About

Kushol Gupta, Ph.D., is a Research Assistant Professor of Biochemistry and Biophysics at the Perelman School of Medicine of the University of Pennsylvania. He is a member of the BMB graduate group and directs the Johnson Foundation Structural Biology and Biophysics Core, a departmental resource serving Penn and the greater region. His expertise lies in structural biology, utilizing techniques such as X-ray crystallography, small-angle X-ray and neutron scattering, light scattering, and analytical ultracentrifugation. His research focuses on three main areas: enzymes involved in inborn errors of metabolism, including phenylalanine hydroxylase, cystathionine beta-synthase, and pantothenate kinase; retroviral integrases, their interactions with host factors, and the development of allosteric integrase inhibitors (ALLINIs) effective against HIV; and biophysical studies of mRNA lipid nanoparticles (mRNA LNPs). Additional research interests include RNA splicing, viral proteins, chromatin, and site-specific recombination, reflecting the collaborative and interdisciplinary research environment at Penn.

Research topics

• Biophysics
• Biology
• Chemistry
• Computational biology
• Cell biology
• Medicine
• Biochemistry

Selected publications

• Resolving heterogeneity of targeted lipid nanoparticles through solution-based biophysical analyses

  bioRxiv (Cold Spring Harbor Laboratory) · 2026-04-02

  articleOpen access

  Targeted lipid nanoparticles (tLNPs) represent the next frontier in nucleic acid therapeutics, enabling cell-specific delivery through covalent attachment of targeting ligands that drive receptor-mediated uptake. tLNPs are particularly promising for pregnancy-associated applications where precise on-target delivery is required to minimize maternal toxicity and protect fetal health. Yet, their rational design is limited by an incomplete understanding of how tLNP physicochemical properties influence biological performance. Conventional LNPs already exhibit pronounced heterogeneity in size, composition, and RNA loading, which is further amplified in tLNPs by variability in ligand attachment and surface density. Because traditional analytical methods report only ensemble-averaged properties, the nanoscale diversity of tLNPs remains unresolved. Here, we find that tLNP functional behavior is governed by previously inaccessible, structurally distinct tLNP subpopulations that are not captured by bulk measurements. We utilize asymmetric flow field-flow fractionation integrated with in-line UV spectral analysis, light scattering, and synchrotron small-angle X-ray scattering (AF4-UV-DLS-MALS-SAXS) to resolve ligand-dependent tLNP subpopulations that differ in size, shape, composition, and relative abundance. We find that protein conjugation preserves the internal lipid-RNA nanostructure of base LNPs but substantially increases particle heterogeneity, particularly for larger and multivalent targeting ligands. Despite increased heterogeneity, tLNPs functionalized with higher-avidity ligands achieve more effective targeted placental RNA delivery in mice, suggesting that binding avidity can offset the functional consequences of polydispersity. Chemometric SAXS analyses reveal that only SAXS-resolved tLNP subpopulations, not ensemble-averaged parameters, correlate with targeted placental transfection in vivo, whereas bulk-derived physicochemical metrics more strongly associate with nonspecific hepatic delivery. Together, this work harnesses a separation-coupled biophysical platform to resolve previously inaccessible tLNP subpopulations and demonstrates that subpopulation nanoscale structure, rather than bulk-averaged properties, dictates targeted RNA delivery. These insights provide a mechanistic foundation for rational engineering of next-generation precision targeted RNA LNP therapeutics.

  PublisherOA PDFDOI

• Introduction to the SAMPREP special issue

  Acta Crystallographica Section F Structural Biology Communications · 2025-09-15

  articleOpen access1st authorCorresponding

  The focused issue on the SAMPREP workshop is introduced. The virtual issue is available at https://journals.iucr.org/special_issues/2025/samprep23/.

  PublisherOA PDFDOI

• Structures of Nucleotide-Bound Redondovirus Rep Protein Link Conformation and Function

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-08-25

  preprintOpen access

  Abstract Circular Rep-encoding single-stranded DNA (CRESS-DNA) virus Rep proteins are multidomain enzymes that mediate viral DNA rolling-circle replication. Reps nick viral DNA to expose a 3’ end for polymerase extension, provide an NTP-dependent helicase activity for DNA unwinding, and join nicked ends to form circular viral genomes. Here, we present the first structures of a Rep protein from the Redondoviridae family, a newly discovered family of human-associated CRESS-DNA viruses that replicates within the oral protozoan Entamoeba gingivalis . Using cryo-EM, we characterized the hexameric structures of a Redondovirus Rep helicase bound with ATPγS, representing the initial ATP-bound state, and with ADP, reflecting the protein state after hydrolysis. The ADP state, but not the ATP state of Rep shows a staircase arrangement of DNA-binding loops that plays a central role in current models for SF3 helicase function. Additionally, we determined a head- to-tail dodecameric structure of ATPγS-bound Rep, in which both the helicase and endonuclease domains are ordered. Conservation of residues involved in stabilizing the dodecamer suggest that this assembly may be functionally relevant for many CRESS-DNA viruses. The positioning of endonuclease domains in the Rep hexamer, combined with our biophysical analyses of Rep oligomerization, provide new insights into Rep function during viral replication.

  PublisherOA PDFDOI

• Insights into mRNA lipid nanoparticle polydispersity and shape using quantitative solution biophysics

  Structural Dynamics · 2025-03-01 · 1 citations

  articleOpen access

  Lipid nanoparticles (LNPs) are the most advanced delivery system currently available for RNA therapeutics. Their development has accelerated rapidly since the success of Patisiran, the first siRNA-LNP therapeutic, and the SARS-CoV-2 mRNA vaccines that emerged during the COVID-19 pandemic. Designing LNPs with specific targeting, high potency, and minimal side effects is crucial for their successful clinical use. However, our understanding of how the composition and mixing methods influence the structure, biophysical properties, and biological activity of the resulting particles remains limited. While microfluidic technologies have significantly improved the speed and uniformity of LNP production, a major challenge that remains is that ~60-80% of mRNA-LNP formulations are unloaded (empty lipid particles). This study tackles this challenge by relating current standard characterization methods with more powerful emerging methods, including 1. multi-wavelength analytical ultracentrifugation (MWL-AUC), 2. In-line multi-angle light scattering (MALS) methods, and 3. synchrotron size-exclusion chromatography in-line with small-angle X-ray scattering (SEC-SAXS) coupled with singular-value decomposition methods (SVD). We will present the strengths and weaknesses of each approach and showcase the increased detail newer advanced methods provide by comparing LNP formulations made using two common small-scale production methods: microfluidic rapid mixing and bulk mixing. The characterization techniques employed here can enhance our understanding of LNP structure-function relationships and enable researchers to define their RNA LNP products more precisely, which can improve LNP quality and potentially accelerate pharmaceutical development.

  PublisherDOI

• Solution conformational differences between conventional and CENP-A nucleosomes are accentuated by reversible deformation under high pressure

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-01-21

  preprintOpen access1st author

  Solution-based interrogation of the physical nature of nucleosomes has its roots in X-ray and neutron scattering experiments, including those that provided the initial observation that DNA wraps around core histones. In this study, we performed a comprehensive small-angle scattering study to compare canonical nucleosomes with variant centromeric nucleosomes harboring the histone variant, CENP-A. We used nucleosome core particles (NCPs) assembled on an artificial positioning sequence (Widom 601) and compared these to those assembled on a natural α-satellite DNA cloned from human centromeres. We establish the native solution properties of octameric H3 and CENP-A NCPs using analytical ultracentrifugation (AUC), small-angle X-ray scattering (SAXS), and contrast variation small-angle neutron scattering (CV-SANS). Using high-pressure SAXS (HP-SAXS), we discovered that both histone identity and DNA sequence have an impact on the stability of octameric nucleosomes in solution under high pressure (300 MPa), with evidence of reversible unwrapping in these experimental conditions. Both canonical nucleosomes harboring conventional histone H3 and their centromeric counterparts harboring CENP-A have a substantial increase in their radius of gyration, but this increase is much less prominent for centromeric nucleosomes. More broadly for chromosome-related research, we note that as HP-SAXS methodologies expand in their utility, we anticipate this will provide a powerful solution-based approach to study nucleosomes and higher-order chromatin complexes.

  PublisherDOI

• Structural Impact of Ex Vivo Resistance Mutations on Hiv-1 Integrase Polymers Induced by Allosteric Inhibitors

  SSRN Electronic Journal · 2025-01-01 · 1 citations

  preprintOpen access
  PublisherDOI

• Understanding the Molecular Mechanism of Redondovirus Rep in Rolling Circle Replication of the Redondoviral Genome

  Structural Dynamics · 2025-03-01

  articleOpen access

  Redondoviridae is a family of the widespread circular Rep-encoding single-stranded (CRESS) DNA viruses. Redondoviruses were identified in human oro-respiratory samples from healthy and diseased individuals and were present at higher levels in samples taken from those with periodontal disease, severe respiratory diseases, critical illness, or COVID-19. Recent studies suggest that redondoviruses infect Entamoeba gingivalis – a parasitic protozoan that colonizes the human oral cavity and is associated with periodontal disease. Like other CRESS DNA viruses, redondoviruses encode a replication-associated protein (Rep) that mediates the rolling circle replication of the viral genome. This dynamic, multi-functional protein consists of an endonuclease domain at the N-terminus and a helicase domain at the C-terminus, linked together by an oligomerization domain. The molecular mechanism of Rep in rolling circle replication of the viral genome is not fully clarified for redondoviruses or any CRESS virus. Here, we report on the structural properties of redondovirus Rep using cryo-electron microscopy (cryo-EM), X-ray crystallography, and a battery of solution biophysical methods. We determined the structure of a hexameric assembly of redondovirus Rep with bound ATPγS at 2.85 Å resolution, revealing an assembly comprised of largely disordered endonuclease domains, and an ordered planar ring containing the oligomeric and ATPase domains. Resolved in our structure are six ATPγS ligands nestled between neighboring ATPase domain subunits. We additionally determined the X-ray crystal structure of the redondovirus Rep endonuclease domain to 1.46 Å resolution, revealing a canonical four-stranded antiparallel β-sheet core flanked by alpha helices. The structure shows high similarity to the Rep endonuclease domain of a wheat dwarf virus from the Geminiviridae family, a CRESS DNA virus family that infects plants. Using analytical ultracentrifugation, mass photometry, and size-exclusion chromatography in line with small-angle X-ray scattering, our analyses indicate that redondovirus Rep exists as a stable hexamer at nanomolar concentrations but transitions into a mixture of lower and higher-order oligomers at concentrations in the 10-100 μM range, particularly when ATP or ADP is present. This oligomeric heterogeneity is also evident in our cryo-EM data analyses. Collectively, these structural findings lay a foundation for understanding the molecular mechanism of redondovirus Rep and provide insights into the dynamics of CRESS virus DNA replication.

  PublisherDOI

• Elucidating lipid nanoparticle properties and structure through biophysical analyses

  Nature Biotechnology · 2025-10-23 · 14 citations

  article
  PublisherDOI

• Solution conformational differences between conventional and CENP-A nucleosomes are accentuated by reversible deformation under high pressure

  Chromosome Research · 2025-06-11 · 2 citations

  articleOpen access1st authorCorresponding

  Solution-based interrogation of the physical nature of nucleosomes has its roots in X-ray and neutron scattering experiments, including those that provided the initial observation that DNA wraps around core histones. In this study, we performed a comprehensive small-angle scattering study to compare canonical nucleosomes with variant centromeric nucleosomes harboring the histone variant, CENP-A. We used nucleosome core particles (NCPs) assembled on an artificial positioning sequence (Widom 601) and compared these to those assembled on a natural α-satellite DNA from human centromeres. We establish the native solution properties of octameric H3 and CENP-A NCPs using analytical ultracentrifugation (AUC), small-angle X-ray scattering (SAXS), and contrast variation small-angle neutron scattering (CV-SANS). Using high-pressure SAXS (HP-SAXS), we discovered that both histone and DNA sequence have an impact on the stability of octameric nucleosomes in solution under high pressure (300 MPa), with evidence of reversible unwrapping in these experimental conditions. Both canonical nucleosomes harboring conventional histone H3 and their centromeric counterparts harboring CENP-A have a substantial increase in their radius of gyration, but this increase is much less prominent for centromeric nucleosomes. More broadly for chromosome-related research, we note that as HP-SAXS methodologies expand in their utility, we anticipate this will provide a powerful solution-based approach to study nucleosomes and higher-order chromatin complexes.

  PublisherOA PDFDOI

• Characterization and structural basis for the brightness of mCLIFY: A novel monomeric and circularly permuted bright yellow fluorescent protein

  Biophysical Journal · 2025-05-22

  article
  PublisherDOI

Frequent coauthors

• Gregory D. Van Duyne

  University of Pennsylvania

  50 shared

• A. Joshua Wand

  Texas A&M University

  18 shared

• Patrick J. Loll

  17 shared

• Brian Fuglestad

  Virginia Commonwealth University

  16 shared

• Barry S. Selinsky

  Villanova University

  15 shared

• Frederic D. Bushman

  14 shared

• Emilia Arturo

  11 shared

• Loraine Silvestro

  American Society for Gastrointestinal Endoscopy

  11 shared