Swapna G. Naik

Research Summary: I have contributed to understanding the impact of METTL3 transcription regulation on melanoma metastasis. Additionally, I have elucidated the role of m6A RNA modification in the adult cerebellum and how its deregulation can lead to conditions such as neurodegeneration and ataxia. My work characterizing the phosphodiesterase enzyme in C. elegans has identified key features of the CEPDE3 enzyme compared to human PDE3, which could help in designing more effective small-molecule modulators of PDE3's catalytic activity with clinical relevance. the

• Member: The American Society for Biochemistry and Molecular Biology (ASBMB), 2025
• Member: The RNA Society, 2025

• Translational science training program, NIH
• Undertaken molecular cell biology and biochemistry courses at the Foundation of Advanced Education In Sciences (FAES) at the NIH.
• Bioinformatics workshop and courses, NIH Library
• USA Science and Engineering Festival, Washington D.C., demonstrated a science project for an audience of >200 participants
• NIH Children’s Inn: Organized and planned events and fundraisers for critically ill patients

 1) Noel, Carter, Director of Studies, noel.carter@sunderland.ac.uk, +44783363729, University of Sunderland, Sunderland, UK

2)  Dr. Heng Ming Ke, NIH- Project Collaborator at University of North Carolina, hengming_ke@med.unc.edu (919)-966-224

3)  Dr. Heng Zhu: Professor, Johns Hopkins, School of Medicine,hzhu4@jhmi.edu, contact person: Ms. Mimi Guercio, mguercio@jhmi.edu, (410)-955-7117

4)   Dr. Sharon Milgram: Director, Graduate Partnership Program, NIH,milgrams@od.nih.gov, (301)-594-2053

5)  Dr. Dan Fabris: Department of Chemistry, UConn, dan.fabris@uconn.edu

6)   Dr. Claudio Alarcon: Department of Pharmacology, Yale University, claudio.alarcon@yale.edu

• Naik, Swapna G. (2017) Molecular cloning, purification, and characterization of the Caenorhabditis elegans Phosphodiesterase 3 (CEPDE3) gene. Doctoral thesis, The University of Sunderland. https://sure.sunderland.ac.uk/id/eprint/16545/
• Emerging phosphodiesterase inhibitors for the treatment of neurodegenerative disorders https://pubmed.ncbi.nlm.nih.gov/38279990/
• Validation of cold shock-based technique for purification of recombinant C. elegans Phosphodiesterase 3 (CEPDE3) protein expressed in E. coli- S. Naik1; A. Samidurai1, B. Sabouri1, D. K. Rhee1, S. Hockman 1, F. Ahmad Khan1,3, N. Carter2, V. C. Manganiello1-2025-(Accepted for publication at Elsevier-Protein Expression and Purification).
• Understanding Phosphorylation of METTL3 at position at s50- (research in progress)
• Impact of METTL3 phosphorylation at s43 on lung metastasis in human metastatic melanoma cells in xenograft model (research in progress)
• Determining the role of m6A in spinocerebellar ataxia using the Hexim1 knockout mice model (research in progress)

     Molecular and Cell Biology

• Investigated the regulation of the METTL3 enzyme and its phosphorylation at serine 50 (S50). Screened CRISPR-Cas9 knock-in cell lines for METTL3 to identify S50A homozygous mutants (non-phosphorylatable) and generated S50E mutants as phosphomimetics.
• Performed CRISPR knock-in in HeLa and WI38 fibroblast cells to mutate serine 50 residues, producing non-phosphorylatable and phosphomimetic cell lines.
• Examined potential kinases and phosphatases involved in phosphorylation and created corresponding cell lines.
• Conducted flow cytometry analysis for single-cell sorting of CRISPR knock-in and knock-out cells performed in HeLa cells
• Explored the role of RNA methylation in the adult cerebellum and its implications in ataxia using a METTL3 D953A knock-in mutation and HEXIM1 Cas-9-CKO mice models.
• Optimized conditions to study the effects of epidermal growth factors (EGF) in mammalian cells under serum starvation and glucose starvation.
• Performed knockdown of endogenous METTL3 in MeWo 2.5 cell lines using lentiviral transfectionthe.
• Developed effective gene silencing methods to target RNA modification enzymes in astrocytoma (U251-MG, U373, and U87-MG) and HeLa cells, to explore the impact of neuromodulators in HIV and drug abuse.
• Optimized mammalian transfection protocols for gene knockdown of DNA helicase subunits in Rhabdomyosarcoma cell lines (RH30, RH41, RD31, and SMSCTR), aiming to enhance the chemotherapy regime in young adults.

      Protein Engineering:

• Recombinant protein production in microbial and baculovirus expression systems
• Recombinant protein expression of the (CEPDE3) gene isoforms in E. coli using His-tag and GST-tag fusion system
• Optimized strategies based on the cold-shock technique to solubilize proteins from inclusion bodies for CEPE3 isoforms.
• Developed protein expression techniques of minichromosomal maintenance complex sub-units (MCM7 and MCM3) in SF9 and Hi5 cells.
• Optimized conditions for high-throughput recombinant protein induction of an extensive collection of > 500 proteins with GST-tag proteins in the yeast for determining biomarkers in cancer
• Optimized and quantified small and large batch protein purification using the GST fusion system and His-tag protein using an IMAC chromatography system.
• Quantified protein expression and purification samples, Lowry, Bradford, protein pierce detecting techniques using SDS-PAGE—analyzed protein based on antibodies using western immunoblotting.
• Analyzed protein purification samples using silver staining and gel staining

      RNA-DNA synthesis

• Manipulation of Nucleic Acids and Biological Samples
• Optimized conditions for RNA extraction using Trizol and QIAGEN RNeasy kits in samplesfrom human blood, saliva, and poliovirus, cells treated with siRNA and neuromodulators to enhance yield and purity. Also optimized workflow with using the Hamilton STAR liquid handling robot to optimize RNA extraction yields.
• Established optimal conditions for mononucleotide digestion by exonucleases of intact RNA from mammalian extracts and siRNA knockdown samples. Developed and optimized protocols for quantifying modified nucleosides (ac4c, m5c) in RNA via Mass Spectrometry.
• Performed dot blot assays using specific antibodies for ac4c and m5c on positively charged nylon membranes.
• Used qPCR and dPCR to assess gene expression levels in gene knockout samples from RNA and cDNA.
• Applied AFA (adaptive focus acoustics) for RNA shearing to facilitate sequence analysis.
• Created low-throughput ultrasonication protocols for shearing 16mer and 58mer in vitro transcribed RNAs, based on timed shearing with Covaris ME220.

      Mass Spectrometry Analysis

• Conducted mass spectrometry for analyzing global RNA post-transcriptional modifications (rPTMs).
• Analyzed rPTM data with a custom Java program to quantify abundance in gene silencing samples treated with NAT10, NSUN2, and ALYREF enzymes.
• Profiling the epitranscriptome of rPTMs from biological samples including saliva, blood, poliovirus RNA, and SARS-CoV-2 RNA.
• Performed high-resolution MS analysis using direct infusion ESI on an LTQ-Orbitrap Velos, with FT-ICR detection and quartz emitters prepared in-house.
• Differentiated isomeric and isobaric uridine and pseudouridine species in native and denatured RNA via TIMS-ToF high-resolution ion mobility mass spectrometry.
• Analyzed epitranscriptomic profiles of global rPTMs in knockdown vs. control cells using LC-MS MS with FT-ICR to quantify target gene knockdown.
• Distinguished RNA PTMs based on treatment conditions using the MODOMICS database and a custom Java program.
• Optimized Western blot protocols for quantifying CEPDE3 protein expression, including samples from gene knockdowns. Quantified RNA levels of rPTM enzymes in knockdown samples via dot blot analysis.
• Evaluated protein and cell gene knockdowns through Western immunoblotting and JESS system analysis.
• Quantification of motor behavior data analysis using GraphPad-Prism software, one-way ANOVA test
• Proficient in using Adobe packages (PSE elements, Illustrator)

     Mice work

• Experimental techniques in mouse models include handling, husbandry, and colony management.
• Euthanasia is performed via cervical dislocation or CO2 inhalation.
• Dissection procedures cover the brain, liver, lungs, kidneys, and pancreas.
• Sample preparation involves transcriptomics, immunohistochemistry, proteomics, ear-tagging, and genotyping.
• Motor behavior is assessed with rotarod and dowel tests.
• Tumor growth measurement is conducted in melanoma models. Injections, such as retroorbital, tail vein, subcutaneous, and cardiac, are carried out in mouse models.

      Case Studies-Master’s Program

• Designed techniques to develop diagnostic tests for the detection of chlamydia infections
• Analysis of Type II restriction enzymes for DNA sequencing
• Detecting the effects of agitation and aeration on the transfer of oxygen in a fermentation vessel

      Selective Panel & Facilitation Experience

• Bio-protocol Ambassador, 2022-2023
• Team Leader: Focus group on cell culture techniques. Met bi-weekly to discuss research topics with team members for hosting the BP roundtable webinar, and to invite speakers.
• Member of RNA tech group for Bio-protocol: Planned speaker events, group discussions on RNA
• Planning committee- NIH research symposium; organized, participated, and recruited five keynote speakers on a panel with research and development in science for an audience of 500+.
• Facilitated science career communications by leading a panel discussion on careers in science.
• Co-coordinator mentorship committee- Yale Postdoctoral Association- June 2023-2024, managed the operations and functioning of 4 sub-committees