Watson-Crick Pairing Research Articles

Watson-Crick Base Pairing of N-Methoxy-1,3-Oxazinane (MOANA) Nucleoside Analogues within Double-Helical DNA.

Hairpin oligodeoxynucleotides incorporating a (2R,3S)-4-(methoxyamino)butane-1,2,3-triol residue in the middle of the double-helical stem and opposite to either one of the canonical nucleobases or an abasic 2-(hydroxymethyl)tetrahydrofuran-3-ol spacer were synthesized. Under mildly acidic conditions, aromatic aldehydes reacted reversibly with these oligonucleotides, converting the (2R,3S)-4-(methoxyamino)butane-1,2,3-triol unit into a 2-aryl-N-methoxy-1,3-oxazinane nucleoside analogue. The equilibrium of this reaction was found to be dependent on both the aldehyde and the nucleobase opposite to the modified residue. 9-Formyl-9-deazaadenine, combining a large stacking surface with an array of hydrogen bond donors and acceptors, showed the highest affinity as well as selectivity consistent with the rules of Watson-Crick base pairing. 5-Formyluracil or indole-3-carbaldehyde, lacking in either stacking or hydrogen bonding ability, were incorporated with a much lower affinity and selectivity.

Hydrogen-Bonding Interactions of 8-Substituted Purine Derivatives.

Hydrogen bonding between nucleobases is a crucial noncovalent interaction for life on Earth. Canonical nucleobases form base pairs according to two main geometries: Watson-Crick pairing, which enables the static functions of nucleic acids, such as the storing of genetic information; and Hoogsteen pairing, which facilitates the dynamic functions of these biomacromolecules. This precisely tuned system can be affected by oxidation or substitution of nucleobases, leading to changes in their hydrogen-bonding patterns. This paper presents an investigation into the intermolecular interactions of various 8-substituted purine derivatives with their hydrogen-bonding partners. The systems were analyzed using nuclear magnetic resonance spectroscopy and density functional theory calculations. Our results demonstrate that the stability of hydrogen-bonded complexes, or base pairs, depends primarily on the number of intermolecular H-bonds and their donor-acceptor alternation. No strong preferences for a particular geometry, either Watson-Crick or Hoogsteen, were found.

Watson-Crick hydrogen-bonded macrocycles self-assembled from Z-shaped dinucleoside monomers

The self-assembly of two lipophilic dinucleoside molecules that have a common structure, comprising a bent “Z-shaped” central block substituted at the edges with complementary G and C nucleobases, are studied in this work by a combination of 1D and 2D NMR experiments, as well as by absorption, fluorescence and circular dichroism measurements as a function of the temperature and the concentration. Watson-Crick pairing affords cyclic tetramer G:C H-bound species that are less stable than those formed by related linear dinucleosides and that, in principle, due to the unusual monomer geometry, can establish an equilibrium between different conformations with the shape of a square and a rhomb.

Molecular modeling and thermodynamics of the interaction between DNA base pairs and radon originated ionizing alpha radiation

Ionizing alpha radiation (He2+) is known to adversely affect human DNA, but the biochemical reasoning is not clear yet. Relatedly, the present computational study was conducted investigating the effects of ionizing alpha radiation onto the Watson-Crick type DNA base pairs (nucleotides) Adenine-Thymine (AT’) and Guanine-Cytosine (GC’). The long-range cation (He2+)−π interactions were modeled for this purpose. A hybrid DFT functional of M06-2X was used with 6-31G(d,p) and 6-311G(d) basis sets at unrestricted level. The results showed that alpha radiation severely changed the considered base pairs’ hydrogen bond lengths and their interaction enthalpies and Gibbs free energies, however, the more drastic changes were observed in GC’ rather than AT’. This observation was also supported by frontier molecular orbital analyses performed. GC’ was more favored to form He2+ complexes (oxidize) than AT’ and consequently these complexes had more exothermic interaction energies (formed more spontaneously) than that of AT’. It could be highlighted that the molecular modeling proposed in this study would contribute to the elucidation of the uncertainty in this field.

Base pairs with 5-chloroorotic acid and comparison with the natural nucleobase. Structural and spectroscopic study, and three suggested antiviral modified nucleosides

A structural and spectroscopic study of 5-chloroorotic acid (5-ClOA) biomolecule was carried out by IR and FT-Raman and the results obtained were compared to those achieved in 5-fluoroorotic acid and 5-aminoorotic acid compounds. The structures of all possible tautomeric forms were determined using DFT and MP2 methods. To know the tautomer form present in the solid state, the crystal unit cell was optimized through dimer and tetramer forms in several tautomeric forms. The keto form was confirmed through an accurate assignment of all the bands. For this purpose, an additional improvement in the theoretical spectra was carried out using linear scaling equations (LSE) and polynomic equations (PSE) deduced from uracil molecule. Base pairs with uracil, thymine and cytosine nucleobases were optimized and compared to the natural Watson-Crick (WC) pairs. The counterpoise (CP) corrected interaction energies of the base pairs were also calculated. Three nucleosides were optimized based on 5-ClOA as nucleobase, and their corresponding WC pairs with adenosine. These modified nucleosides were inserted in DNA:DNA and RNA:RNA microhelices, which were optimized. The position of the -COOH group in the uracil ring of these microhelices interrupts the DNA/RNA helix formation. Because of the special characteristic of these molecules they can be used as antiviral drugs. Communicated by Ramaswamy H. Sarma

Dynamic DNA nanomachines for amplification imaging of diseased cells based on stimuli-responsive mechanism

As a basic functional unit, living cell with sophisticated structures play an indispensable role in life activities. Since the abnormality of important molecules inside cells is closely related to diseases, the dynamic analysis and spatio-temporal monitoring of specific molecules in living cells can provide precious information for the diagnosis and treatment of diseases. More recently, DNA has not only been recognized as the carrier of genetic information, but has also used as a robust building block for the assembly of multitudinous nanoscale structures due to the intrinsic advantages of high programmability of classic Watson–Crick base-pairing rule. Intensive study promotes the rapid progress of nanotechnology in various fields, such as bioimaging, diagnosis, and therapeutics. Among numerous well-defined DNA nanomaterials, DNA nanomachines have been widely exploited in cell imaging owing to their desirable ability to achieve high-resolution temporal and spatial images in response to endogenous or exogenous stimuli. In brief, elaborate DNA nanomachines can undergo structural changes upon the stimuli of target analytes or environmental factors, resulting in rapid increase or reduction of output signals and thereby indirectly reflecting the expression level of targets. DNA nanomachines with high sensitivity and specificity contribute to the recognition of diseased tissues. In this review, we introduce the basic assembly modules of DNA nanomachines and summarize the recent advances in dynamic DNA nanomachines for diseased-cell imaging. Finally, the current challenges and future directions of DNA nanomachines for bioimaging are discussed.

Molecular dynamics of mismatch detection—How MutS uses indirect readout to find errors in DNA

The mismatch repair protein MutS safeguards genomic integrity by finding and initiating repair of basepairing errors in DNA. Single-molecule studies show MutS diffusing on DNA, presumably scanning for mispaired/unpaired bases, and crystal structures show a characteristic “mismatch-recognition” complex with DNA enclosed within MutS and kinked at the site of error. But how MutS goes from scanning thousands of Watson-Crick basepairs to recognizing rare mismatches remains unanswered, largely because atomic-resolution data on the search process are lacking. Here, 10 μs all-atom molecular dynamics simulations of Thermus aquaticus MutS bound to homoduplex DNA and T-bulge DNA illuminate the structural dynamics underlying the search mechanism. MutS-DNA interactions constitute a multistep mechanism to check DNA over two helical turns for its 1) shape, through contacts with the sugar-phosphate backbone, 2) conformational flexibility, through bending/unbending engineered by large-scale motions of the clamp domain, and 3) local deformability, through basepair destabilizing contacts. Thus, MutS can localize a potential target by indirect readout due to lower energetic costs of bending mismatched DNA and identify a site that distorts easily due to weaker base stacking and pairing as a mismatch. The MutS signature Phe-X-Glu motif can then lock in the mismatch-recognition complex to initiate repair.

Design of LNA Analogues Using a Combined Density Functional Theory and Molecular Dynamics Approach for RNA Therapeutics.

Antisense therapeutics treat a wide spectrum of diseases, many of which cannot be addressed with the current drug technologies. In the quest to design better antisense oligonucleotide drugs, we propose five novel LNA analogues (A1-A5) for modifying antisense oligonucleotides and establishing each with the five standard nucleic acids: adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U). Monomer nucleotides of these modifications were considered for a detailed Density Functional Theory (DFT)-based quantum chemical analysis to determine their molecular-level structural and electronic properties. A detailed MD simulation study was done on a 14-mer ASO (5'-CTTAGCACTGGCCT-3') containing these modifications targeting PTEN mRNA. Results from both molecular- and oligomer-level analysis clearly depicted LNA-level stability of the modifications, the ASO/RNA duplexes maintaining stable Watson-Crick base pairing preferring RNA-mimicking A-form duplexes. Notably, monomer MO isosurfaces for both purines and pyrimidines were majorly distributed on the nucleobase region in modifications A1 and A2 and in the bridging unit in modifications A3, A4, and A5, suggesting that A3/RNA, A4/RNA, and A5/RNA duplexes interact more with the RNase H and solvent environment. Accordingly, solvation of A3/RNA, A4/RNA, and A5/RNA duplexes was higher compared to that of LNA/RNA, A1/RNA, and A2/RNA duplexes. This study has resulted in a successful archetype for creating advantageous nucleic acid modifications tailored for particular needs, fulfilling a useful purpose of designing novel antisense modifications, which may overcome the drawbacks and improve the pharmacokinetics of existing LNA antisense modifications.

Sequence-Specific Fluorescence Turn-On Sensing of RNA by DNA Probes Incorporating the Tricyclic Cytidine Analogue DEAtC.

Sequence-specific fluorescent probes for RNA are widely used in microscopy applications such as fluorescence in situ hybridization and a growing number of newer approaches to live-cell RNA imaging. The sequence specificity of most of these approaches relies on differential hybridization of the probe to the correct target. Competing sequences with only one or two base mismatches are prone to causing off-target recognition. Here, we report the sequence-specific fluorescent detection of model RNA targets using a tricyclic cytidine analogue DEAtC that is included as a surrogate for natural cytidine in DNA probe strands and that reports directly on Watson-Crick base pairing. The DEAtC-containing DNA oligonucleotide probes exhibit an average 8-fold increase in fluorescence intensity when hybridized to matched RNA with DEAtC base paired with G and little fluorescence turn-on when DEAtC is base paired with A. Duplex structure determination by NMR, time-resolved fluorescence studies, and Stern-Volmer quenching experiments suggest that the combination of greater π stacking and narrower grooves in the A-form DNA-RNA heteroduplex provides additional shielding and favorable electronic interactions between bases, explaining why DEAtC's fluorescence turn-on response to RNA targets is typically 3-fold greater than for DNA targets.

Fluorescent labeling of RNA and DNA on the Hoogsteen edge using sulfinate chemistry.

We have devised a single pot, low-cost method to add azide groups to unmodified nucleic acids without the need for enzymes or chemically modified nucleoside triphosphates. This involves reacting an azide-containing sulfinate salt with the nucleic acid, leading to replacement of C-H bonds on the nucleobase aromatic rings with C-R, where R is the azide-containing linker derived from the original sulfinate salt. With the addition of azide functional groups, the modified nucleic acid can easily be reacted with any alkyne-labeled compound of interest, including fluorescent dyes as shown in this work. This methodology enables the fluorescent labeling of a wide variety of nucleic acids, including natively folded RNAs, under mild conditions with minimal effects upon biochemical function and ribozyme catalysis. To demonstrate this, we show that a pair of labeled complementary ssDNA oligonucleotides (oligos) can hybridize to form dsDNA, even when labeled with multiple fluorophores per oligo. In addition, we also demonstrate that two different group II introns can splice when prelabeled internally with fluorophores, using our method. Broadly, this demonstrates that sulfinate modification of RNA is compatible with ribozyme function and Watson-Crick pairing, while preserving the labile backbone.

A Comprehensive Review of DNA Origami Stabilization Techniques

In recent years, DNA has emerged as a powerful tool in the field of nanotechnology. The DNA origami technique is largely responsible for this, revolutionizing nanofabrication due to its controllability, precision, and ability to leverage DNA’s unique properties. The technique consists of folding a long, single-stranded DNA (called a scaffold strand) by binding it with shorter staple strands to create almost any shape desired. With a desired structure in mind, researchers can design and assemble scaffold and staple strands using computer software like Cadnano or Tiamat. This is possible because of the Watson-Crick base pairing of DNA strands, which allows for programmable self-assembly of DNA nanostructures and therefore, the synthesis of arbitrary 2D and 3D shapes. Because DNA is a biomolecule,the nanostructures are also biocompatible and can be employed in biological applications including drug delivery. DNA origami nanostructures are not only limited to biological applications; they have also found uses in nanophotonics, plasmonics, and electronics. However, DNA origami still faces many challenges before it can be widely adopted. One such challenge is ensuring stability, and thus guaranteeing the performance of the DNA origami, in the presence of heat, nuclease in organic bodies, and chaotropic agents. This warrants the question: what methodologies can be employed to best stabilize DNA origami structures? This paper further focuses on two methods: covalently binding various molecules by cross-linking and non-binding encapsulation. Detailed analysis and comparison between various molecules used to bind and coat DNA nanostructures is used to evaluate performance and applicability of each method. In the end an oligolysines coating cross-linked with glutaraldehyde was found to have the strongest biological stability, thymine cross-linking had the strongest thermal stability, a silica coating had the best stability against the largest number of factors, and both graphene and Al3O2 coatings had the best mechanical stability.

Parameterization of the miniPEG-Modified γPNA Backbone: Toward Induced γPNA Duplex Dissociation.

γ-Modified peptide nucleic acids (γPNAs) serve as potential therapeutic agents against genetic diseases. Miniature poly(ethylene glycol) (miniPEG) has been reported to increase solubility and binding affinity toward genetic targets, yet details of γPNA structure and dynamics are not understood. Within our work, we parameterized missing torsional and electrostatic terms for the miniPEG substituent on the γ-carbon atom of the γPNA backbone in the CHARMM force field. Microsecond timescale molecular dynamics simulations were carried out on six miniPEG-modified γPNA duplexes from NMR structures (PDB ID: 2KVJ). Three NMR models for the γPNA duplex (PDB ID: 2KVJ) were simulated as a reference for structural and dynamic changes captured for the miniPEG-modified γPNA duplex. Principal component analysis performed on the γPNA backbone atoms identified a single isotropic conformational substate (CS) for the NMR simulations, whereas four anisotropic CSs were identified for the ensemble of miniPEG-modified γPNA simulations. The NMR structures were found to have a 23° helical bend toward the major groove, consistent with our simulated CS structure of 19.0°. However, a significant difference between simulated methyl- and miniPEG-modified γPNAs involved the opportunistic invasion of miniPEG through the minor and major groves. Specifically, hydrogen bond fractional analysis showed that the invasion was particularly prone to affect the second G-C base pair, reducing the Watson-Crick base pair hydrogen bond by 60% over the six simulations, whereas the A-T base pairs decreased by only 20%. Ultimately, the invasion led to base stack reshuffling, where the well-ordered base stacking was reduced to segmented nucleobase stacking interactions. Our 6 μs timescale simulations indicate that duplex dissociation suggests the onset toward γPNA single strands, consistent with the experimental observation of decreased aggregation. To complement the insight of miniPEG-modified γPNA structure and dynamics, the new miniPEG force field parameters allow for further exploration of such modified γPNA single strands as potential therapeutic agents against genetic diseases.

Mutagenic incorporation of inosine into DNA via T:I mismatch formation by human DNA polymerase eta (polη).

Inosine is a key intermediate in de novo purine nucleotide biosynthesis in cells. Inosine is known to be mutagenic when it is present in DNA, in place of adenine via deamination, by facilitating the incorporation of dCTP exclusively, resulting in A:T to G:C mutation. The structural basis for the mutagenicity of inosine bypass has been reported in some DNA polymerases including human DNA polymerase eta (polη). However, the structural and biochemical basis for the mutagenic potential of the incorporation of deoxyinosine triphosphate (dITP) into DNA remains poorly understood. To gain insights into the mutagenic potential of the incorporation of inosine into DNA, we conducted structural and kinetic studies of human polη incorporating dITP across undamaged DNA template containing dC or dT. Polη incorporated dITP opposite dC 14-fold more efficiently than opposite dT, indicating that dITP incorporation by polη can be mutagenic unlike the bypass of inosine by polη, which incorporated dCTP almost exclusively opposite the templating inosine over dTTP (70:1). Polη-dC:dITP crystal structure showed that the incoming dITP formed Watson-Crick base pair along with wobble base pair via 4-imino-2-keto tautomer of cytosine diminishing the catalytic efficiency compared to dGTP incorporation across dC. In addition, the crystal structure of polη-dT:dITP revealed that dT and dITP formed Watson-Crick like base pair via 4-enol-2-keto tautomer of thymine, reinforced by wobble base pair via 4-keto-2-keto tautomer of thymine resulting in the increased mutagenicity of dITP incorporation (14:1 across dC and dT), which is 14-fold higher than dGTP incorporation by polη (190:1 across dC and dT).

Binding and stabilizations effects of RNA triplex poly(U*A-U) by enantiomers of ruthenium(II) polypyridyl complex [Ru(bpy)2(10-OH-dppz)]2+

Two new chiral ruthenium (II) complexes, Δ-[Ru (bpy)2 (10-OH-dppz)]2+ (Δ-1; bpy = 2,2ʹ-bipyridine, 10-OH-dppz = 10-hydroxyl-dipyrido [3,2-a:2′,3′-c]phenazine) and Λ-[Ru (bpy)2 (10-OH-dppz)]2+ (Λ-1), have been synthesized and characterized in this work. The binding properties of the two enantiomers with an RNA triplex poly (U*A-U) [in text, dash (−) denotes the Watson-Crick base pairing and the asterisk (*) denotes the Hoogsteen base pairing] have been studied. Analysis of the absorption and fluorescence spectra suggest that the binding constant of enantiomer Λ-1 toward the triplex is somewhat smaller than that of enantiomer Δ-1, which is further supported by viscosity experiments. Viscosity experiments showed that although the two enantiomers were intercalated to the triplex, the Δ-enantiomer was a better intercalator than the Λ-enantiomer. Furthermore, thermal denaturation experiments demonstrate that although the two enantiomers can significantly increase the triplex stabilization, unlike enantiomer Λ-1, enantiomer Δ-1 stabilizes the Watson-Crick base-paired duplex and the Hoogsteen base-paired third strand of the triplex with obvious selectivity. In addition, the triplex stabilization by enantiomer Δ-1 was significantly stronger than that by Λ-1. The obtained results indicate that the binding ability of enantiomers Δ-1 and Λ-1 to the triplex is dependent on the chirality of enantiomers.

Programmable DNA Interstrand Crosslinking by Alkene-Alkyne [2+2] Photocycloaddition.

Covalent crosslinking of DNA strands provides a useful tool for medical, biochemical, and DNA nanotechnology applications. Here we present a light-induced interstrand DNA crosslinking reaction using the modified nucleoside 5-phenylethynyl-2'-deoxyuridine (PhedU). The crosslinking ability of PhedU was programmed by base pairing and by metal ion interaction at the Watson-Crick base pairing site. Rotation to intrahelical positions was favored by hydrophobic stacking and enabled an unexpected photochemical alkene-alkyne [2 + 2] cycloaddition within the DNA duplex, resulting in efficient formation of a PhedU dimer after short irradiation times of a few seconds. A PhedU-dimer-containing DNA was shown to efficiently bind a helicase complex, but the covalent crosslink completely prevented DNA unwinding, suggesting possible applications in biochemistry or structural biology.

Anionic G•U pairs in bacterial ribosomal rRNAs.

Wobble GU pairs (or G•U) occur frequently within double-stranded RNA helices interspersed between standard G=C and A-U Watson-Crick pairs. Another type of G•U pair interacting via their Watson-Crick edges has been observed in the A site of ribosome structures between a modified U34 in the tRNA anticodon triplet and G + 3 in the mRNA. In such pairs, the electronic structure of the U is changed with a negative charge on N3(U), resulting in two H-bonds between N1(G)…O4(U) and N2(G)…N3(U). Here, we report that such pairs occur in other highly conserved positions in ribosomal RNAs of bacteria in the absence of U modification. An anionic cis Watson-Crick G•G pair is also observed and well conserved in the small subunit. These pairs are observed in tightly folded regions.

The marvelous dna macromolecule: computer modeling of dna 3d structure and diversity of watson-crick duplex conformations

The work reviews the development of ideas about the 3D structure of DNA and the mechanisms of its formation, from the discovery of the Double Helix to the present day. It tracks the methods of modeling the 3D structure at different stages of the study of the main molecule of life. The discussion underscores a marvelous expedient in DNA molecular structure and its adaptability to important biological functions based on the results of calculations of the intra- and intermolecular interactions of macromolecule subunits. The work presents new data about substantial contribution of chemically monotonous and conformationally flexible sugarphosphate backbone to the formation of sequence-dependent 3D structure of DNA. The diversity of the conformational possibilities of DNA is visible both in the formation of duplexes (as well as triplexes and quadruplexes) with different geometric parameters for base pairs and in the formation of duplexes with Watson-Crick nucleoside pairs containing local conformations corresponding to different regions of the torsion angles of the sugar-phosphate backbone (different conformational classes). Based on our calculations, these classes can be divided into two groups. The first group includes local conformations in which the torsion angles are close to one of energy minima of its isolated elemental repeating fragment, and the second group consists of conformations with one or more of these angles deviating from that of the nearest energy minimum by more than 30°. The regularities of the formation of the local 3D structure of these two groups differ significantly.

Unravelling the Drug Encapsulation Ability of Functional DNA Origami Nanostructures: Current Understanding and Future Prospects on Targeted Drug Delivery.

Rapid breakthroughs in nucleic acid nanotechnology have always driven the creation of nano-assemblies with programmable design, potent functionality, good biocompatibility, and remarkable biosafety during the last few decades. Researchers are constantly looking for more powerful techniques that provide enhanced accuracy with greater resolution. The self-assembly of rationally designed nanostructures is now possible because of bottom-up structural nucleic acid (DNA and RNA) nanotechnology, notably DNA origami. Because DNA origami nanostructures can be organized precisely with nanoscale accuracy, they serve as a solid foundation for the exact arrangement of other functional materials for use in a number of applications in structural biology, biophysics, renewable energy, photonics, electronics, medicine, etc. DNA origami facilitates the creation of next-generation drug vectors to help in the solving of the rising demand on disease detection and therapy, as well as other biomedicine-related strategies in the real world. These DNA nanostructures, generated using Watson-Crick base pairing, exhibit a wide variety of properties, including great adaptability, precise programmability, and exceptionally low cytotoxicity in vitro and in vivo. This paper summarizes the synthesis of DNA origami and the drug encapsulation ability of functionalized DNA origami nanostructures. Finally, the remaining obstacles and prospects for DNA origami nanostructures in biomedical sciences are also highlighted.

Crystal structures and identification of novel Cd2+-specific DNA aptamer.

Cadmium (Cd) is one of the most toxic heavy metals. Exposure to Cd can impair the functions of the kidney, respiratory system, reproductive system and skeletal system. Cd2+-binding aptamers have been extensively utilized in the development of Cd2+-detecting devices; however, the underlying mechanisms remain elusive. This study reports four Cd2+-bound DNA aptamer structures, representing the only Cd2+-specific aptamer structures available to date. In all the structures, the Cd2+-binding loop (CBL-loop) adopts a compact, double-twisted conformation and the Cd2+ ion is mainly coordinated with the G9, C12 and G16 nucleotides. Moreover, T11 and A15 within the CBL-loop form one regular Watson-Crick pair and stabilize the conformation of G9. The conformation of G16 is stabilized by the G8-C18 pair of the stem. By folding and/or stabilizing the CBL-loop, the other four nucleotides of the CBL-loop also play important roles in Cd2+binding. Similarly to the native sequence, crystal structures, circular dichroism spectrum and isothermal titration calorimetry analysis confirm that several variants of the aptamer can recognize Cd2+. This study not only reveals the underlying basis for the binding of Cd2+ ions with the aptamer, but also extends the sequence for the construction of novel metal-DNA complex.

Pseudouridine-Modifying Enzymes SapB and SapH Control Entry into the Pseudouridimycin Biosynthetic Pathway.

Pseudouridimycin is a microbial C-nucleoside natural product that specifically inhibits bacterial RNA polymerases by binding to the active site and competing with uridine triphosphate for the nucleoside triphosphate (NTP) addition site. Pseudouridimycin consists of 5'-aminopseudouridine and formamidinylated, N-hydroxylated Gly-Gln dipeptide moieties to allow Watson-Crick base pairing and to mimic protein-ligand interactions of the triphosphates of NTP, respectively. The metabolic pathway of pseudouridimycin has been studied in Streptomyces species, but no biosynthetic steps have been characterized biochemically. Here, we show that the flavin-dependent oxidase SapB functions as a gate-keeper enzyme selecting pseudouridine (KM = 34 μM) over uridine (KM = 901 μM) in the formation of pseudouridine aldehyde. The pyridoxal phosphate (PLP)-dependent SapH catalyzes transamination, resulting in 5'-aminopseudouridine with a preference for arginine, methionine, or phenylalanine as cosubstrates as amino group donors. The binary structure of SapH in complex with pyridoxamine-5'-phosphate and site-directed mutagenesis identified Lys289 and Trp32 as key residues for catalysis and substrate binding, respectively. The related C-nucleoside oxazinomycin was accepted as a substrate by SapB with moderate affinity (KM = 181 μM) and was further converted by SapH, which opens possibilities for metabolic engineering to generate hybrid C-nucleoside pseudouridimycin analogues in Streptomyces.