially for smaller labs. Addressing these shortcomings motivates the development of alternate approaches, such as the liquid-based PBM explored in this study, which seek to preserve the high-throughput, quantitative advantages of PBMs while introducing a more physiologically relevant, dynamic solution environment for measuring TF-COF interactions.

2e. Recent Innovations and Field Trajectory Recent developments have extended the capabilities of traditional PBMs by introducing designs that more closely approximate cellular conditions.

One example is NextPBM, which adapts the PBM platform to measure transcription factor binding in a more cell-specific and context-dependent manner. NextPBM incorporates features such as cell-derived nuclear extracts, modified probe designs, and conditions that allow cooperative interactions between transcription factors to be observed more effectively than in the original universal PBM format. This innovation demonstrates that PBM technology continues to evolve beyond its early rigid framework, moving toward assays that can capture more biologically realistic regulatory behavior. [12] Across the field, there is a broader push to design transcriptional assays that are both highly scalable and more accessible. Traditional PBMs,

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SELEX-based methods, and complex chromatin assays often require large amounts of protein, numerous reagents, and specialized equipment, limiting their use and adoption to well-funded laboratories. Newer platforms seek to reduce these barriers by minimizing material requirements, automating steps of the workflow, and leveraging more efficient detection strategies. High-throughput methods, such as barcoded libraries, microfluidic systems, and rapid profiling tools like those described in recent high-throughput studies, illustrate a shared objective: to generate regulatory binding data at a significantly lower cost and with fewer technical constraints. [13] These efforts underscore the need for assays that maintain precision while improving experimental accessibility.

2f. LEF1 as a Model System To develop and validate the liquid PBM, this project used Lymphoid Enhancer-binding Factor 1 (LEF1) as the model transcription factor.

LEF1 is a member of the HMG-box family of TFs and plays a central role in the Wnt signaling pathway, where it recruits cofactors such as β-catenin to activate target gene programs involved in lymphoid development, cell proliferation, and differentiation. Its well-characterized binding motif and established cofactor interactions make it a tractable model system for assay development: the binding site sequence is known, a validated antibody reagent is available for the relevant cofactor, and prior work in the Siggers Lab provides a reference framework against which liquid PBM performance can be assessed. All binding oligos used in this project were designed around the LEF1 consensus binding site, with a sequence-disrupted mutated version serving as the negative control throughout.

3. Methods 3a. Lab Setting and Assay Design Framework All of the work for this project was carried out at the Siggers Lab at Boston University, a setting equipped for detailed biochemical and

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molecular analysis of transcriptional regulation. Working in this environment provided access to the full range of tools needed for assay development, including facilities for DNA preparation, cell cultivation, controlled incubation, PCR amplification, and gel electrophoresis. The lab’s established protocols and technical support ensured that each experimental setup was performed under conditions that minimized variability and maintained experimental integrity.

The assay was designed by adapting the conceptual framework of a PBM into a liquid-based format that could test TF-COF interactions under more physiologically realistic conditions. This required constructing a multistep workflow modeled on the CoRec proximity ligation framework developed by Inge and colleagues in the Siggers Lab. [2] Each stage of the project design, execution, troubleshooting, and interpretation reflected the process of building an experimental system from the ground up to determine whether the essential strengths of PBMs could be preserved in solution rather than on a fixed microarray surface.

3b. Proximity Ligation Mechanism The liquid PBM employs a proximity ligation strategy to detect TF-COF interactions in solution. [14] The assay is based on two pre-digested DNA oligonucleotides, short synthetic single- or double-stranded DNA sequences designed to bind specific molecular targets, with compatible restriction overhangs: a template oligonucleotide containing the TF binding motif of interest, and an antibody-conjugated oligonucleotide bearing a covalently attached antibody specific to the COF being tested.

When nuclear extract, a cell-derived mixture containing endogenous transcription factors, cofactors, and associated proteins in their native form, is added to the reaction, the TF binds to its assigned motif on the template oligonucleotide. If the COF interacts with the DNA-bound TF, the COF-specific antibody attaches to the COF, thereby bringing the antibody-conjugated oligonucleotide in proximity to the template oligonucleotide. This proximity enables T4 DNA ligase to join the compatible overhangs of the two oligonucleotides, creating a single longer DNA strand. [15] The ligated DNA product serves as the direct

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readout of TF-COF interaction; it is amplified by PCR and visualized by gel electrophoresis. The presence of a band at the expected size for the ligated product indicates that the TF and COF interacted in solution, while band intensity reflects the efficiency of the interaction. In the absence of TF-COF binding, the two oligonucleotides remain spatially separated, and ligation efficiency is minimal, producing little to no PCR product of the desired length.

3c. Component Preparation The following sections describe the preparation of assay components, experimental workflow, and optimization strategy used to develop this liquid PBM platform.

Template and antibody-conjugated DNA oligonucleotides were prepared by restriction digestion to generate the compatible single-stranded overhangs required for proximity ligation. Two restriction enzyme systems, PaqCI and BsaI, were tested in parallel during the early phase of the project (February through March) to compare the quality and consistency of the overhangs each produced. Both are Type IIS enzymes that cut outside their recognition sequence, allowing the generation of defined, asymmetric overhangs without leaving enzyme recognition site sequences at the ligation junction. Early experiments ran PaqCI-cut and BsaI-cut binding oligos in separate tube sets (Tubes 01-06 and 07-12, respectively) to directly compare performance on gel. By June, the BsaI system was selected exclusively based on more consistent digestion efficiency and cleaner overhang generation observed across these parallel comparisons.

Binding oligos used across the project included the wild-type LEF1 binding site (Wild Type BS) and a sequence-disrupted mutated version (Mutated BS), which served as the negative control throughout. A blunt-end ligation variant was also tested in late June and July, using uncut binding oligo (312 bp, 31.4 ng/μL stock) paired with precut antibody-oligo to assess whether blunt-end ligation could substitute for the cohesive-end system. These experiments did not produce a better

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signal than the BsaI-cut cohesive-end approach, and the project returned to precut BsaI binding oligos for all subsequent experiments beginning July 10.

3d. Binding and Ligation Phases In the binding phase of the assay, TFs present in the nuclear extract interact with the template DNA and recruit their associated COFs through native interactions. An antibody-linked oligonucleotide, designed so that its attached antibody recognizes a specific COF, then binds to the recruited COF. This brings the DNA template and the antibody oligo into close spatial proximity. The resulting arrangement creates the physical setup necessary for antibody-mediated proximity ligation and enables the assay to detect TF-COF interactions through later events.

Once the binding complex is formed, T4 DNA ligase and ATP are added to join the compatible overhangs and seal the nicks in the resulting DNA backbone. A central challenge in this stage was distinguishing true

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antibody-mediated ligation events from nonspecific or non-specific ligation that could occur independently of TF-COF interactions. Careful control reactions and repeated testing were therefore essential to confirm that ligation signals reflected biologically meaningful binding rather than non-specific ligation and background noise.

3e. PCR Amplification and Controls Following ligation, the joined DNA product was amplified using PCR. The intensity of the amplified band served as a semi-quantitative measure of the ligation efficiency, and, by extension, the strength of the TF-COF interaction being tested. As part of my optimization of the protocol, I established the appropriate number of PCR cycles through serial dilution of the DNA template. This involved taking a fixed volume of ligation product, diluting it across a range (initially 0.1, 0.25, 0.5, 1.0, 2.0, and 5.0 μL template per reaction in March, later standardized to 1:2 through 1:16 serial dilutions in June onward), and running all dilutions through identical PCR conditions in parallel. The dilution at which the amplification product became faint or absent on the gel defined the upper boundary of the linear range. The template volume that produced a clear band two to three dilution steps above this boundary was selected as the standard input. This procedure was repeated across experiment dates to account for variation in ligation efficiency and product concentration

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between runs. The PCR template volume was ultimately standardized at 0.5 μL from a 1:5 dilution (2 μL ligation into 8 μL water) through July, and later updated to 2 μL from a larger 1:20 dilution (2 μL ligation into 38 μL water) beginning in August to further reduce template concentration and avoid saturation, which would mask differences between specific and nonspecific binding events and obscure meaningful variation in the data.

To distinguish specific TF-mediated binding from background ligation, experiments included both wild-type (WT) DNA sequences containing the authentic TF binding motif and mutated sequences with disrupted binding sites. WT sequences should produce a robust ligation signal, while mutated sequences, which prevent TF binding, serve as negative controls, establishing baseline background ligation levels.

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3f. Gel Analysis and Optimization Strategy PCR products were analyzed by gel electrophoresis to visualize ligation events. Initially, differences in band presence and intensity guided optimization of reagent concentrations, and once those concentrations were established, gel analysis allowed the comparison of wild-type sequences (specific binding) and mutated sequences (nonspecific/background ligation), confirming that the majority of the signal depended on specific protein-DNA interactions rather than nonspecific ligation. Gel imaging provided the final readout of each assay iteration and guided the ongoing optimization of reaction conditions.

The major part of developing the liquid PBM involved systematically testing and adjusting the concentrations of the core reaction components—T4 DNA ligase, the antibody oligonucleotide, and the nuclear extract. Each reagent strongly influenced the strength, clarity, and reproducibility of the results. Experiments were performed in cycles: concentrations were varied across a controlled range, results were evaluated on gels, and the most promising conditions were carried forward into the next round of testing. Through this iterative approach, the assay gradually moved from inconsistent and weak signals toward conditions that produced clearer and more reliable ligation products.

4. Results 4a. Early Development and Inconsistency The first experiments conducted in February 2025 served primarily as proof-of-mechanism tests rather than optimized assays. Using a 50 μL binding volume, a nuclear extract stock of 8.12 μg/μL adjusted to 1 μg/μL final, and both PaqCI and BsaI enzyme systems running in parallel across twelve tubes, these experiments aimed to determine whether any ligation product could be detected at all. ATP was added separately at 1 mM final concentration, and NaCl was adjusted to 110 mM final, accounting for the salt contribution of BufferC (357.7 mM). These early

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gels were inconsistent: bands appeared in some replicates and not others, and the mutated-sequence controls showed nonspecific signals comparable in intensity to the wild-type lanes, making it impossible to claim specificity. The February experiments established that the assay mechanism could produce a PCR product in principle, but that the concentrations, buffer conditions, and ligation parameters required substantial refinement before any result could be interpreted.

The development timeline showed clear progression in assay result quality. Early experiments in June 2025 produced gels with weak bands and substantial variability across replicates, with bands appearing in the mutant oligos as well, making it difficult to distinguish true specific ligation events from chance and background ligation. By late August, after multiple rounds of optimization, the assay generated noticeably stronger band intensities, sharper bands for ligated products, and greater consistency and reproducibility across replicate and adjacent reactions.

This improvement reflected both optimized reagent concentrations and better control over technical variables.

4b. Identified Error Sources As the project progressed, it became clear that several technical errors were contributing to the inconsistent results. One major issue seen earlier

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was primer contamination, where template DNA found its way into the PCR primers and produced false amplification products visible on gel readouts, obfuscating the true ligation signal. Additional variability arose from human error in pipetting, particularly with small volume additions of ligase, which produced fluctuations in band intensity across replicates.

To rule out these confounding factors, the nuclear extract was also tested for endogenous ligase activity, which could, in principle, create background ligation products independent of the intended antibody-mediated ligation. Identifying and controlling these sources of error was essential to achieving the improved reproducibility observed later in the project.

4c. Mid-project Refinements In July, the assay began to show clear improvements as key technical parameters were refined. Serial dilutions were used to determine the optimal number of PCR cycles, ensuring that amplification remained within the linear range and eliminating the signal saturation that had previously obscured meaningful differences between reactions. At the same time, adjustments to the concentration of T4 DNA ligase and the antibody oligonucleotide produced stronger and more consistent bands

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across replicates. These refinements reduced background noise, improved overall signal clarity, and marked a transition from the early stages of the project to a more controlled and interpretable assay workflow.

4d. Late-stage Optimization and Final Working Conditions By late August, the assay showed marked improvement in both reproducibility and interpretability. Reactions performed under the optimized conditions produced consistent banding patterns across instances, and adjustments to the agarose concentration improved band resolution at the relevant size range. A more deliberate organization of lanes, systematically comparing wild-type, mutant, and no-protein controls side by side, made it substantially easier to distinguish proximity-mediated ligation from background signal. Under these conditions, band intensities aligned with what would be expected from genuine antibody-mediated ligation rather than non-specific ligation.

This convergence between predicted and observed results indicated that

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the liquid PBM design was beginning to function as intended, providing preliminary evidence that the assay could capture TF-COF interactions in a controlled and quantitative manner.

Each of the three core reagents (T4 DNA ligase, the antibody-conjugated oligonucleotide, and the nuclear extract) required systematic concentration screening before the assay produced interpretable results.

Identifying working concentrations was not a single decision but an iterative process that unfolded over several months of successive experiments, each round guided by the gel readouts of the previous one.

T4 DNA ligase was the first variable to require careful titration. Early experiments in February used a 400,000 U/mL stock, adding 0.5 µL per tube, a starting point inherited from existing proximity ligation protocols.

By March, this was increased to 2.5 µL per tube after early gels showed insufficient ligation product. By April, a further increase to 3 µL was tested alongside a temperature comparison (25°C for one hour versus 16°C overnight), which revealed that a room-temperature, one-hour incubation produced cleaner results without the risk of overnight evaporation or reagent degradation. A switch to a 20 U/µL working stock in June enabled more precise volume control, and subsequent experiments screened final concentrations of 0 U/µL (no-ligase control), 1 U/µL, and 0.5 U/µL within the 30 µL ligation volume. The 0.5 U/µL condition, established by late July (July 28), consistently yielded the best ratio of specific to nonspecific ligation product and was fixed for all subsequent experiments. Higher concentrations amplified background ligation; lower concentrations failed to drive sufficient ligation of the proximity complex.

The antibody oligonucleotide concentration required a parallel but independent optimization. An initial molar ratio screen in February tested 1X and 10X excess antibody-oligo relative to the binding oligo. In March, this was adjusted to 3X, and by April the screen shifted to a direct volume ratio approach (1:1, 1:0.5, and 1:0.25 binding-to-antibody oligo). Beginning in June, concentrations were tracked explicitly in nanomolar: three conditions (21.9 nM, 2 nM, and 0.2 nM) were run in

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parallel across the LEF1 WT and Mutated binding site designs. Gels from these experiments showed that 21.9 nM produced an elevated background in the mutated controls, suggesting nonspecific antibody-oligo proximity events at higher concentrations, while 0.2 nM produced a weak or absent WT signal. On July 28, a single concentration of 2 nM was tested; one day later, July 29, the concentration was increased to 10 nM following a weak signal in the 2 nM condition, and this value was retained without further variation through all subsequent experiments. The 10 nM condition produced the strongest and most consistent WT-specific signal while keeping the mutant background at interpretable levels.

The nuclear extract concentration was set early at 1 µg/µL final concentration in the binding reaction, a target maintained throughout the project by adjusting the volume added based on the stock concentration of each new batch. Stock concentrations varied across batches (8.12, 9.2, 11.83, 11.28, 9.37, and 8.66 µg/µL across successive preps), requiring recalculation of the additional volume for each new extraction. Higher nuclear extract concentrations were not systematically screened, as the 1 µg/µL target was inherited from prior lab protocols and consistently produced a detectable binding signal. An additional buffer variable, the NaCl concentration, was also corrected late in the project: earlier experiments used 4 µL of 1M NaCl in the 30 µL reaction, but a recalculation in late August that accounted for the salt contribution from the buffer reduced this to 1.87 µL (August 28), correcting what had been a salt excess throughout much of the summer.

Together, these three optimized values (0.5 U/µL T4 DNA ligase, 10 nM antibody-oligonucleotide, and 1 µg/µL nuclear extract at corrected NaCl) defined the final working condition for the liquid PBM and served as the baseline from which replicate designs were subsequently built.

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5. Discussion 5a. Core Findings and Proof of Concept This project has thus far demonstrated that a liquid-based PBM can generate clear and reproducible readouts while potentially requiring substantially less material than a traditional slide-based PBM. Under the optimized conditions identified over the course of the summer, the assay consistently produced ligation signals that reflected TF-COF-dependent interactions rather than background activity. This shift from weak, unstable bands to well-defined, repeatable products indicates that the core concept of using antibody-mediated proximity ligation as the central readout is viable in a solution-based environment. In practical terms, the assay succeeded in preserving a key advantage of PBMs, motif-level specificity, while reducing several of the barriers that limit PBM accessibility, most notably the advanced equipment and potentially the large quantities of purified protein.

5b. Remaining Challenges At the same time, the findings make clear that important challenges remain before the liquid PBM can be considered a fully mature assay.

The most significant barrier is the discrimination between genuine, antibody-mediated ligation events and nonspecific ligation that occurs by chance proximity. Although optimization has improved this discrimination, and rates are trending in the right direction, the assay is still sensitive to minor variations in component quality, incubation conditions, and pipetting accuracy. In particular, small volume errors in T4 DNA ligase addition translate directly into disproportionate changes in ligation efficiency, shifting the ratio of specific to nonspecific ligation products detected on the gel. These dependencies show the central difficulty of translating a surface-immobilized assay into solution: once DNA and proteins are free to diffuse between each other, the kinetic landscape becomes more complex and variable, and unintended interactions become far more likely. In addition, the work repeatedly showed that contamination can also confound real ligation events,

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making stringent reagent handling and control reactions essential for future use. These issues do not invalidate the assay but help highlight where methodological refinement is still required.

5c. Technical Interventions and Path Forward Several technical interventions directly improved the stability and interpretability of the assay, suggesting a clear path forward. Optimizing PCR cycles through dilution gradients substantially limited oversaturation as a source of signal distortion, while inhibiting DNase activity produced minor improvements by reducing DNA degradation.

These improvements show that many of the assay's weaknesses arise not from the conceptual design of the liquid PBM but from technical variables that can be systematically identified and corrected. In this sense, the findings point toward a future prototype that will be fully functional, promising, and increasingly reliable, provided that further work is conducted to refine the ligation efficiency, standardize components, and expand the panel of TF-COF pairs used to validate the system.

5d. Field Positioning Viewed against the current landscape of transcriptional assay development, the liquid PBM represents a promising early-stage prototype rather than a ready, direct substitute for established PBM platforms. Traditional slide PBMs continue to offer unparalleled performance in sequence coverage and highly standardized motif discovery pipelines, and the liquid assay does not replicate those capabilities in full just yet. Instead, it occupies a complementary space, adapting the PBM’s core principle of systematic, quantitative readouts into a format that allows TFs, COFs, and DNA to interact under solution conditions that more closely resemble the biochemical environment in which these complexes normally assemble. This distinction is essential.

Many regulatory interactions, particularly those involving COFs, depend on dynamics, diffusion, and multicomponent assembly that a fixed microarray surface cannot adequately capture. By placing those interactions in solution, the liquid PBM aligns with a growing

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recognition that TF function cannot be understood solely through static binding profiles. [3] 5e. Alignment with Field Trends This shift toward more dynamic assays mirrors broader trends in high-throughput regulatory biology. Recent work emphasizes the need for approaches that map TF-COF networks in a manner sensitive to cellular context, while efforts like NextPBM adapt PBM logic to incorporate cell-derived factors and condition-dependent behavior. The liquid PBM fits clearly within this movement. It participates in the same push toward assays that reduce technical rigidity, incorporate more realistic biochemical conditions, and generate data that connect sequence specificity with the cooperative processes that shape gene regulation. In this sense, the assay developed here is not an outlier but part of a broader methodological shift in the field. [2] 5f. Practical Advantages and Accessibility The technical results from this project suggest that a liquid PBM could offer a low-cost, high-throughput platform for profiling TF-COF interactions. This directly addresses one of the most pressing unmet needs in transcriptional biology: the field has long required tools capable of measuring TF-COF binding systematically across large numbers of protein pairs, but existing platforms impose infrastructure requirements that restrict this kind of profiling to a small number of specialized facilities. By eliminating the need for custom microarrays and large amounts of purified protein, the assay is designed to lower both material and infrastructure requirements. This opens the possibility for laboratories without access to microarray fabrication or dedicated PBM scanners to conduct TF-COF studies using standard molecular biology equipment, such as gel electrophoresis. If refined further, the assay could scale into panels that screen dozens of TF-COF combinations in parallel, offering a more accessible path to mapping regulatory interactions at scale.

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5g. Biomedical Applications These practical advantages point toward important applications in biomedical research. Many diseases, including cancer, Alzheimer's, developmental disorders, and autoimmune conditions, involve disrupted transcription factor networks that can only be meaningfully interrogated with tools capable of profiling many TF-COF combinations in parallel.

[16] A scalable assay capable of quantifying TF-COF interactions could therefore support drug discovery efforts targeting transcriptional regulation, especially small-molecule inhibitors or stabilizers of TF-COF complexes. In oncology, many cancers are driven by aberrant TF-COF interactions that hijack normal gene expression programs, and identifying which specific partnerships are disrupted in a given tumor type could guide the development of more targeted therapeutics with fewer off-target effects than broad transcriptional inhibitors currently in use. In neurodegenerative disease, where transcriptional dysregulation contributes to progressive loss of cell-type-specific gene expression, high-throughput mapping of COF-dependent TF behavior across neuronal subtypes could help pinpoint where regulatory networks begin to break down, opening earlier windows for intervention. In addition, the assay could be applied to comparative studies across cell types or disease states, enabling more systematic evaluation of how COFs alter TF behavior under different biological conditions. Even in its early form, the liquid PBM shows the potential to serve as a versatile tool for dissecting regulatory mechanisms and expanding the experimental reach of transcriptional biology.

Beyond individual applications, the broader societal implications of accessible TF-COF profiling are significant. Current platform barriers concentrate transcriptional regulatory research in well-funded institutions, limiting the diversity of scientific questions being asked. A liquid PBM operable with standard laboratory equipment could enable smaller research groups and lower-resource institutions to contribute to mapping the regulatory networks underlying human development and disease. In the longer term, a comprehensive map of TF-COF interactions across cell types and disease states would serve as a

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foundational resource for precision medicine, where therapeutic strategies are tailored not just to a patient's genome but to the specific regulatory landscape of their disease.

5h. Future Directions Looking to the future, several steps would strengthen and broaden the impact of the liquid PBM platform. A key priority after finalizing the current iteration of the liquid PBM would be to expand testing beyond the LEF1 system to include multiple TF-COF pairs, allowing assessment of whether the assay's performance generalizes across different regulatory complexes. It will also be important to benchmark the liquid PBM directly against the traditional solid-phase PBM, comparing accuracy, reproducibility, and material efficiency under matched conditions to better characterize its performance relative to traditional PBMs. As the assay matures, there is potential to integrate computational modeling, using predicted binding motifs or energetics to guide probe design and interpret ligation data in a more quantitative framework.

Finally, the progress made here provides a foundation for contributing to future grant proposals, where continued refinement of the assay (its sensitivity, throughput, and applicability) could position the liquid PBM as a viable complementary tool for studying transcriptional regulation en masse.

5i. Personal Reflection Working on this project in the Siggers Lab provided my first sustained experience with molecular biology at a professional research level, and my role evolved substantially as I became more comfortable with the techniques and expectations of the lab. Much of my day-to-day work involved PCR reactions, pouring and running gels, making master mixes, and pipetting various components of experiments. Although these steps are largely routine, each one demanded precision, repetition, and an understanding of how small variations could alter the outcome of the entire assay. Learning these techniques as a high school researcher required both technical training and a resilient mindset. Experiments were no longer isolated classroom exercises with predictable outcomes,

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but open-ended investigations in which uncertainty and troubleshooting were core parts of the process.

The collaborative environment of the lab played a crucial role in this learning curve. Graduate students provided a strong support system, and their guidance, answers, and demonstrated abilities shaped how I approach experimental setbacks. Through this mentorship, I came to appreciate how scientific discovery advances through iterative cycles of design, testing, failure, and refinement. This project taught me that progress in research often comes from understanding why an experiment did not produce the expected result and using that to redesign the next attempt, rather than building incrementally on an initial success. The experience deepened my appreciation for the rigor and creativity required in experimental science and strengthened my interest in continuing research at the intersection of molecular biology and regulatory mechanisms.

6. Conclusion This project set out to ask whether the logic of a PBM could be reimagined in solution to study TF-COF interactions in a way that is both technically accessible and statistically informative. Starting from the limitations of solid-phase PBMs (short probe length, high material and equipment demands, and the inability to fully capture dynamic, multicomponent assembly), the work here developed and tested a liquid PBM framework employing proximity ligation with a PCR-based readout. The assay was designed, implemented, and iteratively refined in the Siggers Lab, with each stage of digestion, binding, ligation, amplification, and analysis adjusted to balance specificity, sensitivity, and reproducibility.

Under optimized conditions, the liquid PBM produced consistent ligation signals that reflected TF-COF-dependent interactions, while being designed to use less material and simpler infrastructure than a traditional slide-based PBM. Serial optimization of ligase, antibody oligonucleotide, and nuclear extract concentrations coupled with careful control of PCR

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conditions and contamination converted an initially noisy, unstable system into one capable of generating reproducible, interpretable bands aligned with expected antibody-mediated ligation. At the same time, the work made clear that key challenges remain: the assay is still sensitive to pipetting variability, and fully resolving specific ligation from chance proximity ligation will require further refinement of both chemistry and workflow. The liquid PBM developed here should therefore be understood as an early-stage prototype. It is technically viable, empirically validated, and promising, but not yet a complete complement for established PBM platforms. It demonstrates proof-of-concept for solution-phase PBM methodology but requires further validation before widespread adoption as a complementary platform.

Even in this preliminary form, the assay speaks to a broader scientific and practical goal. By moving PBM principles into a liquid format, this work contributes to ongoing efforts to create high-throughput, quantitative, and more affordable tools for studying gene regulation. A liquid PBM that can be run with standard molecular biology equipment lowers the barrier to systematic TF-COF profiling and opens space for laboratories without access to microarray fabrication or specialized scanners to participate in mapping regulatory frameworks. With further validation across additional TF-COF pairs and benchmarking against solid-phase PBMs, the liquid PBM has the potential to become a useful complementary assay for probing the architecture of transcriptional control. In that sense, in addition to serving as a proof of concept, this project served as a step toward more accessible, scalable models for understanding how TFs, COFs, and their interactions shape gene expression.

7. References [1] Roeder, R. G. (2019). 50+ years of eukaryotic transcription: An expanding universe of factors and mechanisms. Nature Structural & Molecular Biology, 26(9), 783–791. https://doi.org/10.1038/s41594-019-0287-x

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[2] Inge, M., Miller, R., Hook, H., Bray, D., Keenan, J., Zhao, R., Gilmore, T., & Siggers, T. (2024). Rapid profiling of transcription factor–cofactor interaction networks reveals principles of epigenetic regulation. Nucleic Acids Research. https://doi.org/10.1093/nar/gkae706 [3] Berger, M. F., & Bulyk, M. L. (2009). Universal protein-binding microarrays for the comprehensive characterization of the DNA-binding specificities of transcription factors. Nature Protocols, 4(3), 393–411. https://doi.org/10.1038/nprot.2008.195 [4] Lambert, S. A., Jolma, A., Campitelli, L. F., Das, P. K., Yin, Y., Albu, M., Chen, X., Taipale, J., Hughes, T. R., & Weirauch, M. T. (2018). The human transcription factors. Cell, 172(4), 650–665. https://doi.org/10.1016/j.cell.2018.01.029 [5] Richter, W. F., Nayak, S., Iwasa, J., & Taatjes, D. J. (2022). The Mediator complex as a master regulator of transcription by RNA polymerase II. Nature Reviews Molecular Cell Biology, 23(11), 732–749. https://doi.org/10.1038/s41580-022-00498-3 [6] Siggers, T., Duyzend, M. H., Reddy, J., Khan, S., & Bulyk, M. L. (2011). Non-DNA-binding cofactors enhance DNA-binding specificity of a transcriptional regulatory complex. Molecular Systems Biology, 7(1). https://doi.org/10.1038/msb.2011.89 [7] Wang, Z., Wang, P., Li, Y., Peng, H., Zhu, Y., Mohandas, N., & Liu, J. (2021). Interplay between cofactors and transcription factors in hematopoiesis and hematological malignancies. Signal Transduction and Targeted Therapy, 6(1). https://doi.org/10.1038/s41392-020-00422-1 [8] Bannister, A. J., & Kouzarides, T. (2011). Regulation of chromatin by histone modifications. Cell Research, 21(3), 381–395. https://doi.org/10.1038/cr.2011.22 [9] Stormo, G. D., & Zhao, Y. (2010). Determining the specificity of protein-DNA interactions. Nature Reviews Genetics, 11(11), 751–760. https://doi.org/10.1038/nrg2845

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[10] Berger, M. F., Philippakis, A. A., Qureshi, A. M., He, F. S., Estep, P. W., & Bulyk, M. L. (2006). Compact, universal DNA microarrays to comprehensively determine transcription-factor binding site specificities.

Nature Biotechnology, 24(11), 1429–1435. https://doi.org/10.1038/nbt1246 [11] Ritzefeld, M., & Sewald, N. (2012). Real-time analysis of specific protein-DNA interactions with surface plasmon resonance. Journal of Amino Acids, 2012, 816032. https://doi.org/10.1155/2012/816032 [12] Mohaghegh, N., Bray, D., Keenan, J., Penvose, A., Andrilenas, K. K., Ramlall, V., & Siggers, T. (2019). NextPBM: a platform to study cell-specific transcription factor binding and cooperativity. Nucleic Acids Research, 47(6), e31. https://doi.org/10.1093/nar/gkz020 [13] Orenstein, Y., & Shamir, R. (2017). Modeling protein-DNA binding via high-throughput in vitro technologies. Briefings in Functional Genomics, 16(3), 171–180. https://doi.org/10.1093/bfgp/elw030 [14] Hegazy, M., Cohen-Barak, E., Koetsier, J. L., Najor, N. A., Gruber, R., Pfendner, E. G., Uitto, J., Gat, A., Sprecher, E., & Green, K. J. (2020). Proximity ligation assay for detecting protein-protein interactions and protein modifications in cells and tissues in situ. Current Protocols in Cell Biology, 89(1), e115. https://doi.org/10.1002/cpcb.115 [15] Nong, R. Y., Wu, D., Yan, J., Hammond, M., Gu, G. J., Kamali-Moghaddam, M., Landegren, U., & Darmanis, S. (2013).

Solid-phase proximity ligation assays for individual or parallel protein analyses with readout via real-time PCR or sequencing. Nature Protocols, 8(6), 1234–1248. https://doi.org/10.1038/nprot.2013.070 [16] Lee, T. I., & Young, R. A. (2013). Transcriptional regulation and its misregulation in disease. Cell, 152(6), 1237–1251. https://doi.org/10.1016/j.cell.2013.02.014

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[17] Adams, A. (2025). Schematic representation of traditional solid-phase protein-binding microarray methodology [Illustration].

Created with BioRender.com.

[18] Adams, A. (2025). Restriction-digested DNA oligonucleotide bearing a compatible single-stranded overhang [Illustration]. Created with BioRender.com.

[19] Adams, A. (2025). Ligation and PCR amplification of the liquid PBM readout [Illustration]. Created with BioRender.com.

[20] Levine, E. (2025). Transcription factor-cofactor complex bound to double-stranded DNA [Illustration]. Created with BioRender.com.

[21] Levine, E. (2025). Binding phase of the liquid PBM assay [Illustration]. Created with BioRender.com.

[22] Levine, E. (2025). Antibody-mediated proximity ligation in the liquid PBM assay [Illustration]. Created with BioRender.com.

[23] Adams, A. (2025). Agarose gel from the June 16, 2025 antibody oligonucleotide concentration screen [Unpublished experimental data].

Siggers Laboratory, Boston University.

[24] Adams, A. (2025). Serial dilution gel used to establish the linear PCR amplification range [Unpublished experimental data]. Siggers Laboratory, Boston University.

[25] Adams, A. (2025). Agarose gel from the July 2, 2025 blunt-end ligation experiment identifying primer contamination [Unpublished experimental data]. Siggers Laboratory, Boston University.

[26] Adams, A. (2025). Agarose gel of control reactions from August 5, 2025 comparing 1-hour and 4-hour ligation incubation conditions [Unpublished experimental data]. Siggers Laboratory, Boston University.