Just as countries import a wide range of consumer goods across national borders, living cells are engaged in a vibrant import-export business. Their entry ports are sophisticated transport channels embedded in the protective membrane of a cell. Regulation of the types of goods that can cross the boundary zones formed by the two-layered membrane of the cell is essential for its proper functioning and survival.
In new research, Hao Yan, a professor at Arizona State University, along with ASU colleagues and international collaborators from University College London, describes the design and construction of artificial membrane channels, engineered at the using short stretches of DNA. DNA constructs behave like natural cellular channels or pores, providing selective transport of ions, proteins, and other cargoes, with enhanced features unavailable in their natural counterparts.
These innovative DNA nanochannels could one day find application in a variety of scientific fields, ranging from biosensing and drug delivery applications to creating networks of artificial cells capable of autonomously capturing, concentrating, storing and delivering. a microscopic cargo.
“Many biological pores and channels are closed by reversibility to allow ions or molecules to pass through,” says Yan. Here, we mimic these natural processes to engineer DNA nanopores that can be locked and opened in response to “key” or “lock” external molecules.
Professor Yan is the Milton D. Glick Professor Emeritus of Chemistry and Biochemistry at ASU and directs the Biodesign Center for Molecular Design and Biomimetics. He is also a professor in the School of Molecular Sciences at ASU.
The research results appear in the current issue of the journal Nature Communications.
All living cells are enveloped in a unique biological structure, the cell membrane. The scientific term for such membranes is phospholipid bilayer, which means that the membrane is formed from phosphate molecules attached to a fatty or lipid component to form an outer and inner membrane layer.
These inner and outer membrane layers are much like the interior and exterior walls of a room. But unlike normal walls, the space between the inner and outer surfaces is fluid, resembling a sea. Moreover, cell membranes are said to be semi-permeable, allowing the designated entry or exit of cargo from the cell. Such transport typically occurs when transiting cargo binds to another molecule, altering the dynamics of the canal structure to allow entry into the cell, much like the opening of the Panama Canal.
Semi-permeable cell membranes are necessary to protect sensitive ingredients inside the cell from a harsh external environment, while allowing the transit of ions, nutrients, proteins, and other vital biomolecules.
Researchers including Yan have explored the possibility of synthetically creating selective membrane channels, using a technique known as DNA nanotechnology. The basic idea is simple. The double strands of DNA that form the genetic blueprint of all living organisms are held together by the base pairing of the molecule’s 4 nucleotides, labeled A, T, C, and G. A simple rule applies, namely that nucleotides A always pair with T and C with G. Thus, a DNA segment ATTCTCG would form a complementary strand with CAAGAGC.
DNA base pairing allows for the synthetic construction of virtually unlimited network or 2- and 3-D nanostructures. Once a structure has been carefully designed, usually using a computer, the DNA segments can be mixed and will self-assemble in solution into the desired shape.
However, creating a semi-permeable channel using DNA nanotechnology has proven to be a daunting challenge. Conventional techniques have failed to replicate the structure and capabilities of natural membrane channels, and synthetic DNA nanopores typically only allow unidirectional transport of goods.
The new study describes an innovative method, allowing researchers to design and construct a synthetic membrane channel whose pore size allows for the transport of greater cargo than natural cellular channels. Unlike previous efforts to create DNA nanopores attached to membranes, the new technique builds the channel structure step by step, assembling the component DNA segments horizontally to the membrane, rather than vertically. The method allows the construction of nanopores with larger openings, allowing the transport of a greater range of biomolecules.
Additionally, the DNA design allows the channel to be selectively opened and closed by means of a hinged cover, fitted with a lock and key mechanism. The “keys” are made up of sequence-specific DNA strands that bind to the channel lid and trigger it to open or close.
In a series of experiments, the researchers demonstrate the ability of the DNA channel to successfully transport cargoes of varying sizes, ranging from tiny dye molecules to folded protein structures, some larger than the pore dimensions of natural membrane channels.
The researchers used atomic force microscopy and transmission electron microscopy to visualize the resulting structures, confirming that they met the original design specifications of the nanostructures.
Fluorescent dye molecules were used to verify that the DNA channels successfully pierced and inserted through the cell’s lipid bilayer, successfully providing selective entry of carrier molecules. The transport operation was completed within an hour of channel formation, a significant improvement over previous DNA nanopores, which typically require 5-8 hours for complete transit of the biomolecule.
DNA nanochannels can be used to capture and study proteins and look closely at their interactions with the biomolecules they bind to or study the rapid and complex folding and unfolding of proteins. These channels could also be used to exert precise control over biomolecules entering cells, providing a new window into targeted drug delivery. Many other possible applications will likely arise from the new ability to custom design artificial, self-assembled transport channels.
- Swarup Dey, Adam Dorey, Leeza Abraham, Yongzheng Xing, Irene Zhang, Fei Zhang, Stefan Howorka, Hao Yan. A reversible gating protein transport membrane channel made of DNA. Communication Nature, 2022; 13 (1) DOI: 10.1038/s41467-022-28522-2
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