Polyelectrolyte theory of the gene

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The Polyelectrolyte Theory of the Gene proposes that for a linear genetic biopolymer dissolved in water, such as DNA, to undergo Darwinian evolution anywhere in the universe, it must be a polyelectrolyte, a polymer containing repeating ionic charges (Benner 2017). These charges are needed to maintain the uniform physical properties needed for Darwinian evolution, regardless of the information encoded in the genetic biopolymer (Benner 2017). DNA has this property as it has the same physical properties regardless of its nucleic acid sequence.

The Polyelectrolyte Theory of the Gene was first proposed by Steven A. Benner and Daniel Hutter in 2002 and has largely remained a theoretical framework astrobiologists have used to think about how life may be detected throughout the universe. This theory was later linked by Benner (Benner 2017, 2023a) to Erwin Schrödinger's view of the gene as an aperiodic crystal (what is life?) to make a robust generalized theory of a genetic biopolymer.

Benner (Benner and Hutter 2002; Benner 2017, 2023b), and others who build on his work, have proposed methods for how to concentrate and identify genetic biopolymers on other planets using electrophoresis.

Although few have tested the Polyelectrolyte Theory of the Gene, in recent years lab experiments have challenged the universality of this theory (Brown and Brown 2019).  

The physical structure of polyelectrolytes

A polyelectrolyte is a polymer with repeating electrically charged units {create image}. In the context of the Polyelectrolyte Theory of the Gene, this polyelectrolyte is a biopolymer with a repeating charged unit, similar to the genetic biopolymer in modern biology, DNA. RNA may also be considered a genetic biopolymer. Although RNA does not act as a genetic biopolymer in modern biology, except in the case of some viruses such as coronavirus and HIV (Harwig et al. 2015; Brant et al. 2021), the RNA World hypothesis suggests that RNA may have preceded DNA as life’s first genetic biopolymer (Pavlinova et al. 2023). DNA and RNA nucleotide building blocks are connected by negatively charged phosphate groups; these phosphodiester linkages create the repeated negative charges on the molecule’s backbone that give DNA and RNA their polyelectrolyte nature (citation).

Polyelectrolytes in the context of genetic biopolymers

Fundamentally, Darwinian evolution can be summarized as "descent with modification." In other words, Darwinian evolution requires a unit of inheritance capable of imperfect replication, occasionally resulting in a new unit of replication slightly different that from the unit it was copied from, yet still possessing the ability to be replicated(Gregory 2009). This creates the variation on which evolution can act; and to act, evolution needs a selecting environment to winnow that pool of variants to their most evolutionarily fit forms. The Polyelectrolyte Theory of the Gene attempts to understand modern biology’s inheritable unit, the genetic biopolymer DNA, at a basic generalizable level that may be applied to a theoretical framework of life that can be used to search for it throughout the universe. In 2002 Steven A. Benner and Daniel Hutter identified the repeated negative charges in DNA's phosphodiester linkages as crucial to its function as a genetic biopolymer and a generalizable requirement for all water-dissolved genetic biopolymers to undergo Darwinian evolution anywhere in the Cosmos (Benner and Hutter 2002).

This theory works in tandem with the view of the gene as an “Aperiodic Crystal” as proposed by Erwin Schrödinger’s book “What is Life?” published in 1944 (Schrodinger 1944). An aperiodic crystal, as Schrödinger describes it, has a discrete set of molecules in a non-repeating arrangement, like how DNA is composed of the discrete nucleobases (A, T, C, and G) which are arranged based on the information they encode, not in any repeated format. While this idea of an “Aperiodic Crystal” was not initially linked to the polyelectrolyte theory of a gene, Benner, in later work connects the two to develop a more robust generalized view of the unit of genetic inheritance (Benner 2017, 2023a).

Polyelectrolytes remain physically uniform regardless of the information encoded

In biochemistry, the structure of a biomolecule dictates its function, and therefore changes in structure cause changes in function (“2.3” 2017). To work as a unit of inheritance, the genetic biopolymer must maintain shape and therefore physical and chemical consistency regardless of the information that the structure encodes. DNA is such a molecule. No matter what the nucleic acid sequence is, DNA maintains a consistent double helix structure and therefore the consistent physical properties that allow it to remain dissolved in water and be replicated by cellular machinery. For example, thymidine nucleotides (T) are very soluble in water while guanosine nucleotides (G) are insoluble; however, an oligonucleotide composed of only thymine and one composed of only guanine has the same structure and physical properties (Benner 2023b). If changes in nucleic acid sequence, which encodes the genetic information, change the physical properties of DNA, these changes could break down the mechanism by which DNA replicates. This physical uniformity is very rare in nature. Take another biopolymer, for example, proteins. The nucleic acid sequence in DNA codes for the sequence of amino acids that make up proteins. A change to even a single amino acid in the primary sequence of a protein can completely change the physical properties of that protein. For example, the sick-cell trait is caused by a single mutation of an adenine to a thymine at the 17th nucleic acid of the hemoglobin gene causing a switch from a glutamic acid to a valine. This completely changes the three-dimensional structure of hemoglobin and thus changes the physical properties of hemoglobin that lead to the sickle-cell trait (citation). The Polyelectrolyte Theory of the Gene reasons that DNA can maintain its shape regardless of mutations because the negative charges on the phosphate backbone dominate the physical interactions of the molecule to such a degree that changes in the nucleic acid sequence, the encoded information, do not affect the overall physical behavior of the molecule.

Proteins are sensitive to changes in amino acid sequence because the 20 different amino acid side chains form bonds and partial bonds with each other (citation). In addition, the protein backbone has a dipole (having partial positive and partial negative ends) which can further create interactions within the molecule. These side-chain and backbone interactions are sensitive to changes in environmental and amino acid sequence. Because of the sensitivity of the physical nature of proteins to changes in amino acid sequence, it is unlikely they could act as a genetic biopolymer (Benner and Hutter 2002).  

Benner and Hutter initially described this property of DNA as being capable of surviving modifications in constitution without loss of properties essential for replication (COSMIC-LOPER). It should be noted that although RNA is often described as a genetic biopolymer because of its theorized role as life’s first unit of inheritance, it is not entirely COSMIC-LOPER. RNA, especially sequences high in guanine (G), is capable of folding and performing enzyme-type chemistry (Talini et al. 2011). Folding in G-rich RNA sequences prevents the templating ability of RNA and thus its ability to be replicated in an RNA-world scenario (Benner and Hutter 2002).  

Repeated ionic charges increase solubility in water

The repeated negative charges increase the solubility of DNA and RNA in water. Because ionic charges are highly soluble in water, having ionic charges on the molecule's backbone increases the molecule's solubility (Benner 2010). If the backbone of a hypothetical genetic biopolymer were linked together in a non-ionic fashion, the solubility of the whole molecule would decrease.

Repeated ionic charges promote Watson-Crick base pairing specificity

The repeated negative charges of the DNA backbone repel each other, preventing interactions both within and between DNA strands. This repulsion promotes specific interactions along the Watson-Crick 'edge' of the nucleobases, promoting Watson-Crick base pairing—A pairs with T and C pairs with G—with specificity (Benner and Hutter 2002).

Repeated ionic charges prevent folding

The repeated negative charges on the backbone keep DNA and RNA molecules from folding and allow them to act as templates. In water, molecules take on a conformation that is the most energetically favorable, meaning it has the lowest Gibbs free energy (Voet D, Voet JG, Pratt CW (2016). Principles of Biochemistry (Fifth ed.). Wiley). This configuration maximizes favorable interactions (hydrogen bonding, positive-negative charge interactions, van der Waals interactions) and minimizes unfavorable interactions (ie hydrophilic-hydrophobic interactions and like charge interactions). In the case of double-stranded DNA and RNA, the most energetically favorable form is the linear double helix configuration because it maximizes interactions between the base pairs and between the negatively charged backbone and the surrounding water molecules while minimizing interactions between the negatively charged phosphodiester linkages of the backbone. If the double-stranded DNA or RNA molecule folded it would have into exchange favorable water-backbone interaction for unfavorable backbone-backbone interactions. A biopolymer without an ionically charged backbone, like proteins, would not produce unfavorable backbone-backbone interaction during folding and thus would readily fold and aggregate. This inherent tendency towards linearity improves DNA’s ability to act as a template for replication (Benner and Hutter 2002). It should be noted that single-stranded RNA can and does fold in modern biology, especially when it has a G-rich sequence (Benner and Hutter 2002).  

Lab experiments

Lab experiments conducted with non-electrolyte analogs of DNA and RNA initially inspired Benner and Hutton to publish on the Polyelectrolyte Theory of the Gene (Benner 2023a). During the late ‘80s and '90s, scientists developed synthetic DNA-like molecules to bind to and silence unwanted mRNA gene products as a way to treat disease (Crooke 2000; Benner and Hutter 2002). As part of this exploratory research, researchers developed a variety of non-electrolyte RNA and DNA analogs that would be able to cross the cell membrane, which DNA and RNA are incapable of doing because of their charged backbone. One of these analogs substituted a sulphone (SO₂) for the natural phosphodiester (PO₂⁻) linkage(fig). While initial experiments showed the sulphone analog to have very similar properties to DNA as a dimer, two nucleotides linked together, when longer sulphone analogs were synthesized they folded, lost Watson-Crick base pair specificity, and had dramatic changes in physical properties on small changes in nucleic acid sequence. The reduction in the quality of the traits that make DNA a good genetic molecule was seen with all the nonionic linkers that were tested as of 2002 (Benner and Hutton ref 16). The closest non-electrolyte analog to maintain the qualities of DNA was the polyamide-linked nucleic acid analog (PNA) that replaced the phosphodiester linkage of DNA with an uncharged N-(2-aminoethyl)glycine linkage (fig). Even Benner and Hutter questioned if PNA might disprove their polyelectrolyte hypothesis; however, even though PNA maintained the qualities of DNA up to a length of 20 nucleotides, beyond that length the molecules started to lose Watson-Crick base pair specificity, aggregated, and become sensitive to changes in nucleic acid sequence (Benner and Hutter 2002).

Lab experiments that challenge The Polyelectrolyte Theory of the Gene

In 2019, a group led by Philipp Holliger at Cambridge developed non-electrolyte P-alkylphosphonate nucleic acids (phNA) DNA analogs that were able to undergo templated synthesis and directed evolution (Arangundy-Franklin et al. 2019). The phNA analogs substituted the charged oxygen on DNA’s phosphate backbone with an uncharged methyl or ethyl group (fig). While other DNA analogs have been shown to undergo templated synthesis and directed evolution, this discovery was the first time a non-electrolyte DNA analog had been shown to have these properties and the first time the Polyelectrolyte Theory of the Gene had been challenged in the lab (Brown and Brown 2019). However, it should be noted that template-directed synthesis of phNA was only performed up to a length of 72 nucleotides (Arangundy-Franklin et al. 2019).

The Polyelectrolyte Theory of the Gene as an Agnostic Biosignature

From its inception, the polyelectrolyte theory of a gene has been put in the context of searching for life in the universe. This theory, combined with Schrödinger's view of a gene as an aperiodic crystal, provides a potential agnostic biosignature (Benner 2023a), a sign of life that does not pre-suppose any biochemistry (Kaufman 2019). In other words, a generalized view of life that should hold anywhere in the universe because the polyelectrolyte and aperiodic crystal theory of the gene are based on the universal laws of chemistry and physics (Benner 2023a).

Since the theorized genetic polyelectrolyte biomolecules could be charged either positively or negatively, as in the case of DNA and RNA, they can be concentrated in water with an electric field using electrophoresis or electrodialysis. This hypothetical concentration device has been called an agnostic life-finding device (Benner 2023). Similar to how electrophoresis works to separate DNA molecules, negatively charged molecules, like DNA or RNA, would be attracted to a positively charged anode, and positively charged genetic biomolecule would be attracted to a negatively charged cathode (Benner 2023b).  

Once the polyelectrolyte biomolecule has been concentrated, Benner suggests the molecules be tested for size and shape uniformity. In addition they should be tested for the usage of limited number of building blocks arranged in a non-repeating fashion, an aperiodic crystal structure (Benner 2023). Benner has suggested that this could be done using matrix-assisted laser desorption ionization (MALDI) paired with an orbitrap high-resolution mass spectrometer (Špaček and Benner 2022; Benner 2023b). Another suggested approach has been to use nanopore sequencing technology, although questions of whether the solar radiation experienced during transit and on-site would affect the functionality of the device remain (Sutton et al. 2019). While space agencies have yet to use any of these proposed systems for agnostic life detection, they may be used in the future for Mars (Špaček and Benner 2022), Enceladus (Benner 2017), and Europa (Sutton et al. 2019) exploration.

Despite the Polyelectrolyte Theory of the Gene and the aperiodic crystal view of the gene being described as agnostic biosignatures, these theories are terra-, or earth-life, centric. It is unknown what life on another world might be, while it is often stated that life of any kind needs biomolecules and water, this may not be true (Benner 2010).    

Incompatibility with the RNA World hypothesis

Main article: RNA World

The RNA World hypothesis is a popular hypothesis among origin-of-life scientists. It proposes life emerged as a single biopolymer that acted both as the genetic unit of inheritance as well as the catalyst that could perform replication (Pavlinova et al. 2023). This dual role a genetic biopolymer would have to play in an RNA World-like scenario is largely incompatible with the Polyelectrolyte Theory of the Gene. One of the pillars supporting the Polyelectrolyte Theory of the Gene claims that the repeated negative charges on the backbone prevent the biopolymer from folding, but for a single biopolymer system to work, that biopolymer would have to be capable of folding to perform catalysis and remain linearized to act as a template for replication. In addition, this biopolymer would need to be sensitive to changes in encoded information so it could explore evolutionary space and evolve fitter catalysts, but be insensitive to changes in encoded information to maintain the physical properties required for replicated (Benner and Hutter 2002).