De Novo Negatively Charged Binders Targeting MMLVRT Nucleic Acid Binding Sites Overcome Stability-Activity Trade-offs

Abstract

The limited thermostability and storage-induced inactivation of Moloney murine leukemia virus reverse transcriptase (MMLV RT) have constrained its applications. In this study, a high-stability mutant, MMLV RT-SV, was generated through a multi-site mutagenesis strategy targeting the nucleic acid-binding pocket. Through de novo protein design, three highly negatively charged binders with affinities of 45 nM, 362 nM, and 829 nM were developed to specifically target the positively charged nucleic acid-binding region on the surface of MMLV RT-SV. Experimental results demonstrated that these binders formed complexes with the enzyme via electrostatic interaction, significantly enhancing the thermostability and long-term storage stability of MMLV RT-SV while not affecting its RNA- dependent DNA polymerase function. This work pioneers the rational design of negatively charged binders targeting strongly positively charged nucleic acid-binding sites, overcoming the traditional stability-activity trade-off inherent in site-directed mutagenesis and directed evolution. The proposed strategy not only provides an innovative solution to address the thermal sensitivity and storage instability of reverse transcriptase but also establishes a novel paradigm for De Novo-based precision engineering of enzyme functions, demonstrating significant potential in molecular diagnostics, gene editing, and related fields.