Engineered CJ protein crystals for rapid nucleic acid removal in PCR sample preparation

Overview

This technology leverages engineered CJ protein crystals as a green, high-precision biomaterial for rapid removal of free nucleic acids from samples prior to PCR testing. The crystals feature a dense array of 13-nanometer nanopores that irreversibly adsorb free DNA and RNA under nearly all solution conditions, effectively concentrating nucleic acids within the crystal volume. This approach is economical, requiring only micrograms of material per assay, and the crystals are non-cytotoxic, edible, and straightforward to produce at scale.

The solution addresses a critical sample preparation challenge in molecular diagnostics, microbiology testing, and any workflow where free nucleic acid interference must be minimized before amplification-based detection. By clearing free DNA and RNA from samples, the technology improves assay specificity and reliability.

Technical specifications

Key features:

• Nanopore architecture: Dense arrays of 13-nanometer pores within crosslinked CJ crystals create a molecular trap with negligible off-rate for captured nucleic acids
• Rapid capture kinetics: Small DNA molecules (<2000 bp) are removed from solution within minutes across a wide pH range (pH 4–9)
• High capacity: 100 μg of CJ protein corresponds to approximately 68 million microcrystals, each 4 μm in diameter and 0.5 μm in height
• Robust stability: Crosslinked crystals resist solvent, desiccation, heat, and protease attack
• Scalable production: A single 1L E. coli shake flask expression yields approximately 100 mg of raw CJ protein
• Irreversible adsorption: Once captured, nucleic acids remain bound within the crystal interior

How it works:

The nanopore arrays within CJ crystals serve as molecular traps that concentrate free nucleic acids from comparatively large sample volumes into the small volume of the crystals. Confocal microscopy has confirmed strong DNA uptake into large crystals, and microcrystal suspensions have demonstrated collective DNA binding affinity by quantifying DNA remaining in solution.

Technology readiness level

The technology is at an early-to-mid stage of development. Key preliminary results have demonstrated DNA and RNA capture under diverse solution conditions, with confocal microscopy directly confirming DNA uptake and quantitative assays showing removal of DNA ladder fragments within five minutes for smaller molecules.

Future validation plans include:

• Quantitative isotherm binding experiments using a custom cuvette to measure free DNA reduction
• Validation via qPCR and ddPCR to assess impact on apparent nucleic acid concentration
• Scale-up of CJ protein yield using yeast secretion and microcrystal production via liquid handling robotics
• Testing for interference with microbiological testing protocols
• Development of magnetic nanoparticle-doped CJ crystals and immobilized crystal formats for separation if needed

The underlying CJ crystal platform is protected by US Patent 10590176 B2, providing a foundation for further co-development and commercialization partnerships.