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Left-handed Z-RNA and Right-Handed A-RNA. The front ribbon of the Z-RNA Double-Helix points upwards to the left. In A-RNA and in Watson-Crick B-DNA the foremost ribbon points upwards to the right. Left handed Z-RNA can form from right-handed A-RNA by flipping the steps of the double helix upside down. The steps can be flipped by holding, then twisting each end of the helix. ©Alan Herbert

The finding of the Z-RNA nanoswitch is also remarkable because it is based on the unusual left-handed double-stranded RNA conformation. Normally, dsRNA is right-handed like the right-handed Watson-Crick B-DNA double helix. Since the discovery of the left-handed double helix, scientists have wondered if this structure has a function in biology. This paper confirms that the left-handed Z-RNA has a key role in regulating immune responses and details the mechanism by how that happens. The question whether flipon switches regulate other genetic programs is currently under active investigation. The paper also highlights the way self-made RNAs are tagged with sequences encoded by so-called “junk” DNA. “Junk” DNA received this name because it does not code for protein. Instead, all “junk DNA” does is “copy and paste” itself throughout a host genome. By doing so, the RNA sequences encoded by “junk DNA” become a way to distinguish self-made RNAs from foreign RNAs which lack these sequences. By forming Z-RNA, the “junk” sequence acts as flipons that turn “off” immune responses against self. These “junk” elements, once an existential threat to the human genome now serve to protect normal cells from damage by preventing inappropriate activation of the immune system. Z-RNA formation is important in directing attacks to pathogens and other evolving viral threats.

How does the switch turn “off”?
The answer illustrates how nature works at the nanoscale. Double-stranded RNA (dsRNA) is formed by pairing of two RNA strands that have matched sequences. Some dsRNA can adopt different three-dimensional conformations under physiological conditions. The sequences are called flipons. The Z-RNA nanoswitch is made from dsRNAs that adopt either a right-handed double-helix (called A-RNA) or a left-handed double helix (called Z-RNA). The two flipon conformations represent the “on” and “off” setting for the switch. The switch is “on” when it is A-RNA. In this state, the switch allows the assembly of proteins that drive the inflammatory response. The switch is “off” when it flips to Z-RNA. The flip to Z-RNA promotes the release of pro-inflammatory proteins from self dsRNA. It also localizes a protein, called ADAR1, that binds the Z-RNA flipon conformation. After modification by ADAR1, the self dsRNA becomes single-stranded and is no longer capable of activating an immune response.

What causes the nanoswitch to change to the Z-RNA conformation?
The pro-inflammatory proteins twist the dsRNA as they assemble on it. As a result of twisting, the bound dsRNA shortens in length, stretching the adjacent dsRNA segment. One way to relieve the tension is to flip a portion of the unbound dsRNA segment into the left-handed Z-RNA conformation. The flip relieves tension because the Z-RNA helix is 4.56 nanometers long whereas the A-RNA helix is only 2.46 nanometers in length. By being left-handed, the longer Z-RNA helix also offsets the right-handed twist caused by the pro-inflammatory proteins. The extra-slack produced by the Z-RNA flip causes these proteins to fall off the dsRNA, ending the immune response. The Z-RNA nanoswitch works similarly to an electrical “pull-switch” used to operate a light or a fan. In the case of a “pull-switch”, tugging on the cord with sufficient force twists the internal components to the “off” position. With the Z-RNA nanoswitch, the action of ADAR1 prevents the switch from being turned back “on” as the changes made by the enzyme are irreversible.

How does the Z-RNA nanoswitch protect normal cells?
“The switch is based on flipon sequences present in the human genome, but absent from viruses and other disease-causing organisms. Rather surprisingly, the flipons are encoded by what was formerly called “junk DNA”. They arise from “junk DNA” elements called Alu repeats that make up about 11% of the human genome. Many Alu repeats are in reverse orientation to each other. They are called inverted repeats. When copied into RNA, the inverted repeats fold back on themselves to form A-RNA. The Alu inverted repeat dsRNA contain a Z-RNA forming element called a Z-Box that Is highly conserved. The Z-box enables the switch to turn “off” an immune response against self RNA. These repeats are absent in viruses and serve to protect the host against attack.

The reported findings synthesize the discoveries of many scientists from different disciplines over the last 40 years. The Z-RNA nanoswitch is the first example of a flipon where an alternative nucleic conformations where there is direct genetic evidence for biological function. It is an example of a genetically encoded binary switch. Other types of flipons such as G-quartets and triplets are more varied in type than Z-flipons. Their different roles in cellular pathways are also being actively investigated. The new appreciation of the important role played by flipons in biology represents quite a paradigm shift.