Interaction models matter: an efficient, flexible computational framework for model-specific investigation of epistasis.

PURPOSE: Epistasis, the interaction between two or more genes, is integral to the study of genetics and is present throughout nature. Yet, it is seldom fully explored as most approaches primarily focus on single-locus effects, partly because analyzing all pairwise and higher-order interactions requires significant computational resources. Furthermore, existing methods for epistasis detection only consider a Cartesian (multiplicative) model for interaction terms. This is likely limiting as epistatic interactions can evolve to produce varied relationships between genetic loci, some complex and not linearly separable. METHODS: We present new algorithms for the interaction coefficients for standard regression models for epistasis that permit many varied models for the interaction terms for loci and efficient memory usage. The algorithms are given for two-way and three-way epistasis and may be generalized to higher order epistasis. Statistical tests for the interaction coefficients are also provided. We also present an efficient matrix based algorithm for permutation testing for two-way epistasis. We offer a proof and experimental evidence that methods that look for epistasis only at loci that have main effects may not be justified. Given the computational efficiency of the algorithm, we applied the method to a rat data set and mouse data set, with at least 10,000 loci and 1,000 samples each, using the standard Cartesian model and the XOR model to explore body mass index. RESULTS: This study reveals that although many of the loci found to exhibit significant statistical epistasis overlap between models in rats, the pairs are mostly distinct. Further, the XOR model found greater evidence for statistical epistasis in many more pairs of loci in both data sets with almost all significant epistasis in mice identified using XOR. In the rat data set, loci involved in epistasis under the XOR model are enriched for biologically relevant pathways. CONCLUSION: Our results in both species show that many biologically relevant epistatic relationships would have been undetected if only one interaction model was applied, providing evidence that varied interaction models should be implemented to explore epistatic interactions that occur in living systems.

Cluster Analysis reveals Socioeconomic Disparities among Elective Spine Surgery Patients.

This work demonstrates the use of cluster analysis in detecting fair and unbiased novel discoveries. Given a sample population of elective spinal fusion patients, we identify two overarching subgroups driven by insurance type. The Medicare group, associated with lower socioeconomic status, exhibited an over-representation of negative risk factors. The findings provide a compelling depiction of the interwoven socioeconomic and racial disparities present within the healthcare system, highlighting their consequential effects on health inequalities. The results are intended to guide design of fair and precise machine learning models based on intentional integration of population stratification.

Aliro: an automated machine learning tool leveraging large language models.

MOTIVATION: Biomedical and healthcare domains generate vast amounts of complex data that can be challenging to analyze using machine learning tools, especially for researchers without computer science training. RESULTS: Aliro is an open-source software package designed to automate machine learning analysis through a clean web interface. By infusing the power of large language models, the user can interact with their data by seamlessly retrieving and executing code pulled from the large language model, accelerating automated discovery of new insights from data. Aliro includes a pre-trained machine learning recommendation system that can assist the user to automate the selection of machine learning algorithms and its hyperparameters and provides visualization of the evaluated model and data. AVAILABILITY AND IMPLEMENTATION: Aliro is deployed by running its custom Docker containers. Aliro is available as open-source from GitHub at: https://github.com/EpistasisLab/Aliro.

Plasticity of circulating tumor cells in small cell lung cancer.

Small cell lung cancer (SCLC) is an aggressive neuroendocrine tumor with low five-year survival rates. Recently described molecular phenotypes of SCLC exhibit differential vulnerabilities heralding potential for stratified treatment. Whilst tumor biopsy in SCLC is challenging, circulating tumor cells in the liquid biopsy are prevalent and can be repeatedly sampled accommodating the dynamic plasticity of SCLC phenotypes. The aim of this study was to characterize the heterogeneity of rare circulating cells with confirmed tumor origin and to explore a liquid biopsy approach for future clinical trials of targeted therapies. This study applied the 3rd generation of a previously validated direct imaging platform to 14 chemo-naive SCLC patients and 10 non-cancerous normal donor (ND) samples. Phenotypic heterogeneity of circulating rare cells in SCLC was observed and a patient-level classification model was established to stratify SCLC patients from non-cancerous donors. Eight rare cell groups, with combinations of epithelial, endothelial, and mesenchymal biomarker expression patterns, were phenotypically characterized. The single-cell genomic analysis confirmed the cancer cell plasticity in every rare cell group harboring clonal genomic alterations. This study shows rare cell heterogeneity and confirms cellular plasticity in SCLC providing a valuable resource for better opportunities to discover novel therapeutic targets in SCLC.

Characterization of BCMA Expression in Circulating Rare Single Cells of Patients with Plasma Cell Neoplasms.

B-cell maturation antigen (BCMA), a key regulator of B-cell proliferation and survival, is highly expressed in almost all cases of plasma cell neoplasms and B-lymphoproliferative malignancies. BCMA is a robust biomarker of plasma cells and a therapeutic target with substantial clinical significance. However, the expression of BCMA in circulating tumor cells of patients with hematological malignancies has not been validated for the detection of circulating plasma and B cells. The application of BCMA as a biomarker in single-cell detection and profiling of circulating tumor cells in patients' blood could enable early disease profiling and therapy response monitoring. Here, we report the development and validation of a slide-based immunofluorescence assay (i.e., CD138, BCMA, CD45, DAPI) for enrichment-free detection, quantification, and morphogenomic characterization of BCMA-expressing cells in patients (N = 9) with plasma cell neoplasms. Varying morphological subtypes of circulating BCMA-expressing cells were detected across the CD138(+/-) and CD45(+/-) compartments, representing candidate clonotypic post-germinal center B cells, plasmablasts, and both normal and malignant plasma cells. Genomic analysis by single-cell sequencing and correlation to clinical FISH cytogenetics provides validation, with data showing that patients across the different neoplastic states carry both normal and altered BCMA-expressing cells. Furthermore, altered cells harbor cytogenetic events detected by clinical FISH. The reported enrichment-free liquid biopsy approach has potential applications as a single-cell methodology for the early detection of BCMA+ B-lymphoid malignancies and in monitoring therapy response for patients undergoing anti-BCMA treatments.

Multianalyte liquid biopsy to aid the diagnostic workup of breast cancer.

Breast cancer (BC) affects 1 in every 8 women in the United States and is currently the most prevalent cancer worldwide. Precise staging at diagnosis and prognosis are essential components for the clinical management of BC patients. In this study, we set out to evaluate the feasibility of the high-definition single cell (HDSCA) liquid biopsy (LBx) platform to stratify late-stage BC, early-stage BC, and normal donors using peripheral blood samples. Utilizing 5 biomarkers, we identified rare circulating events with epithelial, mesenchymal, endothelial and hematological origin. We detected a higher level of CTCs in late-stage patients, compared to the early-stage and normal donors. Additionally, we observed more tumor-associated large extracellular vesicles (LEVs) in the early-stage, compared to late-stage and the normal donor groups. Overall, we were able to detect reproducible patterns in the enumeration of rare cells and LEVs of cancer vs. normal donors and early-stage vs. late-stage BC with high accuracy, allowing for robust stratification. Our findings illustrate the feasibility of the LBx assay to provide robust detection of rare circulating events in peripheral blood draws and to stratify late-stage BC, early-stage BC, and normal donor samples.

Modular mixing of benzene-1,3,5-tricarboxamide supramolecular hydrogelators allows tunable biomimetic hydrogels for control of cell aggregation in 3D.

Few synthetic hydrogels can mimic both the viscoelasticity and supramolecular fibrous structure found in the naturally occurring extracellular matrix (ECM). Furthermore, the ability to control the viscoelasticity of fibrous supramolecular hydrogel networks to influence cell culture remains a challenge. Here, we show that modular mixing of supramolecular architectures with slow and fast exchange dynamics can provide a suitable environment for multiple cell types and influence cellular aggregation. We employed modular mixing of two synthetic benzene-1,3,5-tricarboxamide (BTA) architectures: a small molecule water-soluble BTA with slow exchange dynamics and a telechelic polymeric BTA-PEG-BTA with fast exchange dynamics. Copolymerisation of these two supramolecular architectures was observed, and all tested formulations formed stable hydrogels in water and cell culture media. We found that rational tuning of mechanical and viscoelastic properties is possible by mixing BTA with BTA-PEG-BTA. These hydrogels showed high viability for both chondrocyte (ATDC5) and human dermal fibroblast (HDF) encapsulation (>80%) and supported neuronal outgrowth (PC12 and dorsal root ganglion, DRG). Furthermore, ATDC5s and human mesenchymal stem cells (hMSCs) were able to form spheroids within these viscoelastic hydrogels, with control over cell aggregation modulated by the dynamic properties of the material. Overall, this study shows that modular mixing of supramolecular architectures enables tunable fibrous hydrogels, creating a biomimetic environment for cell encapsulation. These materials are suitable for the formation and culture of spheroids in 3D, critical for upscaling tissue engineering approaches towards cell densities relevant for physiological tissues.

Identification of epithelial and mesenchymal circulating tumor cells in clonal lineage of an aggressive prostate cancer case.

Little is known about the complexity and plasticity of circulating tumor cell (CTC) biology in different compartments of the fluid microenvironment during tumor metastasis. Here we integrated phenomics, genomics, and targeted proteomics to characterize CTC phenotypic and genotypic heterogeneity in paired peripheral blood (PB) and bone marrow aspirate (BMA) from a metastatic prostate cancer patient following the rapid disease progression, using the High-Definition Single Cell Assay 3.0 (HDSCA3.0). Uniquely, we identified a subgroup of genetically clonal CTCs that acquired a mesenchymal-like state and its presence was significantly associated with one subclone that emerged along the clonal lineage. Higher CTC abundance and phenotypic diversity were observed in the BMA than PB and differences in genomic alterations were also identified between the two compartments demonstrating spatial heterogeneity. Single cell copy number profiling further detected clonal heterogeneity within clusters of CTCs (also known as microemboli or aggregates) as well as phenotypic variations by targeted proteomics. Overall, these results identify epithelial and mesenchymal CTCs in the clonal lineage of an aggressive prostate cancer case and also demonstrate a single cell multi-omic approach to deconvolute the heterogeneity and association of CTC phenotype and genotype in multi-medium liquid biopsies of metastatic prostate cancer.

Enrichment-Free Single-Cell Detection and Morphogenomic Profiling of Myeloma Patient Samples to Delineate Circulating Rare Plasma Cell Clones.

Multiple myeloma is an incurable malignancy that initiates from a bone marrow resident clonal plasma cell and acquires successive mutational changes and genomic alterations, eventually resulting in tumor burden accumulation and end-organ damage. It has been recently recognized that myeloma secondary genomic events result in extensive sub-clonal heterogeneity both in localized bone marrow areas and circulating peripheral blood plasma cells. Rare genomic subclones, including myeloma initiating cells, could be the drivers of disease progression and recurrence. Additionally, evaluation of rare myeloma cells in blood for disease monitoring has numerous advantages over invasive bone marrow biopsies. To this end, an unbiased method for detecting rare cells and delineating their genomic makeup enables disease detection and monitoring in conditions with low abundant cancer cells. In this study, we applied an enrichment-free four-plex (CD138, CD56, CD45, DAPI) immunofluorescence assay and single-cell DNA sequencing for morphogenomic characterization of plasma cells to detect and delineate common and rare plasma cells and discriminate between normal and malignant plasma cells in paired blood and bone marrow aspirates from five patients with newly diagnosed myeloma (N = 4) and monoclonal gammopathy of undetermined significance (n = 1). Morphological analysis confirms CD138+CD56+ cells in the peripheral blood carry genomic alterations that are clonally identical to those in the bone marrow. A subset of altered CD138+CD56- cells are also found in the peripheral blood consistent with the known variability in CD56 expression as a marker of plasma cell malignancy. Bone marrow tumor clinical cytogenetics is highly correlated with the single-cell copy number alterations of the liquid biopsy rare cells. A subset of rare cells harbors genetic alterations not detected by standard clinical diagnostic methods of random localized bone marrow biopsies. This enrichment-free morphogenomic approach detects and characterizes rare cell populations derived from the liquid biopsies that are consistent with clinical diagnosis and have the potential to extend our understanding of subclonality at the single-cell level in this disease. Assay validation in larger patient cohorts has the potential to offer liquid biopsy for disease monitoring with similar or improved disease detection as traditional blind bone marrow biopsies.

Platelet-Coated Circulating Tumor Cells Are a Predictive Biomarker in Patients with Metastatic Castrate-Resistant Prostate Cancer.

Metastatic castration-resistant prostate cancer (mCRPC) includes a subset of patients with particularly unfavorable prognosis characterized by combined defects in at least two of three tumor suppressor genes: PTEN, RB1, and TP53 as aggressive variant prostate cancer molecular signature (AVPC-MS). We aimed to identify circulating tumor cells (CTC) signatures that could inform treatment decisions of patients with mCRPC with cabazitaxel-carboplatin combination therapy versus cabazitaxel alone. Liquid biopsy samples were collected prospectively from 79 patients for retrospective analysis. CTCs were detected, classified, enumerated through a computational pipeline followed by manual curation, and subjected to single-cell genome-wide copy-number profiling for AVPC-MS detection. On the basis of immunofluorescence intensities, detected rare cells were classified into 8 rare-cell groups. Further morphologic characterization categorized CTC subtypes from 4 cytokeratin-positive rare-cell groups, utilizing presence of mesenchymal features and platelet attachment. Of 79 cases, 77 (97.5%) had CTCs, 24 (30.4%) were positive for platelet-coated CTCs (pc.CTCs) and 25 (38.5%) of 65 sequenced patients exhibited AVPC-MS in CTCs. Survival analysis indicated that the presence of pc.CTCs identified the subset of patients who were AVPC-MS-positive with the worst prognosis and minimal benefit from combination therapy. In AVPC-MS-negative patients, its presence showed significant survival improvement from combination therapy. Our findings suggest the presence of pc.CTCs as a predictive biomarker to further stratify AVPC subsets with the worst prognosis and the most significant benefit of additional platinum therapy. IMPLICATIONS: HDSCA3.0 can be performed with rare cell detection, categorization, and genomic characterization for pc.CTC identification and AVPC-MS detection as a potential predictive biomarker of mCRPC.

Large Extracellular Vesicle Characterization and Association with Circulating Tumor Cells in Metastatic Castrate Resistant Prostate Cancer.

Liquid biopsies hold potential as minimally invasive sources of tumor biomarkers for diagnosis, prognosis, therapy prediction or disease monitoring. We present an approach for parallel single-object identification of circulating tumor cells (CTCs) and tumor-derived large extracellular vesicles (LEVs) based on automated high-resolution immunofluorescence followed by downstream multiplexed protein profiling. Identification of LEVs >6 µm in size and CTC enumeration was highly correlated, with LEVs being 1.9 times as frequent as CTCs, and additional LEVs were identified in 73% of CTC-negative liquid biopsy samples from metastatic castrate resistant prostate cancer. Imaging mass cytometry (IMC) revealed that 49% of cytokeratin (CK)-positive LEVs and CTCs were EpCAM-negative, while frequently carrying prostate cancer tumor markers including AR, PSA, and PSMA. HSPD1 was shown to be a specific biomarker for tumor derived circulating cells and LEVs. CTCs and LEVs could be discriminated based on size, morphology, DNA load and protein score but not by protein signatures. Protein profiles were overall heterogeneous, and clusters could be identified across object classes. Parallel analysis of CTCs and LEVs confers increased sensitivity for liquid biopsies and expanded specificity with downstream characterization. Combined, it raises the possibility of a more comprehensive assessment of the disease state for precise diagnosis and monitoring.

Competitive Supramolecular Associations Mediate the Viscoelasticity of Binary Hydrogels.

Supramolecular polymers are known to form strong and resilient hydrogels which can take up large amounts of water while exhibiting ease of processing and self-healing. They also possess similarities with networks of biological macromolecules. The combination of these features makes supramolecular polymers ideal candidates for studying mechanisms and consequences of self-assembly, which are relevant to biological materials. At the same time, this renders investigations of mixed hydrogels based on different supramolecular compounds necessary, since this substantially widens their applicability. Here, we address unusual viscoelastic properties of a class of binary hydrogels made by mixing fibrillar supramolecular polymers that are formed from two compounds: 1,3,5-benzene-tricarboxamide decorated with aliphatic chains terminated by tetra(ethylene glycol) (BTA) and a 20 kg/mol telechelic poly(ethylene glycol) decorated with the same hydrogen bonding BTA motif on both ends (BTA-PEG-BTA). Using a suite of experimental and simulation techniques, we find that the respective single-compound-based supramolecular systems form very different networks which exhibit drastically different rheology. More strikingly, mixing the compounds results in a non-monotonic dependence of modulus and viscosity on composition, suggesting a competition between interactions of the two compounds, which can then be used to fine-tune the mechanical properties. Simulations offer insight into the nature of this competition and their remarkable qualitative agreement with the experimental results is promising for the design of mixed hydrogels with desired and tunable properties. Their combination with a sensitive dynamic probe (here rheology) offer a powerful toolbox to explore the unique properties of binary hydrogel mixtures.

PEG Analogs Synthesized by Ring-Opening Metathesis Polymerization for Reversible Bioconjugation.

Poly(ethylene glycols) (PEGs) with protein-reactive end-groups are widely utilized in bioconjugation reactions. Herein, we describe the use of ring-opening metathesis polymerization (ROMP) to synthesize unsaturated protein-reactive PEG analogs. These ROMP PEGs (rPEGs) contained terminal aldehyde functionality and ranged in molecular weight from 6 to 20 kDa. The polymers were readily conjugated to free amines on the protein hen egg-white lysozyme (Lyz). Biocompatibility of the unsaturated PEGs was assessed in vitro, revealing the polymers to be nontoxic up to concentrations of at least 1 mg/mL in human dermal fibroblasts (HDFs). The resulting unsaturated rPEG-lysozyme conjugates underwent metathesis-based depolymerization, resulting in decreased molecular weight of the conjugate.

Polymorphism in Benzene-1,3,5-tricarboxamide Supramolecular Assemblies in Water: A Subtle Trade-off between Structure and Dynamics.

In biology, polymorphism is a well-known phenomenon by which a discrete biomacromolecule can adopt multiple specific conformations in response to its environment. The controlled incorporation of polymorphism into noncovalent aqueous assemblies of synthetic small molecules is an important step toward the development of bioinspired responsive materials. Herein, we report on a family of carboxylic acid functionalized water-soluble benzene-1,3,5-tricarboxamides (BTAs) that self-assemble in water to form one-dimensional fibers, membranes, and hollow nanotubes. Interestingly, one of the BTAs with the optimized position of the carboxylic group in the hydrophobic domain yields nanotubes that undergo reversible temperature-dependent dynamic reorganizations. SAXS and Cryo-TEM data show the formation of elongated, well-ordered nanotubes at elevated temperatures. At these temperatures, increased dynamics, as measured by hydrogen-deuterium exchange, provide enough flexibility to the system to form well-defined nanotube structures with apparently defect-free tube walls. Without this flexibility, the assemblies are frozen into a variety of structures that are very similar at the supramolecular level, but less defined at the mesoscopic level.

Dynamic diversity of synthetic supramolecular polymers in water as revealed by hydrogen/deuterium exchange.

Numerous self-assembling molecules have been synthesized aiming at mimicking both the structural and dynamic properties found in living systems. Here we show the application of hydrogen/deuterium exchange (HDX) mass spectrometry (MS) to unravel the nanoscale organization and the structural dynamics of synthetic supramolecular polymers in water. We select benzene-1,3,5-tricarboxamide (BTA) derivatives that self-assemble in H2O to illustrate the strength of this technique for supramolecular polymers. The BTA structure has six exchangeable hydrogen atoms and we follow their exchange as a function of time after diluting the H2O solution with a 100-fold excess of D2O. The kinetic H/D exchange profiles reveal that these supramolecular polymers in water are dynamically diverse; a notion that has previously not been observed using other techniques. In addition, we report that small changes in the molecular structure can be used to control the dynamics of synthetic supramolecular polymers in water.

Exposing Differences in Monomer Exchange Rates of Multicomponent Supramolecular Polymers in Water.

The formation of multicomponent and bioactive supramolecular polymers is a promising strategy for the formation of biomaterials that match the dynamic and responsive nature of biological systems. In order to fully realize the potential of this strategy, knowledge of the location and behavior of bioactive components within the system is crucial. By employing synthetic strategies to create multifunctional monomers, coupled with FRET and STORM techniques, we have investigated the formation and behavior of a bioactive and multicomponent supramolecular polymer. By creating a peptide-dye-monomer conjugate, we were able to measure high degrees of monomer incorporation and to visualize the equal distribution of monomers within the supramolecular polymer. Furthermore, by tracking the movement of monomers, we uncovered small differences in the dynamics of the bioactive monomers.

Dual pH- and Temperature-Responsive Protein Nanoparticles.

Multiply responsive protein nanoparticles are interesting for a variety of applications. Herein, we describe the synthesis of a vault nanoparticle that responds to both temperature and pH. Specifically, poly(N-isopropylacrylamide-co-acrylic acid) with a pyridyl disulfide end group was prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization. The polymer had a lower critical solution temperature (LCST) of 31.9 °C at pH 5, 44.0 °C at pH 6 and above 60 °C at pH 7. The polymer was conjugated to human major vault protein (hMVP), and the resulting nanoparticle was analyzed by UV-Vis, dynamic light scattering (DLS) and electron microscopy. The data demonstrated that poly(N-isopropylacrylamide-co-acrylic acid)-vault conjugate did not respond to temperatures below 60 °C at pH 7, while the nanoparticles reversibly aggregated at pH 6. Furthermore, it was shown that the vault nanoparticle structure remained intact for at least three heat and cooling cycles. Thus, these dually responsive nanoparticles may serve as a platform for drug delivery and other applications.

Synthesis of Nanogel-Protein Conjugates.

The covalent conjugation of bovine serum albumin (BSA) to disulfide cross-linked polymeric nanogels is reported. Polymeric nanogel precursors were synthesized via a reversible addition-fragmentation chain transfer (RAFT) random copolymerization of poly(ethylene glycol) methyl ether methacrylate (PEGMA) and pyridyl disulfide methacrylate (PDSMA). Reaction of the p(PEGMA-co-PDSMA) with dithiothreitol resulted in the formation of nanogels. PDSMA serves as both a crosslinking agent and a reactive handle for the surface modification of the nanogels. Lipophilic dye, DiI, was sequestered within the nanogels by performing the crosslinking reaction in the presence of the hydrophobic molecule. Thiol-enriched BSA was conjugated to nanogels loaded with DiI via a disulfide reaction between the BSA and the surface exposed nanogel pyridyl disulfides. Conjugation was confirmed by fast protein liquid chromatography, dynamic light scattering, and agarose and polyacrylamide gel electrophoresis. We expect that this methodology is generally applicable to the preparation of nanogel-protein therapeutics.

Smart vaults: thermally-responsive protein nanocapsules.

Synthetic modification of a recombinant protein cage called a vault with stimuli-responsive smart polymers provides access to a new class of biohybrid materials; the polymer nanocapsules retain the structure of the protein cage and exhibit the responsive nature of the polymer. Vaults are naturally occurring ubiquitous ribonucleoprotein particles 41 × 41 × 72.5 nm composed of a protein shell enclosing multiple copies of two proteins and multiple copies of one or more small untranslated RNAs. Recombinant vaults are structurally identical but lack the vault content. Poly(N-isopropylacrylamide) (pNIPAAm), a polymer responsive to heat, was conjugated to recombinant vaults that were composed of ~78 copies of the major vault protein (MVP) modified to contain a cysteine rich region at the N-terminus (CP-MVP). The polymer was synthesized using reversible addition-fragmentation chain transfer (RAFT) polymerization to have a dansyl group at the alpha end and modified to have a thiol-reactive pyridyl disulfide at the omega end, which readily coupled to CP-MVP vaults. The resulting vault nanocapsules underwent reversible aggregation upon heating above the lower critical solution temperature (LCST) of the polymer as determined by electron microscopy (EM), dynamic light scattering experiments, and UV-vis turbidity analysis. The vault structure remained entirely intact throughout the phase transition; suggesting its use in a myriad of biomedical and biotechnology applications.

Aminooxy and Pyridyl Disulfide Telechelic Poly(Polyethylene Glycol Acrylate) by RAFT Polymerization.

An efficient method to synthesize telechelic, bio-reactive polymers is described. Homotelechelic polymers were synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization in one step by employing bifunctional chain transfer agents (CTAs). A bis-carboxylic acid CTA was coupled to N-BOC-aminooxy ethanol or pyridyl disulfide ethanol resulting in a bis-N-BOC-aminooxy CTA and a bis-pyridyl disulfide CTA, respectively. RAFT polymerization of polyethylene glycol (PEG) acrylate in the presence of both CTAs resulted in a series of polymers over a range of molecular weights (~8.4 kDa to 35.2 kDa; polydispersity indices, PDIs of 1.11 to 1.44) with retention of end-groups post-polymerization. The polymers were characterized by 1H NMR spectroscopy and gel permeation chromatography (GPC). Conjugations of small molecules and peptides resulted in homotelechelic polymer conjugates.