Core Technology: The ezRNA Platform

At the center of EnPlusOne Bio's operations is its proprietary ezRNA platform. The platform replaces harsh chemical synthesis with nature-inspired enzymatic catalysis operating in benign aqueous conditions.

Primary Technological Components

1. Universal Enzyme Engine EnPlusOne Bio utilizes a versatile, engineered universal enzyme capable of performing template-independent nucleic acid synthesis. This enzyme operates across natural nucleobases, industry-standard chemical modifications, and over 50 novel chemistries.

2. Unprotected Nucleoside Triphosphate (NTP) Building Blocks Unlike traditional chemical synthesis that requires complex protection and deprotection chemical reactions on nucleosides, the ezRNA platform processes unprotected NTP building blocks directly in water. This drastically improves atom economy and reduces chemical side reactions.

3. Template-Free Enzymatic Synthesis The platform synthesizes custom RNA strands without requiring a DNA or RNA template strand. This capability allows precise, site-specific addition of modified or unmodified nucleotides directly onto growing RNA chains.

Capabilities and Manufacturing Solutions

EnPlusOne Bio provides end-to-end RNA synthesis solutions spanning early-stage discovery, optimization, and scale-up for Chemical, Manufacturing, and Controls (CMC) applications.

1. De Novo Full-Length RNA Synthesis

The platform enables complete step-by-step assembly of RNA oligonucleotides. Higher cycle efficiencies inherent to enzymatic assembly allow the production of longer RNA sequences with superior purity relative to conventional phosphoramidite chemistry.

2. Blockmer Assembly and Ligation

To construct large RNA molecules (such as guide RNAs and messenger RNAs), EnPlusOne employs a blockmer strategy. Enzymatically synthesized shorter sub-units (blockmers) are ligated together, creating an efficient enzymatic route to full-length, complex RNA structures.

3. Hybrid Synthesis and Payload Extension

EnPlusOne's enzymatic technology can perform single or multi-nucleotide extensions off existing therapeutic payloads. This enables targeted modification or tailing of pre-existing RNA molecules, enabling hybrid manufacturing workflows that integrate existing chemistries with enzymatic terminal modification.

Chemical Customization and Modification Portfolio

Nature utilizes more than 200 distinct chemical modifications in RNA, whereas traditional chemical synthesis historically provides efficient access to only a fraction of that diversity. EnPlusOne's ezRNA platform dramatically expands the accessible chemical design space.

Base Modifications

• Standard nucleobases: Adenine (A), Cytosine (C), Guanine (G), and Uracil (U).
• Non-standard and novel nucleobases: Modified nucleobases incorporating fluorophores, alkyl groups, halogens, biotin, and specific chemical conjugation handles.

2'-Sugar Modifications

• Essential chemistries for small interfering RNA (siRNA) and antisense oligonucleotides (ASOs), including 2'-OH, 2'-H (DNA), 2'-OMe (2'-O-methyl), and 2'-F (2'-fluoro).
• Advanced sugar architectures, such as locked nucleic acids (LNAs) and halogenated sugars.

3'-Sugar Modifications

• Functional terminal handles including 3'-dideoxy nucleotides, click chemistry handles, linkers, and chain terminators.

Backbone Chemistries

• Standard phosphodiester backbones.
• Therapeutically essential modified backbones, including phosphorothioate (PS) linkages and boronated backbones to enhance nuclease resistance and pharmacological stability.

Therapeutic Applications and Product Offerings

EnPlusOne Bio collaborates with biopharmaceutical partners and researchers across discovery and development phases. The platform supports a broad array of therapeutic RNA modalities:

• Small Interfering RNA (siRNA): High-purity duplexes with customized sugar and backbone modifications for targeted gene silencing.
• Antisense Oligonucleotides (ASOs): Single-stranded oligonucleotides engineered for mRNA binding, splice modulation, or degradation.
• Guide RNA (gRNA): Long, highly modified single-guide RNAs (sgRNAs) for CRISPR-based gene editing applications.
• Messenger RNA (mRNA) and Emerging Modalities: Long-chain RNA constructs, circular RNAs, and next-generation nucleic acid structures featuring custom modifications.

Environmental Sustainability and Manufacturing Advantages

EnPlusOne Bio addresses environmental and economic sustainability in RNA manufacturing:

• Reduction of Organic Solvents: Eliminates the vast majority of hazardous organic solvents (such as acetonitrile and dichloromethane) required by chemical synthesizers, performing reactions in aqueous buffers.
• Improved Atom Economy: Direct use of unprotected building blocks reduces synthesis steps, purification burden, and raw material waste.
• Scalable Production: Designed to scale seamlessly from milligram discovery quantities to multi-kilogram commercial requirements necessary to supply large patient populations.