Harvard-Cambridge team uncovers plausible pathway for RNA function without enzymes

New research suggests a "plausible pathway" for the emergence of functional ribonucleic acid (RNA) before an RNA copying process was in place. This new research could help develop hypotheses for the origin of biological life on early Earth.

The researchers investigated how RNA molecules might self-replicate, without enzymes, using only physics. This would make possible an RNA world as a hypothetical early stage in evolution, where RNA was able to store genetic material. The idea has been under discussion and debate by scientists for many years.

Recent work by a group of scientists at the Department of Molecular Biology and the Department of Genetics at Harvard University, and the MRC Laboratory of Molecular Biology in Cambridge, United Kingdom, appears in bioRxiv, a preprint server for biology papers, Nov. 21, 2021.

RNA hairpin loops

The researchers looked at the prevailing secondary structure in functional RNA, known as RNA hairpin loops, which allow RNA chains to fold into complex structures.

Lead author Longfei Wu describes the hairpin loops as analogous to the arches in a cathedral:

"RNA hairpin loops are the most prevailing secondary structures of functional RNAs," he wrote. "If we think of the assembly of structured, functional RNA like building a cathedral, RNA hairpins could be the functional arch structures, windows and doors for example, to the cathedral."

"To build a cathedral, one must know how to build those stable arches, just as one needs hairpin loops for functional RNAs, " Wu added, noting that in both situations one usually must use templates, or scaffolding.

"The scaffolding used for building a cathedral arch can be removed after finishing the work, of course by human labor or intervention," Wu continued. "However, the template for building a longer, structured RNA (using RNA joining or ligation) cannot be removed afterward because it binds the structure more tightly than before."

The ligation forms a double-stranded helix, Wu noted, which cannot be removed afterward.

An outside-the-box solution

As an analogy, "Imagine that the scaffolding to build an arch is made of wood, and the arch is built by stones," Wu said. "In that case you could simply burn the wood and the arch will remain for the cathedral. Similarly, cycles of heating and cooling were proposed and explored to get rid of the RNA template partially, without destroying them. But then we explored a crazy idea by thinking outside the box [and asked] why not build structured RNAs directly, without templates?

"We were pleased to find out," Wu added, "that the inherent structural features of RNA hairpins are sufficient to enable their self-assembly without templates, by using the loop-closing for of ligation we describe in our paper."

The new work, Wu said, "offers a plausible, template-free way to assemble these important RNA structural units. This, in turn, could facilitate the emergence of structured, functional RNA at the very beginning, before more sophisticated replication/mutation/selection processes became available," he added.

The conventional way of joining short RNA pieces, Wu said, has been by using templated ligation chemistry, called nicked-duplex ligation or splint ligation. But that template impedes the RNA folding necessary for it to develop functions. To go back to the cathedral analogy, Wu said, it would be as though the arch scaffolding could not be removed, so the doors and windows would not function.

Instead, the loop-closing ligation described in the paper enables a template-free assembly process for RNA hairpins by making use of their structural features. The researchers found "the transfer RNA-like structures and full-length functional RNAs--usually having one or more hairpin structures--could be assembled from their constituent fragments without the help of external templates," Wu said.

"The loop-closing ligation not only offers a direct connection of short, unstructured RNAs to full-length structured RNAs, but also suggests a conceptual shift to focusing on RNA structures rather than mere RNA sequences while assembling functional RNAs," Wu added.

Next steps

Now that the research team has established the possibility of loop-closing ligation, it plans to examine other loop sizes and sequences. Wu noted that the group he works with at Harvard was "using high-throughput deep-sequencing technology to explore the sequence-activity relationship of the RNA loop-closing chemistry."

Their experiments will probe two questions. The first is the whether loop-closing ligation property is inherent in different loop sizes and sequences, and second, "what are the most efficient RNA loop-closing constructs in terms of loop-sizes, loop-sequences, and their ways of disconnection and connection?" Wu said. "The information will help to guide us to join RNA pieces into hairpin structures more efficiently in an engineering point of view." 

The lab is also exploring a plausible prebiotic activation chemistry "to construct hairpin loops in situ in a continuous manner. This will really make the chemistry more powerful and more prebiotic(ally) plausible, which is of great importance from both an engineering and an origin-of-life view." 

The research group is at Harvard's Szostak Lab, associated with Nobel laureate Jack Szostak, who has pursued different ways that life on Earth might have originated. 

"The Szostak Lab, along with others, pioneered the in vitro selection of functional RNAs," Wu said. "I think that evolving an RNA loop-closing ligase using RNA in-vitroselection method would be another interesting extension based on the present discovery."

An international collaboration

Wu started working on this problem as a postdoctoral candidate at the Laboratory of Molecular Biology at Cambridge, under the direction of professor John Sutherland. "I’m super-interested in how the genetic coding system had originated, as is Sutherland, who has been interested in this since the beginning of his career," Wu said. 

What inspired him in the new work is the previous discovery of nicked-loop acyl transfer chemistry and "a potential self-acylating mechanism of a transfer RNA-like molecule."

Sutherland recommended that Wu join the Szostak Lab to continue the work of applying chemistry to construct functional ribozymes and pursue a future career studying the origin of life. "The Szostak Lab is one of the leading groups in the field, working widely on various topics in the origin of life field including non-enzymatic RNA copying, functional ribozymes, in vitro evolution and protocells," Wu said. 

The concept of the loop-closing ligation was conceived and built in the Sutherland Lab, Wu stressed, and this formed the foundation for the ribozyme work done in the Szostak Lab.

"It turned out that the loop-closing strategy worked well in constructing various ribozyme systems, work done in the Szostak Lab," Wu said. "I must say it was only possible by benefiting from the expertise and super-collaborative environment in the Szostak Lab, that the work of applying the loop-closing strategy on assembly ribozymes could be built so quickly and smoothly."

Sutherland's advice affected Wu deeply who concluded, "Do science that surprises your peers, do science that is important and do it well. It’s a privilege to work on such a collaborative project and merge the expertise of the two labs together."

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L.-F. Wu, "Nonenzymatic loop-closing ligation generates RNA hairpins and is a template-free way to assemble functional RNAs."bioRxiv, Nov. 21, 2021.

https://doi.org/10.1101/2021.11.19.469337