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Leakless end-to-end transport of small molecules through micron-length DNA nanochannels.

ABSTRACT: Designed and engineered protein and DNA nanopores can be used to sense and characterize single molecules and control transmembrane transport of molecular species. However, designed biomolecular pores are less than 100 nm in length and are used primarily for transport across lipid membranes. Nanochannels that span longer distances could be used as conduits for molecules between nonadjacent compartments or cells. Here, we design micrometer-long, 7-nm-diameter DNA nanochannels that small molecules can traverse according to the laws of continuum diffusion. Binding DNA origami caps to channel ends eliminates transport and demonstrates that molecules diffuse from one channel end to the other rather than permeating through channel walls. These micrometer-length nanochannels can also grow, form interconnects, and interface with living cells. This work thus shows how to construct multifunctional, dynamic agents that control molecular transport, opening ways of studying intercellular signaling and modulating molecular transport between synthetic and living cells.

SUBMITTER: Li Y

PROVIDER: S-EPMC9451144 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

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Leakless end-to-end transport of small molecules through micron-length DNA nanochannels.

Li Yi Y Maffeo Christopher C Joshi Himanshu H Aksimentiev Aleksei A Ménard Brice B Schulman Rebecca R

Science advances 20220907 36

Designed and engineered protein and DNA nanopores can be used to sense and characterize single molecules and control transmembrane transport of molecular species. However, designed biomolecular pores are less than 100 nm in length and are used primarily for transport across lipid membranes. Nanochannels that span longer distances could be used as conduits for molecules between nonadjacent compartments or cells. Here, we design micrometer-long, 7-nm-diameter DNA nanochannels that small molecules ...[more]

PMID: 36070388

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Project description:The electrokinetic behavior of molecules in nanochannels (<100?nm in length) have generated interest due to the unique transport properties observed that are not seen in microscale channels. These nanoscale dependent transport properties include transverse electromigration arising from partial electrical double layer overlap, enhanced solute/wall interactions due to the small channel diameter, and field-dependent intermittent motion produced by surface roughness. In this study, the electrokinetic transport properties of deoxynucleotide monophosphates (dNMPs) were investigated, including the effects of electric field strength, surface effects, and composition of the carrier electrolyte (ionic concentration and pH). The dNMPs were labeled with a fluorescent reporter (ATTO 532) to allow tracking of the electrokinetic transport of the dNMPs through a thermoplastic nanochannel fabricated via nanoimprinting (110?nm?×?110?nm, width?×?depth, and 100??m in length). We discovered that the transport properties in plastic nanochannels of the dye-labeled dNMPs produced differences in their apparent mobilities that were not seen using microscale columns. We built histograms for each dNMP from their apparent mobilities under different operating conditions and fit the histograms to Gaussian functions from which the separation resolution could be deduced as a metric to gage the ability to identify the molecule based on their apparent mobility. We found that the resolution ranged from 0.73 to 2.13?at pH?=?8.3. Changing the carrier electrolyte pH?>?10 significantly improved separation resolution (0.80-4.84) and reduced the standard deviation in the Gaussian fit to the apparent mobilities. At low buffer concentrations, decreases in separation resolution and increased standard deviations in Gaussian fits to the apparent mobilities of dNMPs were observed due to the increased thickness of the electric double layer leading to a partial parabolic flow profile. The results secured for the dNMPs in thermoplastic nanochannels revealed a high identification efficiency (>99%) in most cases for the dNMPs due to differences in their apparent mobilities when using nanochannels, which could not be achieved using microscale columns.

Project description:Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease with available treatments only marginally slowing progression or improving survival. A hexanucleotide repeat expansion mutation in the C9ORF72 gene is the most commonly known genetic cause of both sporadic and familial cases of ALS and frontotemporal dementia (FTD). The C9ORF72 expansion mutation produces five dipeptide repeat proteins (DPRs), and while the mechanistic determinants of DPR-mediated neurotoxicity remain incompletely understood, evidence suggests that disruption of nucleocytoplasmic transport and increased DNA damage contributes to pathology. Therefore, characterizing these disturbances and determining the relative contribution of different DPRs is needed to facilitate the development of novel therapeutics for C9ALS/FTD. To this end, we generated a series of nucleocytoplasmic transport "biosensors", composed of the green fluorescent protein (GFP), fused to different classes of nuclear localization signals (NLSs) and nuclear export signals (NESs). Using these biosensors in conjunction with automated microscopy, we investigated the role of the three most neurotoxic DPRs (PR, GR, and GA) on seven nuclear import and two export pathways. In addition to other DPRs, we found that PR had pronounced inhibitory effects on the classical nuclear export pathway and several nuclear import pathways. To identify compounds capable of counteracting the effects of PR on nucleocytoplasmic transport, we developed a nucleocytoplasmic transport assay and screened several commercially available compound libraries, totaling 2714 compounds. In addition to restoring nucleocytoplasmic transport efficiencies, hits from the screen also counteract the cytotoxic effects of PR. Selected hits were subsequently tested for their ability to rescue another C9ALS/FTD phenotype-persistent DNA double strand breakage. Overall, we found that DPRs disrupt multiple nucleocytoplasmic transport pathways and we identified small molecules that counteract these effects-resulting in increased viability of PR-expressing cells and decreased DNA damage markers in patient-derived motor neurons. Several HDAC inhibitors were validated as hits, supporting previous studies that show that HDAC inhibitors confer therapeutic effects in neurodegenerative models.

Dataset Information

Leakless end-to-end transport of small molecules through micron-length DNA nanochannels.

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Leakless end-to-end transport of small molecules through micron-length DNA nanochannels.

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