Biology subjects

Mallikaratchy, P.

Publications and source records attributed to Mallikaratchy, P..

4 recordsLinked to original sources

bioRxiv2026

Precision Aptamers Against a Native GPCR through Ligand-Guided Selection

G protein-coupled receptors (GPCRs) constitute the largest and most diverse class of membrane receptors encoded in the human genome. They detect a wide range of chemical and physical stimuli and transduce these signals into intracellular responses through highly regulated pathways. Reflecting their central role in physiology, GPCRs are among the most prominent targets in drug discovery. However, identifying ligands that recognize GPCRs in their native conformational and membrane context remains a significant challenge. Here, we report an expanded aptamer discovery platform based on ligand-guided selection (LIGS) to isolate aptamers against GPCRs in their native cellular state. Using the {beta}2-adrenergic receptor ({beta}2AR) as a model system and employing agonists and antagonists as competing ligands, we identified three aptamers with high specificity for {beta}2AR. These aptamers exhibit selective binding to cell-surface {beta}2AR, showing higher apparent affinity towards cell-membrane bound {beta}2AR than toward the purified receptor, which is consistent with recognition of native receptor context. Beyond target recognition, we show that the selected aptamers induce rapid internalization, indicating functional engagement. Together, these findings establish ligand-guided selection as a generalizable strategy for the discovery of conformationally sensitive aptamers targeting GPCRs in their native membrane environments. SignificanceThe ability to discover ligands for receptors that undergo dynamic conformational changes is essential for advancing targeted therapeutics. G protein-coupled receptors (GPCRs), among the most sought-after drug targets, exist in transient and heterogeneous conformational states that are difficult to replicate in purified or artificial systems. Here, we introduce a ligand discovery platform that leverages native receptor interactions with agonists and antagonists to enable the selection of nucleic acid ligands (aptamers) directly against GPCRs in their cellular context, eliminating the need for purified receptors. The resulting aptamers exhibit selective binding to membrane-bound receptors and display intracellular functionality, highlighting a broadly applicable strategy for discovering ligands that recognize and modulate GPCRs in their native environments.

biochemistry↗

bioRxiv2025

The modification of DNA with indole-linked nucleotides alters its sensitivity to enzymatic cleavage

We describe the synthesis of C-5 indole-tagged pyrimidine and C-8 indole-tagged purine nucleoside phosphoramidites and their incorporation into double-stranded DNA 15 base pairs in length. Of the 23 sequence modifications tested, two induced the DNA duplex to adopt a Z-like left-handed conformation under physiological salt conditions, bypassing the specific sequences typically required for a left-handed Z-DNA structure. The impact of these modifications varied with the linker type: flexible propyl linkers exhibited distinct effects compared to rigid propargyl linkers. Notably, modifications positioned directly on or near a restriction site emphasized the pivotal role of linker rigidity in controlling DNA conformation. Specifically, the conformational change induced by the flexible linker impacted nuclease and restriction endonuclease cleavage, reducing sequence specificity. In contrast, the rigid linker suppressed this effect. Furthermore, our findings indicate that nucleic acid duplexes modified with indole-linked nucleotides using a flexible propyl linker have a pronounced tendency to form BZ or Z-like regions in longer DNA sequences. A higher density of modifications may even induce a full Z-like conformation throughout the duplex. These modified nucleotides hold potential for the development of novel antisense therapeutics and introducing valuable tools for in vitro screening of small molecules targeting distorted B-DNA, BZ-DNA, and Z-DNA structures.

biochemistry↗

bioRxiv2025

The biochemical function of bivalent aptamer assemblies against B-cell markers CD19 and CD20

Aptamers are synthetic oligonucleotides that bind to their specific receptors with high specificity, offering immense potential for the development of molecular tools. Using recently introduced Ligand-Guided Selection (LIGS), a variant of the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) we previously identified anti-CD19 and anti-CD20 aptamers with specificity and affinity. Given their high expression levels, B-cell markers CD19 and CD20 are widely utilized in the diagnosis of B-cell-related malignancies and autoimmune diseases. Here, we report the design and functional characterization of bivalent aptamer assemblies targeting CD19 and CD20 expressed in B-cell lymphomas. Using a strategic approach, we synthesized dimeric constructs of these aptamers with polyethylene glycol (PEG) linkers of varying lengths to tether the two aptamer units. The bivalent aptamers demonstrated enhanced binding affinity and specificity, with an optimal linker length of [~]3.96 nm. Functional studies revealed that dimeric CD19 aptamers selectively internalized in CD21-negative B-cells, while CD20 aptamers exhibited improved antigen binding without triggering calcium release. These findings highlight the potential of bivalent aptamers in engineering cost-effective, stable, and precise therapeutic agents for B-cell-related malignancies, such as diffuse large B-cell lymphoma (DLBCL). This work advances the development of aptamer-based synthetic therapeutics with promising clinical applications.

biochemistry↗

bioRxiv2020

A homodimeric aptamer variant generated from ligand-guided selection activates T-cell receptor cluster of differentiation three complex

Recently, immunotherapeutic modalities with engineered cells and monoclonal antibodies have been effective in treating several malignancies. However, growing evidence suggests that immune-related adverse events (irAE) lead to severe and long-term side effects. Most iRAEs involve prolonged circulation of antibodies. To address this problem, nucleic acid aptamers can serve as alternative molecules to design immunotherapeutics with high functional diversity and predictable circulation times. Here, we report the first synthetic prototype consisting of DNA aptamers, which can activate T-cell receptor cluster of differentiation 3 (TCR-CD3) complex in cultured T-cells. We show that activation potential is similar to that of a monoclonal antibody (mAb) against TCR-CD3, suggesting the potential of aptamers in developing efficacious synthetic immunomodulators. The synthetic prototype of anti-TCR-CD3{varepsilon}, as described herein, was designed using aptamer ZUCH-1 against TCR-CD3{varepsilon}, generated by Ligand Guided Selection (LIGS). Aptamer ZUCH-1 was truncated and modified with nuclease-resistant RNA analogs to enhance stability. Several dimeric analogs with truncated and modified variants were designed with variable linker lengths to investigate the activation potential of each construct. Among them, dimeric aptamer with approximate dimensions similar to those of an antibody showed the highest T-cell-activation, suggesting the importance of optimizing linker lengths in engineering functional aptamers. The observed activation potential of dimeric aptamers shows the vast potential of aptamers in designing synthetically versatile immunomodulators with tunable pharmacokinetic properties, expanding immunotherapeutic designs with the use of nucleic acid-based ligands such as aptamers.

biochemistry↗