Foundation for Applied Molecular Evolution

Under the RNA first hypothesis for the origin of life, RNA that emerges from prebiotic chemistry performed both catalytic and informational roles. Ribose is the only sugar in RNA; thus, many have sought to understand how ribose might have emerged on a prebiotic Earth. Ribose can be formed from formaldehyde with small amounts of glycolaldehyde by formose-like processes. However, under the strongly alkaline conditions of the reaction, ribose is consumed as it is formed. Here, we show that borate significantly decreases the consumption of the ribose formed in the formose reaction, which results in higher amounts of ribose that remained as the reaction progressed. Given a longer timescale of prebiotic chemical reactions governed by geological processes, borate-rich environments could have contributed to accumulating ribose on prebiotic Earth. Borate could be available on proto-continents and is known to contribute to ribonucleoside synthesis, ribose 5-phosphate synthesis, and nucleoside phosphorylation. Therefore, such environments might have promoted chemical reactions to RNA.

"ALternative Isoinformational ENgineered" (ALIEN) DNA is a biomimetic polymer composed of four entirely anthropogenic nucleotides. These alternative nucleosides form base pairs orthogonal to canonical bases and fold into the familiar B-form DNA double-helix, endowing ALIEN DNA with valuable biotechnological applications. The ability to sequence ALIEN DNA is essential for its continued development. However traditional sequencing approaches rely on chemical recognition of ACGT-DNA and cannot be easily adapted to ALIEN DNA. Here we demonstrate de novo nanopore sequencing of DNA comprised entirely of the four anthropogenic DNA bases. We show direct, label-free, single-molecule sequencing of such nucleic acids without the requirements of fluorescent labels, transliteration, amplification, or enzymatic synthesis. This paves the way for routine, accessible, and high-accuracy sequencing of DNA beyond A, C, G, and T.

This Perspective addresses the unresolved, and stillhotly contested,question of how enzymes transition from stable enzyme–substrate(ES) complexes to successful, femtosecond barrier crossings. By extendingMarcus theory to enzyme-catalyzed reactions, we argue that environmentalreorganization of the protein scaffold, together with associated watermolecules, achieves the intersection of reactant and product potentialenergy surfaces. After discussing the experimentally demonstratedimportance of reduced activation enthalpy in enzyme-catalyzed transformations,we describe new methodologies that measure the temperature dependenceof (i) time-averaged hydrogen/deuterium exchange into backbone amidesand (ii) time-dependent Stokes shifts to longer emission wavelengthsin appended chromophores at the protein/water interface. These methodsnot only identify specific pathways for the transfer of thermal energyfrom solvent to the reacting bonds of bound substrates but also suggestthat collective thermally activated protein restructuring must occurvery rapidly (on the ns–ps time scale) over long distances.Based on these findings, we introduce a comprehensive model for howbarrier crossing takes place from the ES complex. This exploits thestructural preorganization inherent in protein folding and subsequentconformational sampling, which optimally positions essential catalyticcomponents within ES ground states and correctly places reactive bondsin the substrate(s) relative to embedded energy transfer networksconnecting the protein surface to the active site. The existence ofthese anisotropic energy distribution pathways introduces a new dimensioninto the ongoing quest for improved de novo enzymedesign.

DNA-joining by ligase and polymerase enzymes has provided the foundational tools for generating recombinant DNA and enabled the assembly of gene and genome-sized synthetic products. Xenobiotic nucleic acid (XNA) analogues of DNA and RNA with alternatives to the canonical bases, so-called ‘unnatural’ nucleobase pairs (UBP-XNAs), represent the next frontier of nucleic acid technologies, with applications as novel therapeutics and in engineering semi-synthetic biological organisms. To realise the full potential of UBP-XNAs, researchers require a suite of compatible enzymes for processing nucleic acids on a par with those already available for manipulating canonical DNA. In particular, enzymes able to join UBP-XNA will be essential for generating large assemblies and also hold promise in the synthesis of single-stranded oligonucleotides. Here, we review recent and emerging advances in the DNA-joining enzymes, DNA polymerases and DNA ligases, and describe their applications to UBP-XNA manipulation. We also discuss the future directions of this field which we consider will involve two-pronged approaches of enzyme biodiscovery for natural UBP-XNA compatible enzymes, coupled with improvement by structure-guided engineering.

We present the draft genome of the bacterium Thermoanaerobacter thermohydrosulfuricus strain AK152, a thermophilic, endospore-spore-forming, anaerobe isolated from a hot spring in Grensdalur, in Southwestern Iceland. This assembled genome will lay the foundation for identifying the carboxylic and amino acid fermentation pathways, suggesting biotechnological applications for this strain.

Adding synthetic nucleotides to DNA increases the linear information density of DNA molecules. Here we report that it also can increase the diversity of their three-dimensional folds. Specifically, an additional nucleotide (dZ, with a 5-nitro-6-aminopyridone nucleobase), placed at twelve sites in a 23-nucleotides-long DNA strand, creates a fairly stable unimolecular structure (that is, the folded Z-motif, or fZ-motif) that melts at 66.5 °C at pH 8.5. Spectroscopic, gel and two-dimensional NMR analyses show that the folded Z-motif is held together by six reverse skinny dZ−:dZ base pairs, analogous to the crystal structure of the free heterocycle. Fluorescence tagging shows that the dZ−:dZ pairs join parallel strands in a four-stranded compact down–up–down–up fold. These have two possible structures: one with intercalated dZ−:dZ base pairs, the second without intercalation. The intercalated structure would resemble the i-motif formed by dC:dC+-reversed pairing at pH ≤ 6.5. This fZ-motif may therefore help DNA form compact structures needed for binding and catalysis.

Pathological hyperphosphorylation and aggregation of microtubule-associated Tau protein contribute to Alzheimer’s Disease (AD) and other related tauopathies. Currently, no cure exists for Alzheimer’s Disease. Aptamers offer significant potential as next-generation therapeutics in biotechnology and the treatment of neurological disorders. Traditional aptamer selection methods for Tau protein focus on binding affinity rather than interference with pathological Tau. In this study, we developed a new selection strategy to enrich DNA aptamers that bind to surviving monomeric Tau protein under conditions that would typically promote Tau aggregation. Employing this approach, we identified a set of aptamer candidates. Notably, BW1c demonstrates a high binding affinity (Kd = 6.6 nM) to Tau protein and effectively inhibits arachidonic acid (AA)-induced Tau protein oligomerization and aggregation. Additionally, it inhibits GSK3β-mediated Tau hyperphosphorylation in cell-free systems and okadaic acid-mediated Tau hyperphosphorylation in cellular milieu. Lastly, retro-orbital injection of BW1c tau aptamer shows the ability to cross the blood brain barrier and gain access to neuronal cell body. Through further refinement and development, these Tau aptamers may pave the way for a first-in-class neurotherapeutic to mitigate tauopathy-associated neurodegenerative disorders.

Aptamers developed using in vitro Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technology are single-stranded nucleic acids 10-100 nucleotides in length. Their targets, often with specificity and high affinity, range from ions and small molecules to proteins and other biological molecules as well as larger systems, including cells, tissues, and animals. Aptamers often rival conventional antibodies with improved performance, due to aptamers' unique biophysical and biochemical properties, including small size, synthetic accessibility, facile modification, low production cost, and low immunogenicity. Therefore, there is sustained interest in engineering and adapting aptamers for many applications, including diagnostics and therapeutics. Recently, aptamers have shown promise as early diagnostic biomarkers and in precision medicine for neurodegenerative and neurological diseases. Here, we critically review neuro-targeting aptamers and their potential applications in neuroscience research, neuro-diagnostics, and neuro-medicine. We also discuss challenges that must be overcome, including delivery across the blood-brain barrier, increased affinity, and improved in vivo stability and in vivo pharmacokinetic properties.

The 4-letter DNA alphabet (A, T, G, C) as found in Nature is an elegant, yet non-exhaustive solution to the problem of storage, transfer, and evolution of biological information. Here, we report on strategies for both writing and reading DNA with expanded alphabets composed of up to 12 letters (A, T, G, C, B, S, P, Z, X, K, J, V). For writing, we devise an enzymatic strategy for inserting a singular, orthogonal xenonucleic acid (XNA) base pair into standard DNA sequences using 2′-deoxy-xenonucleoside triphosphates as substrates. Integrating this strategy with combinatorial oligos generated on a chip, we construct libraries containing single XNA bases for parameterizing kmer basecalling models for commercially available nanopore sequencing. These elementary steps are combined to synthesize and sequence DNA containing 12 letters – the upper limit of what is accessible within the electroneutral, canonical base pairing framework. By introducing low-barrier synthesis and sequencing strategies, this work overcomes previous obstacles paving the way for making expanded alphabets widely accessible.

A number of biologists and philosophers have noted the diversity of interpretations of evolvability in contemporary evolutionary research. Different clusters of research defined by cocitation patterns or shared methodological orientation sometimes concentrate on distinct conceptions of evolvability. We examine five different activities where the notion of evolvability plays conceptual roles in evolutionary biological investigation: setting a research agenda, characterization, explanation, prediction, and control. Our analysis of representative examples demonstrates how different conceptual roles of evolvability are quasi-independent and yet exhibit important relationships across scientific activities. It also provides us with the resources to detail two distinct strategies for how evolvability can help to synthesize disparate areas of research and thereby potentially serve as a unifying concept in evolutionary biology.