Author interview: Pulak Ghosh completed his MS-PhD at IISER Pune under the guidance of Prof. S. G. Srivatsan, working on ‘Environment-sensitive nucleotide probes to study the activity of nucleic acid processing enzymes’. He is currently an Alexander von Humboldt postdoctoral fellow in the Department of Biochemistry at LMU Munich, Germany.
Lab: Prof. S. G. Srivatsan, IISER Pune
Research Summary: This study showcases the potency of environment-sensitive nucleotide probes to investigate TdT activity in real-time, offering new tools for diagnostics, therapeutics, and understanding fundamental enzyme mechanisms.
What was the core problem you aimed to solve with this research?
Terminal deoxynucleotidyl transferase (TdT) is a multifaceted enzyme with the unique ability to incorporate nucleotides at the 3′ end of single-stranded DNA in a template-independent manner. This property is essential for the evolution of the vertebrate adaptive immune system and has been used in many biotechnology applications, including de novo synthesis of oligonucleotides, biosensing, and aptamer development. More recently, the ability of the TdT enzyme to accept a range of modified nucleotide substrates has expanded its applications into DNA functionalization, nanotechnology, and even DNA-based data storage. Biologically, TdT plays a key role in promoting antibody repertoire diversity by facilitating the generation of T-cell receptors and immunoglobulins during the V(D)J recombination process. Clinically, elevated levels of TdT expression are associated with several types of leukemia, including acute lymphocytic leukemia and acute myeloid leukemia, positioning TdT as a potential biomarker and therapeutic target. Therefore, there has been a surge in efforts to develop robust TdT detection tools that obviate the problems (high cost, time-consuming, and hazardous) of standard detections based on immunoglobulin and gel electrophoresis assays that rely on the incorporation of radiolabeled nucleotides by TdT.
Given these diverse roles, the core problem we aimed to solve was to harness the processivity of TdT and the inherent responsiveness of our heterocycle-modified nucleotide analogs for multilayered applications. Specifically, we sought to (i) synthesize 3’-end functionalized DNA ON probes, (ii) develop a real-time fluorescence detection technique to estimate the binding event and monitor the TdT activity, and (iii) create a platform to identify potential inhibitors of TdT activity. Achieving these goals would advance both fundamental understanding of TdT and its broader applications in diagnostics, therapeutics, and biotechnology.
How did you go about solving this problem?
To address these challenges, we focused on two key aspects. First, we aimed to design modified nucleotides that are efficiently recognized and incorporated by the TdT enzyme, enabling a systematic study of its incorporation mechanism. Second, the modified nucleotides needed to be inherently fluorescent and sensitive enough to distinguish between their free and incorporated states through changes in their photophysical properties. This design allowed us to develop a fluorescence-based method for real-time monitoring of TdT activity.
In our lab, we have developed several environment-sensitive fluorescent nucleoside analogs by conjugating a heterocyclic moiety at the C5 positions of pyrimidine bases. These molecules are non-invasive and have moderate quantum yield, and the presence of a connecting biaryl bond between the heterocyclic moiety and uridine ring makes it a molecular rotor element (Figure 1A). High sensitivity to local viscosity, polarity, and stacking interactions with neighboring bases made them highly valuable in probing nucleic acid folding and recognition processes. In our previous studies, we observed that our modified analogs demonstrate good substrate efficacy for enzymatic incorporation by various template-dependent DNA and RNA polymerases. In my recent work, we also demonstrated that these analogs could effectively detect the enzymatic activity of certain template-dependent DNA polymerases through fluorescence. Based on these findings, we identified the best candidate analogs to employ for this study.
Our discoveries on TdT activity and recognition, and the versatile utility of our probe system, are expected to aid in advancing TdT-based DNA labeling, diagnostic, and therapeutic strategies.
How would you explain your research outcomes (Key findings) to the non-scientific community?
Terminal deoxynucleotidyl transferase (TdT), an enzyme known for its unique ability to add nucleotides to DNA without needing a template strand. TdT plays a pivotal role in immune system development and is also overexpressed in certain cancers, particularly leukemia, making it both a biological curiosity and a clinical target. However, studying TdT’s behavior, such as how it recognizes, processes, and incorporates nucleotides, has traditionally been challenging, especially in real time. To address this, we developed a new class of fluorescently labeled nucleotide analogs. These are specially designed molecules that not only fit into TdT’s active site but also respond sensitively to changes in their local environment, altering their fluorescence upon incorporation into DNA.
Using these environment-sensitive probes, we could observe TdT activity directly, gaining new insights into how structural features like steric bulk and stacking interactions affect the enzyme’s efficiency and substrate preference. Our probes revealed that a fine balance between nucleotide size and interaction with the primer end determines successful incorporation. Importantly, the fluorescent behavior of our probes allowed us to build a real-time assay platform, enabling not only the continuous monitoring of TdT activity but also the rapid screening of potential inhibitors, including both nucleotide analogs and non-nucleotide molecules.
What are the potential implications of your findings for the field and society?
Understanding the key interaction between nucleic acids and nucleic acid processing enzymes at the molecular level is of utmost importance for developing specific drug molecules, diagnostic tools, and nucleic acid-based therapeutic purposes. Therefore, the advancement of methods or techniques is gaining attention in the current scenario. Recent developments in high-throughput biopolymer crystallography and biomolecular NMR spectroscopy have helped in gaining structural insights into protein-DNA/RNA complex formation. However, most of the techniques are complicated and costly, and time-consuming. It is noteworthy that many of these methods are conducted under non-physiological conditions and lack real-time analysis capabilities. All around the globe, scientists are working on finding a simple and robust tool. To address this gap, various fluorescence techniques have been developed, offering insights into nucleic acid-protein interactions. However, the challenge lies in establishing an efficient platform that seamlessly correlates fluorescence, NMR, and X-ray crystallography techniques. Such a system could offer a comprehensive understanding of the steps involved in the enzymatic incorporation mediated by different polymerases and transferases. In my work, we tried to address this problem and develop such simple and robust methods. We hope our work advances the molecular understanding of enzyme activity and opens new pathways for its use in DNA functionalization, diagnostic tool development, and possibly even therapeutic intervention.
What was the exciting moment during your research?
In our effort to develop a fluorescence-based technique to study TdT activity, we encountered and overcame several challenges. One major hurdle was controlling the TdT elongation reaction to achieve a single-nucleotide modification. During our initial experiments, TdT incorporated multiple modified nucleotides, complicating the analysis. To address this, we proposed using a modified dideoxy nucleotide, which, lacking a 3′-OH group, would prevent further incorporation. However, previous studies indicated that dideoxy analogs are often poor substrates for many high-processivity DNA polymerases, raising concerns. Our eureka moment came when we discovered that our dideoxy-modified analogs were not only well accepted by TdT but were even preferred over the natural thymidine analogs. Moreover, these dideoxy analogs exhibited distinct fluorescent changes between their free and incorporated states. This pivotal finding gave us the confidence to further develop a fluorescence assay for detecting TdT activity and monitoring the inhibition process.
Paper reference: Ghosh, P.; Phadte, A. A.; Bhojappa, B.; Palani, S.; Srivatsan, S. G. Template-independent enzymatic functionalization of DNA oligonucleotides with environment-sensitive nucleotide probes using terminal deoxynucleotidyl transferase, Nucleic Acids Res., 2025, 53, gkaf108. ( https://doi.org/10.1093/nar/gkaf108)
