Characterization\nof Guest DNA Transport and Adsorption\nwithin Host Porous Protein Crystals

Nucleic acid transport through protein-based pores is\na well-characterized\nphenomenon due in part to advancements in nanopore sequencing. A less\nstudied area is nucleic acid transport through extended protein-based\nchannels, where the additional surface area and increased contact\ntime allow for the study of prolonged binding interactions. Porous\nprotein crystals composed of “CJ”, a putative polyisoprenoid-binding\nprotein from Campylobacter jejuni,\nrepresent a favorable, highly ordered material for studying DNA transport\nand binding/unbinding along protein-based channels. These crystals\nadopt a hexagonal prism habit and contain a densely packed hexagonal\narray of 13 nm diameter axial nanopores that run from the top to the\nbottom of the crystal. After cross-linking, the crystals are easily\nmanipulated for experimentation. An adsorption isotherm between host\ncrystals and guest double-stranded 8 base pair DNA (8mer) revealed\na high equilibrium adsorption constant of 206 ± 30 L/g. Fluorescence\nconfocal microscopy tracked the loading of guest DNA into host crystals\npredominately along the major axial crystal nanopores. Four different\ncomputational models based on the finite volume (FV) method were assessed\nto model the transport process for guest 8mer and 15mer dsDNA loading\ninto empty host crystals in terms of fundamental parameters, such\nas the intrapore diffusion constant. Fitting the models to the data\nrevealed that the most basic FV model sufficed to describe the observed\nloading behavior, characterized by a single effective diffusion coefficient.\nLeveraging Fick’s first law, we more directly fit a numerical\nrange for the observed intrapore diffusion coefficient as a function\nof time, position within the crystal, and relative guest concentration.\nThis new transport analysis strategy was applied to both out-of-equilibrium\nloading and fluorescence recovery after photobleaching (FRAP) experiments.\nThe intrapore diffusion constants are comparable between 8mer and\n15mer dsDNA and were found to be 2 orders of magnitude faster for\nDNA loading into empty crystals than that observed in FRAP experiments,\nwhich averaged (10 ± 4) × 10<sup>–11</sup> cm<sup>2</sup>/s.