Alan Herbert

Questions arise about how well early bacterial models of gene regulation generalize to eukaryotes and whether metazoan repetitive sequences are merely genomic spandrels. The repeat RNAs play multiple roles in the cell through sequence-specific interactions with proteins and other RNAs. Other repeat subsets, called flipons, adopt non-B-DNA conformations under physiological conditions without any sequence change or backbone cleavage. Other low-complexity sequences encode intrinsically disordered regions (IDRs) of proteins, promoting the formation of membrane-less condensates with specific cellular functions. Here, I propose that metazoan repeats enable a fast readout of genetic information by facilitating massively parallel searches of large genomes, far exceeding what diffusion alone can achieve. The locally transcribed repeats and flipon subsets seed condensates that display information about local DNA microstates on their surfaces, creating a 2-dimensional holographic hash of their contents. The hash enables transcription factors to rapidly discover critical cognate binding sites, facilitating rapid cellular responses to perturbation. The information displayed on condensate surfaces is constantly updated by both outward- and inward-directed processes. The rewrite further optimizes responses and increases the phenotypic variability on which natural selection acts. Applying the Principle of Least Action to the Lagrangian demonstrates that this strategy minimizes search times across arbitrary volumes.

Encoding genetic information involves both structure and sequence. Non-protein-coding (NCR) RNAs modulate host immune responses by guiding and assembling stress-specific protein effectors, with flipons enabling dynamic transitions between states. Small NCRs transcribed by both RNA Polymerases 2 and 3 normally modulate these pathways. The miRNAs, tRNA fragments, mitrons, piRNAs, and endo-small-interfering RNA products target the guide effector complexes to their targets. These NCRs filter the subset of RNAs that are translated, a process modulated by other NCRs that fine-tune RNA stability, localization, and kinetics through the modifications they direct. When these RNAs, or their targets, are dysregulated, host interferon responses are activated through the accumulation of uncapped triphosphorylated RNAs, dsRNAs, or flipon-encoded Z-RNAs. These responses are initially suppressed to avoid attacks on self-RNAs, but above a threshold, highly immunogenic pathways are unleashed. These same responses resist viral and other infections without pathogen-specific effectors or prior exposure. They also eliminate dysfunctional cells. These universal responses, when unresolved, contribute to a range of pathologies, increasing cancer, inflammatory diseases, and senescence. The review focuses on recent findings showing how these integrated host responses are driven by, and exploit, the retrotransposon invasion of animal genomes.

The p150 isoform of the double-stranded RNA editing enzyme ADAR1 binds Z-DNA and Z-RNA through the conserved winged helix-turn-helix Zα domain. Here, we describe an inverse computational design strategy to map protein interactors of Zα. We used RFdiffusion and ProteinMPNN to generate ∼10,000 synthetic binders optimized for the Zα recognition surface, then used their sequences as structural templates for BLASTp searches against the human proteome. Multi-stage screening of ∼1,200 candidate regions from 298 proteins via ColabFold pDockQ identified 79 candidates for high-resolution AlphaFold3 modeling, which revealed the m6A reader YTHDC1 as the top-ranked interactor. AlphaFold3 predicts that a glutamate-rich poly-E disordered region of YTHDC1 (residues 199–254) docks into the basic recognition pocket of Zα through a charge-complementary mechanism that mimics the phosphate backbone of Z-RNA. Microsecond molecular dynamics simulations confirmed stability of the binary ADAR1p150–YTHDC1 complex, with the Zα–poly-E interface maintaining RMSD below 3 Å throughout. Ternary complex simulations showed that dsRNA acts as a co-anchoring scaffold stabilizing simultaneous engagement of both proteins in a catalytically dormant conformation. YTHDC1 localizes to transcription-associated YT bodies where nascent RNAs undergo m6A modification and negative supercoiling promotes Z-DNA formation, suggesting that YTHDC1 recruits ADAR1p150 to promote editing of intron-containing substrates prior to splicing. Short Statement: ADAR1 can edit RNAs after they are made, changing the message they carry and removing double-stranded RNAs that can activate inflammatory responses. Using AI-driven protein design, we discovered that ADAR1 physically interacts with YTHDC1, a protein that recognizes newly made self-RNAs. The interaction allows ADAR to edit RNAs as they are made. This unexpected connection between two fundamental RNA modification systems opens new avenues for understanding and potentially targeting autoimmune disorders and cancers driven by the mis-editing of RNA transcripts.

While it is well established that the Zα domains of ADAR1 and ZBP1 proteins bind Z-form-prone nucleic acids (Z-NAs), it has also been shown that the Zα domain of ADAR1 binds DNA G-quadruplexes (GQs). However, no binding partner of the structurally homologous Zβ domain of ADAR1 has been identified to date. Based on AlphaFold and molecular dynamics simulations, it has recently been suggested that Zβ of ADAR1 targets its substrate by recognizing GQs. Here, we provide the first experimental evidence for Zβ binding to select GQ RNA and DNA in vitro, with structural specificity and nanomolar affinity. We also demonstrate that the Zα domains of ZBP1 and ADAR1 bind to both DNA and RNA GQs with similar affinity. These findings extend the range of potential functional roles for these proteins and open new hypotheses for testing in cells.

Unusual DNA structures and misfolded proteins: Alan Herbert, Founder and President of InsideOutBio, discusses the evolution of biological concepts related to DNA and protein structures, highlighting how traditional models have evolved due to new data and discoveries. Much of modern life depends on precisely engineered, highly reliable machines. Such concepts originally shaped our view of biology, beginning with the famous ‘watchmaker’, whom William Paley anointed in 1802 as the creator of life. This concept of ideals was continued by the lock-and-key model of antibody-bound toxins proposed by Ehrlich in 1900, and by the modern view that proteins control the readout of genetic information through the highly specific recognition of nucleotide sequences. All these proposals assume that the world as we see it today has always been this way. They ignore the past worlds that existed before these innovations arose. Each model was based on limited data or on the selective use of that data. They failed when the volume of contradictory information generated by technological advances was overwhelming. These transitions occurred as the Linnean catalog of life forms in the 1700s expanded, as new chemical elements were discovered in the 1800s, and as the zoo of exotic subatomic particles was discovered in the early 1900s. A similar transformation is now occurring in biology, where the old concepts of just a few years ago no longer align with the data.

Triplexes (TRX) are a class of flipons that can form due to the interaction of RNA with B-DNA. While many proteins have been proposed to bind triplexes, structural models of these interactions do not exist. Here, I present AlphaFold V3 (AF3) models that reveal interactions between the high-mobility group protein B1 (HMGB1), HNRNPU (SAF-A), TP53, ARGONAUTE (AGO), and REL domain proteins. The TRXs result from the sequence-specific docking of RNAs to DNA via Hoogsteen base pairing. The RNA and DNA strands in apolar TRX are oriented in the opposite 5′ to 3′ direction, while copolar TRX have RNA and DNA strands pointing in the same 5′ to 3′ direction. TRXs can incorporate different RNA classes, including long noncoding RNAs (lncRNAs), short RNAs, such as miRNAs, piRNAs, and tRNAs, nascent RNA fragments, and non-canonical base triplets. Many pathways regulated by TRX formation have evolved to constrain retroelements (EREs), which are both an existential threat to the host and a source of genotypic variation. TRXs help set the boundaries of active chromatin, repressing the expression of most EREs, while depending on other flipons to modulate cellular programs. The TRXs help nucleate folding of intrinsically disordered proteins.

The role of alternative nucleic acid structures (ANS) in biology is an area of increasing interest. These non-canonical structures include the Z-DNA and Z-RNA duplexes (ZNA), the three-stranded triplex, the four-stranded G-quadruplex (GQ), and i-motifs. Previously, the biological relevance of ANS was dismissed. Their formation in vitro often required non-physiological conditions, and there was no genetic evidence for their function. Further, structural studies confirmed that sequence-specific transcription factors (TFs) bound B-DNA. In contrast, ANS are formed dynamically by a subset of repeat sequences, called flipons. The flip requires energy, but not strand cleavage. Flipons are enriched in promoters where they modulate transcription. Here, computational modeling based on AlphaFold V3 (AF3), under optimized conditions, reveals that known B-DNA-binding TFs also dock to ANS, such as ZNA and GQ. The binding of HLH and bZIP homodimers to Z-DNA is promoted by methylarginine modifications. Heterodimers only bind preformed Z-DNA. The interactions of TFs with ANS likely enhance genome scanning to identify cognate B-DNA-binding sites in active genes. Docking of TF homodimers to Z-DNA potentially facilitates the assembly of heterodimers that dissociate and are stabilized by binding to a cognate B-DNA motif. The process enables rapid discovery of the optimal heterodimer combinations required to regulate a nearby promoter.

Expansion of d(GGGGC)_n repeat in the C9ORF72 gene is causal for Amyotrophic Lateral Sclerosis (ALS) and Frontal Temporal Dementia (FTD). Proposed mechanisms include Repeat-Associated Non-AUG translation or the formation of G-quadruplexes (GQ) that disrupt translation, induce protein aggregation, sequester RNA processing factors, or alter RNA editing. Here, I show, using AlphaFold V3 (AF3) modeling, that the TAR DNA-binding protein (TDP-43) docks to a complex of GQ and hemin. TDP-43 methionines lie over hemin and likely squelch the generation of superoxide by the porphyrin-bound Fe. These TDP-43 methionines are frequently altered in ALS patients. Tau protein, a variant of which causes ALS, also binds to GQ and heme and positions methionines to detoxify peroxides. Full-length Tau, which is often considered prone to aggregation and a prion-like disease agent, can bind to an array composed of multiple GQs as a fully folded protein. In ALS and FTD, loss-of-function variants cause an uncompensated surplus of superoxide, which sparks neuronal cell death. In Alzheimer’s Disease (AD) patients, GQ and heme complexes bound by β-amyloid 42 (Aβ4) are also likely to generate superoxides. Collectively, these neuropathologies have proven difficult to treat. The current synthesis provides a framework for designing future therapeutics.

1 While it is well established that the Zα domains of ADAR1 and ZBP1 proteins bind Z-form-prone nucleic acids (Z-NAs), it has also been shown that the Zα domain of ADAR1 binds DNA G-Quadruplexes (GQ). However, no binding partner of the structurally homologous Zβ domain of ADAR1 has been identified to date. Based on AlphaFold and molecular dynamics simulations, it has recently been suggested that the Zβ domain of ADAR1 targets its substrate by recognizing GQs. Here, we provide the first experimental evidence for Zβ domain binding to select G-quadruplex RNA and DNA in vitro , with structural specificity and low micromolar affinity. We also demonstrate that the Zα domains of ZBP1 bind to both DNA and RNA GQs with similar affinity. These findings extend the range of potential functional roles for these proteins and open new hypotheses for testing in cells.

Sequences called flipons can adopt discrete, alternative nucleic acid conformations, such as the left-handed Z-DNA and Z-RNA double helices (referred to collectively as ZNA), and the four-stranded RNA and DNA G-quadruplexes. Each flipon conformation encodes different information. For example, the base-specific interactions of proteins with B-DNA enable sequence-specific recognition. In contrast, the higher energy Z-DNA and G-quadruplexes facilitate the speedy scanning of chromosomes to locate active regions of the genome. Results synthesized from small-scale benchside and large-scale computational experimental approaches provide compelling evidence that zinc-finger protein domains (ZFDs) not only engage in base-specific recognition of B-DNA, but also bind directly to Z-DNA and G-quadruplexes. The findings address the long-standing speed–stability paradox of how high-affinity ZFPs with multiple zinc fingers can rapidly localize to a specific binding site. The energy gap between different DNA interaction modes enables fast off-rates during the scanning of Z-DNA for cognate binding sites, and a slow off-rate following engagement of the B-DNA conformer. ZFPs represent the most prominent human transcription factor family with 804 annotated members. The coevolution of flipons and ZFP enhances suppression of retroelements and enables rapid, context-specific responses. ZNA and GQ binding proteins are consequently more frequent in the proteome than currently conceded.