A team of researchers from institutions including the Arc Institute and UC Berkeley discovered that certain mobile genetic elements found extensively in bacteria and archaea known as IS110 insertion sequences or MGEs express a structured non-coding RNA (ncRNA) that interacts with their recombinase. This unique RNA, called “bridge” RNA, contains two loops that specifically interact with both the target DNA and the donor DNA of the IS110 element. This RNA can be independently reprogrammed to facilitate sequence-specific recombination, enabling the insertion, removal, or inversion of specific DNA segments. This represents a modular and highly specific approach to DNA rearrangement, expanding upon traditional methods like CRISPR.
The IS110 family, which includes the IS621, employs a distinct DEDD catalytic motif for a cut-and-paste mechanism that lacks terminal inverted repeats. Instead of the usual transposases that recognize terminal inverted repeats, IS110 utilizes a catalytic feature called a DEDD motif and a specialized recombinase to carry out a “cut-and-paste” mechanism. In this process, the recombinase binds to a specific ncRNA derived from the ends of the IS621. This ncRNA bridges the recombinase and the DNA during the integration process. The conserved nature of the ncRNA across IS110 elements aiding in sequence-specific recombination reveals the potential for programmable DNA targeting.
The researchers developed an extensive sequence database from various public databases and used tools such as MMseqs2, IQtree2, and MST to cluster and analyze the data for conserved residues and phylogeny. They identified IS110 element boundaries and predicted “bridge” RNA structures. The use of small RNA-seq, in vitro transcription, and plasmid recombination assays in E.coli demonstrated the high specificity and programmability of this new method. It was found that the system preferred exact matches with low tolerance for mismatches, indicating high fidelity. The expression of bridge RNAs separately (in trans) resulted in significantly improved recombination efficiencies.
The discovery of bridge RNA represents a new approach in RNA-guided genetic manipulation. Unlike single-strand RNA guides, bridge RNAs are bispecific molecules that orchestrate the precise alignment of donor and target DNA sequences for efficient recombination. This mechanism allows IS110 elements to execute diverse DNA rearrangements with minimal dependence on protein-DNA interactions. Structural studies revealed a modular RNA-protein complex capable of catalyzing intricate DNA manipulations. These discoveries suggest promising potential for bridge RNAs as powerful tools for genome engineering beyond conventional RNA-guided systems with potential for applications in therapeutic and biotechnological context due to minimized off-target effects.
