Abiotic Ribose Synthesis Under Aqueous Environments with Various Chemical Conditions.

Ribose is the defining sugar in ribonucleic acid (RNA), which is often proposed to have carried the genetic information and catalyzed the biological reactions of the first life on Earth. Thus, abiological processes that yield ribose under prebiotic conditions have been studied for decades. However, aqueous environments required for the formation of ribose from materials available in quantity under geologically reasonable models, where the ribose formed is not immediately destroyed, remain unclear. This is due in large part to the challenge of analysis of carbohydrates formed under a wide range of aqueous conditions. Thus, the formation of ribose on prebiotic Earth has sometimes been questioned. We investigated the quantitative effects of pH, temperature, cation, and the concentrations of formaldehyde and glycolaldehyde on the synthesis of diverse sugars, including ribose. The results suggest a range of conditions that produce ribose and that ribose could have formed in constrained aquifers on prebiotic Earth.

Enzyme-assisted high throughput sequencing of an expanded genetic alphabet at single base resolution.

With just four building blocks, low sequence information density, few functional groups, poor control over folding, and difficulties in forming compact folds, natural DNA and RNA have been disappointing platforms from which to evolve receptors, ligands, and catalysts. Accordingly, synthetic biology has created "artificially expanded genetic information systems" (AEGIS) to add nucleotides, functionality, and information density. With the expected improvements seen in AegisBodies and AegisZymes, the task for synthetic biologists shifts to developing for expanded DNA the same analytical tools available to natural DNA. Here we report one of these, an enzyme-assisted sequencing of expanded genetic alphabet (ESEGA) method to sequence six-letter AEGIS DNA. We show how ESEGA analyses this DNA at single base resolution, and applies it to optimized conditions for six-nucleotide PCR, assessing the fidelity of various DNA polymerases, and extending this to AEGIS components with functional groups. This supports the renewed exploitation of expanded DNA alphabets in biotechnology.

Aptamer Technologies in Neuroscience, Neuro-Diagnostics and Neuro-Medicine Development.

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.

Rapid detection of West Nile and Dengue viruses from mosquito saliva by loop-mediated isothermal amplification and displaced probes.

Arthropod-borne viruses are major causes of human and animal disease, especially in endemic low- and middle-income countries. Mosquito-borne pathogen surveillance is essential for risk assessment and vector control responses. Sentinel chicken serosurveillance (antibody testing) and mosquito pool screening (by RT-qPCR or virus isolation) are currently used to monitor arbovirus transmission, however substantial time lags of seroconversion and/or laborious mosquito identification and RNA extraction steps sacrifice their early warning value. As a consequence, timely vector control responses are compromised. Here, we report on development of a rapid arbovirus detection system whereby adding sucrose to reagents of loop-mediated isothermal amplification with displaced probes (DP-LAMP) elicits infectious mosquitoes to feed directly upon the reagent mix and expectorate viruses into the reagents during feeding. We demonstrate that RNA from pathogenic arboviruses (West Nile and Dengue viruses) transmitted in the infectious mosquito saliva was detectable rapidly (within 45 minutes) without RNA extraction. Sucrose stabilized viral RNA at field temperatures for at least 48 hours, important for transition of this system to practical use. After thermal treatment, the DP-LAMP could be reliably visualized by a simple optical image sensor to distinguish between positive and negative samples based on fluorescence intensity. Field application of this technology could fundamentally change conventional arbovirus surveillance methods by eliminating laborious RNA extraction steps, permitting arbovirus monitoring from additional sites, and substantially reducing time needed to detect circulating pathogens.

Functional Selection of Tau Oligomerization-Inhibiting Aptamers.

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 GSK3ß-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.

Overcoming resolution attenuation during tilted cryo-EM data collection.

Structural biology efforts using cryogenic electron microscopy are frequently stifled by specimens adopting "preferred orientations" on grids, leading to anisotropic map resolution and impeding structure determination. Tilting the specimen stage during data collection is a generalizable solution but has historically led to substantial resolution attenuation. Here, we develop updated data collection and image processing workflows and demonstrate, using multiple specimens, that resolution attenuation is negligible or significantly reduced across tilt angles. Reconstructions with and without the stage tilted as high as 60° are virtually indistinguishable. These strategies allowed the reconstruction to 3 Å resolution of a bacterial RNA polymerase with preferred orientation, containing an unnatural nucleotide for studying novel base pair recognition. Furthermore, we present a quantitative framework that allows cryo-EM practitioners to define an optimal tilt angle during data acquisition. These results reinforce the utility of employing stage tilt for data collection and provide quantitative metrics to obtain isotropic maps.

A unified Watson-Crick geometry drives transcription of six-letter expanded DNA alphabets by E. coli RNA polymerase.

Artificially Expanded Genetic Information Systems (AEGIS) add independently replicable unnatural nucleotide pairs to the natural G:C and A:T/U pairs found in native DNA, joining the unnatural pairs through alternative modes of hydrogen bonding. Whether and how AEGIS pairs are recognized and processed by multi-subunit cellular RNA polymerases (RNAPs) remains unknown. Here, we show that E. coli RNAP selectively recognizes unnatural nucleobases in a six-letter expanded genetic system. High-resolution cryo-EM structures of three RNAP elongation complexes containing template-substrate UBPs reveal the shared principles behind the recognition of AEGIS and natural base pairs. In these structures, RNAPs are captured in an active state, poised to perform the chemistry step. At this point, the unnatural base pair adopts a Watson-Crick geometry, and the trigger loop is folded into an active conformation, indicating that the mechanistic principles underlying recognition and incorporation of natural base pairs also apply to AEGIS unnatural base pairs. These data validate the design philosophy of AEGIS unnatural basepairs. Further, we provide structural evidence supporting a long-standing hypothesis that pair mismatch during transcription occurs via tautomerization. Together, our work highlights the importance of Watson-Crick complementarity underlying the design principles of AEGIS base pair recognition.

Enzymatic synthesis and nanopore sequencing of 12-letter supernumerary DNA.

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.

DNA Structure Design Is Improved Using an Artificially Expanded Alphabet of Base Pairs Including Loop and Mismatch Thermodynamic Parameters.

We show that in silico design of DNA secondary structures is improved by extending the base pairing alphabet beyond A-T and G-C to include the pair between 2-amino-8-(1'-ß-d-2'-deoxyribofuranosyl)-imidazo-[1,2-a]-1,3,5-triazin-(8H)-4-one and 6-amino-3-(1'-ß-d-2'-deoxyribofuranosyl)-5-nitro-(1H)-pyridin-2-one, abbreviated as P and Z. To obtain the thermodynamic parameters needed to include P-Z pairs in the designs, we performed 47 optical melting experiments and combined the results with previous work to fit free energy and enthalpy nearest neighbor folding parameters for P-Z pairs and G-Z wobble pairs. We find G-Z pairs have stability comparable to that of A-T pairs and should therefore be included as base pairs in structure prediction and design algorithms. Additionally, we extrapolated the set of loop, terminal mismatch, and dangling end parameters to include the P and Z nucleotides. These parameters were incorporated into the RNAstructure software package for secondary structure prediction and analysis. Using the RNAstructure Design program, we solved 99 of the 100 design problems posed by Eterna using the ACGT alphabet or supplementing it with P-Z pairs. Extending the alphabet reduced the propensity of sequences to fold into off-target structures, as evaluated by the normalized ensemble defect (NED). The NED values were improved relative to those from the Eterna example solutions in 91 of 99 cases in which Eterna-player solutions were provided. P-Z-containing designs had average NED values of 0.040, significantly below the 0.074 of standard-DNA-only designs, and inclusion of the P-Z pairs decreased the time needed to converge on a design. This work provides a sample pipeline for inclusion of any expanded alphabet nucleotides into prediction and design workflows.

Nicotinamide Riboside, a Promising Vitamin B3 Derivative for Healthy Aging and Longevity: Current Research and Perspectives.

Many studies have suggested that the oxidized form of nicotinamide adenine dinucleotide (NAD+) is involved in an extensive spectrum of human pathologies, including neurodegenerative disorders, cardiomyopathy, obesity, and diabetes. Further, healthy aging and longevity appear to be closely related to NAD+ and its related metabolites, including nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN). As a dietary supplement, NR appears to be well tolerated, having better pharmacodynamics and greater potency. Unfortunately, NR is a reactive molecule, often unstable during its manufacturing, transport, and storage. Recently, work related to prebiotic chemistry discovered that NR borate is considerably more stable than NR itself. However, immediately upon consumption, the borate dissociates from the NR borate and is lost in the body through dilution and binding to other species, notably carbohydrates such as fructose and glucose. The NR left behind is expected to behave pharmacologically in ways identical to NR itself. This review provides a comprehensive summary (through Q1 of 2023) of the literature that makes the case for the consumption of NR as a dietary supplement. It then summarizes the challenges of delivering quality NR to consumers using standard synthesis, manufacture, shipping, and storage approaches. It concludes by outlining the advantages of NR borate in these processes.

Overcoming Resolution Attenuation During Tilted Cryo-EM Data Collection.

Structural biology efforts using cryogenic electron microscopy are frequently stifled by specimens adopting "preferred orientations" on grids, leading to anisotropic map resolution and impeding structure determination. Tilting the specimen stage during data collection is a generalizable solution but has historically led to substantial resolution attenuation. Here, we develop updated data collection and image processing workflows and demonstrate, using multiple specimens, that resolution attenuation is negligible or significantly reduced across tilt angles. Reconstructions with and without the stage tilted as high as 60° are virtually indistinguishable. These strategies allowed the reconstruction to 3 Å resolution of a bacterial RNA polymerase with preferred orientation. Furthermore, we present a quantitative framework that allows cryo-EM practitioners to define an optimal tilt angle for dataset acquisition. These data reinforce the utility of employing stage tilt for data collection and provide quantitative metrics to obtain isotropic maps.

DNA Structure Design Is Improved Using an Artificially Expanded Alphabet of Base Pairs Including Loop and Mismatch Thermodynamic Parameters.

We show that in silico design of DNA secondary structures is improved by extending the base pairing alphabet beyond A-T and G-C to include the pair between 2-amino-8-(1'-ß-D-2'-deoxyribofuranosyl)-imidazo-[1,2- a ]-1,3,5-triazin-(8 H )-4-one and 6-amino-3-(1'-ß-D-2'-deoxyribofuranosyl)-5-nitro-(1 H )-pyridin-2-one, simply P and Z. To obtain the thermodynamic parameters needed to include P-Z pairs in the designs, we performed 47 optical melting experiments and combined the results with previous work to fit a new set of free energy and enthalpy nearest neighbor folding parameters for P-Z pairs and G-Z wobble pairs. We find that G-Z pairs have stability comparable to A-T pairs and therefore should be considered quantitatively by structure prediction and design algorithms. Additionally, we extrapolated the set of loop, terminal mismatch, and dangling end parameters to include P and Z nucleotides. These parameters were incorporated into the RNAstructure software package for secondary structure prediction and analysis. Using the RNAstructure Design program, we solved 99 of the 100 design problems posed by Eterna using the ACGT alphabet or supplementing with P-Z pairs. Extending the alphabet reduced the propensity of sequences to fold into off-target structures, as evaluated by the normalized ensemble defect (NED). The NED values were improved relative to those from the Eterna example solutions in 91 of 99 cases where Eterna-player solutions were provided. P-Z-containing designs had average NED values of 0.040, significantly below the 0.074 of standard-DNA-only designs, and inclusion of the P-Z pairs decreased the time needed to converge on a design. This work provides a sample pipeline for inclusion of any expanded alphabet nucleotides into prediction and design workflows.

Multiplex Surveillance Kit Using Sweetened Solid Support to Detect Arboviruses Isothermally in Mosquito Saliva from the Field.

Recently reported "displaceable probe" loop amplification (DP-LAMP) architecture has shown to amplify viral RNA from SARS-CoV-2 with little sample processing. The architecture allows signals indicating the presence of target nucleic acids to be spatially separated, and independent in sequence, from the complicated concatemer that LAMP processes create as part of their amplification process. This makes DP-LAMP an attractive molecular strategy to integrate with trap and sampling innovations to detect RNA from arboviruses carried by mosquitoes in the field. These innovations include (a) development of organically produced carbon dioxide with ethylene carbonate as a bait deployable in mosquito trap, avoiding the need for dry ice, propane tanks, or inorganic carbonates and (b) a process that induces mosquitoes to lay virus-infected saliva on a quaternary ammonium-functionalized paper (Q-paper) matrix, where (c) the matrix (i) inactivates the deposited viruses, (ii) releases their RNA, and (iii) captures viral RNA in a form that keeps it stable for days at ambient temperatures. We report this integration here, with a surprisingly simple workflow. DP-LAMP with a reverse transcriptase was found to amplify arboviral RNA directly from Q-paper, without requiring a separate elution step. This capture-amplification-detection architecture can be multiplexed, with the entire system integrated into a device that can support a campaign of surveillance, in the wild outdoors, that reports the prevalence of arboviruses from field-captured mosquitoes.

Synthetic Biology Pathway to Nucleoside Triphosphates for Expanded Genetic Alphabets.

One horizon in synthetic biology seeks alternative forms of DNA that store, transcribe, and support the evolution of biological information. Here, hydrogen bond donor and acceptor groups are rearranged within a Watson-Crick geometry to get 12 nucleotides that form 6 independently replicating pairs. Such artificially expanded genetic information systems (AEGIS) support Darwinian evolution in vitro. To move AEGIS into living cells, metabolic pathways are next required to make AEGIS triphosphates economically from their nucleosides, eliminating the need to feed these expensive compounds in growth media. We report that "polyphosphate kinases" can be recruited for such pathways, working with natural diphosphate kinases and engineered nucleoside kinases. This pathway in vitro makes AEGIS triphosphates, including third-generation triphosphates having improved ability to survive in living bacterial cells. In α-32P-labeled forms, produced here for the first time, they were used to study DNA polymerases, finding cases where third-generation AEGIS triphosphates perform better with natural enzymes than second-generation AEGIS triphosphates.

Assessing Readability of an 8-Letter Expanded Deoxyribonucleic Acid Alphabet with Nanopores.

Chemists have now synthesized new kinds of DNA that add nucleotides to the four standard nucleotides (guanine, adenine, cytosine, and thymine) found in standard Terran DNA. Such "artificially expanded genetic information systems" are today used in molecular diagnostics; to support directed evolution to create medically useful receptors, ligands, and catalysts; and to explore issues related to the early evolution of life. Further applications are limited by the inability to directly sequence DNA containing nonstandard nucleotides. Nanopore sequencing is well-suited for this purpose, as it does not require enzymatic synthesis, amplification, or nucleotide modification. Here, we take the first steps to realize nanopore sequencing of an 8-letter "hachimoji" expanded DNA alphabet by assessing its nanopore signal range using the MspA (Mycobacterium smegmatis porin A) nanopore. We find that hachimoji DNA exhibits a broader signal range in nanopore sequencing than standard DNA alone and that hachimoji single-base substitutions are distinguishable with high confidence. Because nanopore sequencing relies on a molecular motor to control the motion of DNA, we then assessed the compatibility of the Hel308 motor enzyme with nonstandard nucleotides by tracking the translocation of single Hel308 molecules along hachimoji DNA, monitoring the enzyme kinetics and premature enzyme dissociation from the DNA. We find that Hel308 is compatible with hachimoji DNA but dissociates more frequently when walking over C-glycoside nucleosides, compared to N-glycosides. C-glycocide nucleosides passing a particular site within Hel308 induce a higher likelihood of dissociation. This highlights the need to optimize nanopore sequencing motors to handle different glycosidic bonds. It may also inform designs of future alternative DNA systems that can be sequenced with existing motors and pores.

Crystal structures of 'ALternative Isoinformational ENgineered' DNA in B-form.

The first structural model of duplex DNA reported in 1953 by Watson & Crick presented the double helix in B-form, the form that genomic DNA exists in much of the time. Thus, artificial DNA seeking to mimic the properties of natural DNA should also be able to adopt B-form. Using a host-guest system in which Moloney murine leukemia virus reverse transcriptase serves as the host and DNA as the guests, we determined high-resolution crystal structures of three complexes including 5'-CTTBPPBBSSZZSAAG, 5'-CTTSSPBZPSZBBAAG and 5'-CTTZZPBSBSZPPAAG with 10 consecutive unnatural nucleobase pairs in B-form within self-complementary 16 bp duplex oligonucleotides. We refer to this ALternative Isoinformational ENgineered (ALIEN) genetic system containing two nucleobase pairs (P:Z, pairing 2-amino-imidazo-[1,2-a]-1,3,5-triazin-(8H)-4-one with 6-amino-5-nitro-(1H)-pyridin-2-one, and B:S, 6-amino-4-hydroxy-5-(1H)-purin-2-one with 3-methyl-6-amino-pyrimidin-2-one) as ALIEN DNA. We characterized both position- and sequence-specific helical, nucleobase pair and dinucleotide step parameters of P:Z and B:S pairs in the context of B-form DNA. We conclude that ALIEN DNA exhibits structural features that vary with sequence. Further, Z can participate in alternative stacking modes within a similar sequence context as captured in two different structures. This finding suggests that ALIEN DNA may have a larger repertoire of B-form structures than natural DNA. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.

Rethinking nucleic acids from their origins to their applications.

Reviewed are three decades of synthetic biology research in our laboratory that has generated alternatives to standard DNA and RNA as possible informational systems to support Darwinian evolution, and therefore life, and to understand their natural history, on Earth and throughout the cosmos. From this, we have learned that: • the core structure of nucleic acids appears to be a natural outcome of non-biological chemical processes probably in constrained, intermittently irrigated, sub-aerial aquifers on the surfaces of rocky planets like Earth and/or Mars approximately 4.36 ± 0.05 billion years ago; • however, this core is not unique. Synthetic biology has generated many different molecular systems able to support the evolution of molecular information; • these alternatives to standard DNA and RNA support biotechnology, including DNA synthesis, human diagnostics, biomedical research and medicine; • in particular, they support laboratory in vitro evolution (LIVE) with performance to generate catalysts at least 104-105 fold better than standard DNA libraries, enhancing access to receptors and catalysts on demand. Coupling nanostructures to the products of LIVE with expanded DNA offers new approaches for disease therapy; and • nevertheless, a polyelectrolyte structure and size regular building blocks are required for any informational polymer to support Darwinian evolution. These features serve as universal and agnostic biosignatures, useful for seeking life throughout the Solar System. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.

Enzyme-Assisted High Throughput Sequencing of an Expanded Genetic Alphabet at Single Base Resolution.

Many efforts have sought to apply laboratory in vitro evolution (LIVE) to natural nucleic acid (NA) scaffolds to directly evolve functional molecules. However, synthetic biology can move beyond natural NA scaffolds to create molecular systems whose libraries are far richer reservoirs of functionality than natural NAs. For example, "artificially expanded genetic information systems" (AEGIS) add up to eight nucleotides to the four found in standard NA. Even in its simplest 6-letter versions, AEGIS adds functional groups, information density, and folding motifs that natural NA libraries lack. To complete this vision, however, tools are needed to sequence molecules that are created by AEGIS LIVE. Previous sequencing approaches, including approaches from our laboratories, exhibited limited performance and lost many sequences in diverse library mixtures. Here, we present a new approach that enzymatically transforms the target AEGIS DNA. With higher transliteration efficiency and fidelity, this Enzyme-Assisted Sequencing of Expanded Genetic Alphabet (ESEGA) approach produces substantially better sequences of 6-letter (AGCTZP) DNA than previous transliteration approaches. Therefore, ESEGA facilitates precise analysis of libraries, allowing 'next-generation deep sequencing' to accurately quantify the sequences of 6-letter DNA molecules at single base resolution. We then applied ESEGA to three tasks: (a) defining optimal conditions to perform 6-nucleotide PCR (b) evaluating the fidelity of 6-nucleotide PCR with various DNA polymerases, and (c) extending that evaluation to AEGIS components functionalized with alkynyl and aromatic groups. No other approach at present has this scope, allowing this work to be the next step towards exploiting the potential of expanded DNA alphabets in biotechnology.

In vitro evolution of ribonucleases from expanded genetic alphabets.

The ability of nucleic acids to catalyze reactions (as well as store and transmit information) is important for both basic and applied science, the first in the context of molecular evolution and the origin of life and the second for biomedical applications. However, the catalytic power of standard nucleic acids (NAs) assembled from just four nucleotide building blocks is limited when compared with that of proteins. Here, we assess the evolutionary potential of libraries of nucleic acids with six nucleotide building blocks as reservoirs for catalysis. We compare the outcomes of in vitro selection experiments toward RNA-cleavage activity of two nucleic acid libraries: one built from the standard four independently replicable nucleotides and the other from six, with the two added nucleotides coming from an artificially expanded genetic information system (AEGIS). Results from comparative experiments suggest that DNA libraries with increased chemical diversity, higher information density, and larger searchable sequence spaces are one order of magnitude richer reservoirs of molecules that catalyze the cleavage of a phosphodiester bond in RNA than DNA libraries built from a standard four-nucleotide alphabet. Evolved AEGISzymes with nitro-carrying nucleobase Z appear to exploit a general acid-base catalytic mechanism to cleave that bond, analogous to the mechanism of the ribonuclease A family of protein enzymes and heavily modified DNAzymes. The AEGISzyme described here represents a new type of catalysts evolved from libraries built from expanded genetic alphabets.

New Insights into Boron Essentiality in Humans and Animals.

Boron (B) is considered a prebiotic chemical element with a role in both the origin and evolution of life, as well as an essential micronutrient for some bacteria, plants, fungi, and algae. B has beneficial effects on the biological functions of humans and animals, such as reproduction, growth, calcium metabolism, bone formation, energy metabolism, immunity, and brain function. Naturally organic B (NOB) species may become promising novel prebiotic candidates. NOB-containing compounds have been shown to be essential for the symbiosis between organisms from different kingdoms. New insights into the key role of NOB species in the symbiosis between human/animal hosts and their microbiota will influence the use of natural B-based colon-targeting nutraceuticals. The mechanism of action (MoA) of NOB species is related to the B signaling molecule (autoinducer-2-borate (AI-2B)) as well as the fortification of the colonic mucus gel layer with NOB species from B-rich prebiotic diets. Both the microbiota and the colonic mucus gel layer can become NOB targets. This paper reviews the evidence supporting the essentiality of the NOB species in the symbiosis between the microbiota and the human/animal hosts, with the stated aim of highlighting the MoA and targets of these species.