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Despite being made from a relatively simple set of building blocks, ribonucleic acid (RNA) has a broad array of complex responsibilities. From providing structure to carrying the instructions for regulating genes and translating them into proteins, RNA is critical to cellular function.

Now, researchers at Penn State have revealed a potential explanation for how RNAs can be so functionally diverse: a "shifted wobble" of two components in the structure of some molecules of RNA. A paper describing the research is in the journal Nucleic Acids Research.

The team applied an approach, called "cheminformatics," to identify and characterize the shifted wobble in a large database of 3D RNA structures. They confirmed their work experimentally and found that the shifted wobbles are more common among bacteria, making them a potential target for drugs that could specifically impact bacteria with fewer off-target effects.

"Although it's a close relative of DNA, RNA can do much more than carry genetic information," said Philip Bevilacqua, distinguished professor of chemistry and of biochemistry and molecular biology in the Eberly College of Science at Penn State and the leader of the research team.

"It can catalyze reactions as an enzyme, act as a small molecule sensor or provide structure to cellular organelles. This functional diversity has led to the hypothesis that RNA might have been key to origins of life on Earth, but the question remains: 'How is RNA so functionally versatile given its limited molecular diversity?'"

Like DNA, RNA is a long molecule composed of a sugar backbone with four functional sidechain bases. The sidechains are split into two categories: the larger adenine (A) and guanine (G), and the smaller cytosine (C) and uracil (U). Similarly, the DNA alphabet uses A, G and C, but uses thymine (T) instead of U.

Unlike DNA—which is double-stranded forming a helical structure where an A on one strand always pairs with a T on the other, and G with C—RNA is single-stranded. RNA's 3D structure forms by the molecule folding back on itself forming short regions of base pairing, similar to DNA, but it can also form loops, bulges and pseudoknots.

"We know of some changes to RNA structure—called covalent modifications—that can enhance RNA functional flexibility through the addition of a chemical tag like a methyl group, but noncovalent modifications are less well studied," said Md Sharear Saon, postdoctoral scholar in chemistry at Penn State and first author of the paper.

"These noncovalent modifications involve changes in the molecular structure of the sidechain bases allowing unconventional hydrogen bonding which can lead to structural and functional diversity."

Noncovalent modifications can include one of the sidechain bases gaining or losing a proton, leading it to carry a positive or negative charge, or a proton moving to a new position within the base, creating a structure known as a tautomer, which can alter the binding relationship between bases. The team focused on identifying changes where a G binds to a U, instead of a C.

"Because of the nature of how RNAs form their 3D structures, there isn't always perfect alignment of Gs with Cs and As with Us like we see in DNA," Saon said. "When RNA folding leads to a G matched up with a U instead of its usual partner, it is referred to as a wobble because of how the mismatched pair arrange themselves in the molecule.

"We were interested in identifying and characterizing places in RNA structure where noncovalent modifications to the bases cause this wobble to shift into an alternative 'fully shifted' wobble."

In a shifted wobble, the G still pairs with the U, but the G is in a different position than in a standard wobble. The team developed cheminformatics methods to search a database of more than 3,000 high-resolution models of RNA structures for these shifted G-U wobbles. The methods extracted distances and angles between aligned Gs and Us, from which the team could infer their molecular arrangement.

Their analysis found more than 1,000 examples of shifted wobbles, but stringent filtering for redundancy or other potential issues reduced the number to 41 unique shifted G-U wobbles for further study.

"We had to invent methods that use the language of chemistry as search terms," Saon said. "We were searching for hydrogen bond distances and angles in a database of structures."

The team provided experimental support for the existence of the shifted G-U wobbles using a chemical compound, DMS. The compound usually only reacts with C and A bases, but it can also react with U in its shifted wobble positioning. For all the shifted G-U wobbles with moderately strong base pairing tested, DMS reacted with U.

"Our computational identification combined with experimental support suggest that these shifted G-U wobbles do exist in nature and likely add to the functional diversity of RNAs," Bevilacqua said.

"Because of their unique conformation, and the fact that we see more of these shifted wobbles in bacteria than in eukaryotes, which of course includes humans, they could be good targets to design drugs that disrupt the RNAs function while limiting off-target effects."