Preferential Pairing Research Articles

Specific recognition of cytosine by hypoxanthine in pyrrolidinyl peptide nucleic acid

Hypoxanthine is an unnatural base that can potentially pair with all natural nucleobases. While hypoxanthine in DNA exhibits marginal preference for pairing with cytosine (C), little is known about its pairing behavior in other DNA analogues. In this study, we synthesized a hypoxanthine-containing monomer and incorporated it into pyrrolidinyl peptide nucleic acid with α/β-peptide backbone derived from D-prolyl-(1S,2S)-2-aminocyclopentanecarboxylic acid (acpcPNA). DNA binding studies clearly revealed that hypoxanthine in acpcPNA behaves like G-analogue because it can specifically form a stable base pair with dC in DNA. The ability to replace G by hypoxanthine without affecting the base pairing properties of acpcPNA can solve a number of problems associated with G-rich acpcPNA including difficult synthesis, formation of secondary structures and fluorescence quenching.

Statistical analysis of combinatorial transcriptional regulatory motifs in human intron-containing promoter sequences

The combinatorial regulation is an important feature of transcriptional regulation of genes. Although a number of researches have been done, the general properties of combinatorial transcriptional regulatory elements, including their patterns and distributions, are still not well understood. Specially, the understanding for laws of transcriptional regulation involving introns is challenging. Here, we performed statistical analyses on potentially interacting motif pairs in human housekeeping genes and tissue-specific genes of some tissues. We first identified transcriptional regulatory motifs in promoter sequences, including the first introns, using a sequence-based approach which incorporates several different sequence backgrounds. And then we predicted the potentially interacting motif pairs using Hypergeometric distribution and Mann–Whitney U-test. The validation of the predicted interacting motif pairs has been carried out via analyses of orthologous conservation and enrichment. The distribution characteristics of the identified motif pairs were examined using a probability model. We found that interacting motif pairs have some significant preferences in location, distance and orientation in housekeeping and tissue-specific genes. The results suggest that the features of transcriptional regulation in housekeeping genes are different from the tissue-specific genes. Besides, location preferences of the motif pairs provide clues to the way of the introns participating in transcriptional regulation in different tissue-specific genes.

Meiotic chromosome pairing in Actinidia chinensis var. deliciosa

Polyploids are defined as either autopolyploids or allopolyploids, depending on their mode of origin and/or chromosome pairing behaviour. Autopolyploids have chromosome sets that are the result of the duplication or combination of related genomes (e.g., AAAA), while allopolyploids result from the combination of sets of chromosomes from two or more different taxa (e.g., AABB, AABBCC). Allopolyploids are expected to show preferential pairing of homologous chromosomes from within each parental sub-genome, leading to disomic inheritance. In contrast, autopolyploids are expected to show random pairing of chromosomes (non-preferential pairing), potentially leading to polysomic inheritance. The two main cultivated taxa of Actinidia (kiwifruit) are A. chinensis (2x and 4x) and A. chinensis var. deliciosa (6x). There is debate whether A. chinensis var. deliciosa is an autopolyploid derived solely from A. chinensis or whether it is an allopolyploid derived from A. chinensis and one or two other Actinidia taxa. To investigate whether preferential or non-preferential chromosome pairing occurs in A. chinensis var. deliciosa, the inheritance of microsatellite alleles was analysed in the tetraploid progeny of a cross between A. chinensis var. deliciosa and the distantly related Actinidia eriantha Benth. (2x). The frequencies of inherited microsatellite allelic combinations in the hybrids suggested that non-preferential chromosome pairing had occurred in the A. chinensis var. deliciosa parent. Meiotic chromosome analysis showed predominantly bivalent formation in A. chinensis var. deliciosa, but a low frequency of quadrivalent chromosome formations was observed (1 observed in 20 pollen mother cells).

The simulation of meiosis in diploid and tetraploid organisms using various genetic models

BackgroundWhile the genetics of diploid inheritance are well studied and software for linkage mapping, haplotyping and QTL analysis are available, for tetraploids the available tools are limited. In order to develop such tools it would be helpful if simulated populations based on a variety of models of the tetraploid meiosis would be available.ResultsHere we present PedigreeSim, a software package that simulates meiosis in both diploid and tetraploid species and uses this to simulate pedigrees and cross populations. For tetraploids a variety of models can be used, including both bivalent and quadrivalent formation, varying degrees of preferential pairing of hom(oe)ologous chromosomes, different quadrivalent configurations and more. Simulation of quadrivalent meiosis results as expected in double reduction and recombination between more than two hom(oe)ologous chromosomes. The results are shown to match theoretical predictions.ConclusionsThis is the first simulation software that implements all features of meiosis in tetraploids. It allows to generate data for tetraploid and diploid populations, and to investigate different models of tetraploid meiosis. The software and manual are available from http://www.plantbreeding.nl/UK/software_pedigreeSim.html and as Additional files 1, 2, 3 and 4 with this publication.

Strong contributions from vertical triads to helix-partner preferences in parallel coiled coils.

Pairing preferences in heterodimeric coiled coils are determined by complementarities among side chains that pack against one another at the helix-helix interface. However, relationships between dimer stability and interfacial residue identity are not fully understood. In the context of the "knobs-into-holes" (KIH) packing pattern, one can identify two classes of interactions between side chains from different helices: "lateral", in which a line connecting the adjacent side chains is perpendicular to the helix axes, and "vertical", in which the connecting line is parallel to the helix axes. We have previously analyzed vertical interactions in antiparallel coiled coils and found that one type of triad constellation (a'-a-a') exerts a strong effect on pairing preferences, while the other type of triad (d'-d-d') has relatively little impact on pairing tendencies. Here, we ask whether vertical interactions (d'-a-d') influence pairing in parallel coiled-coil dimers. Our results indicate that vertical interactions can exert a substantial impact on pairing specificity, and that the influence of the d'-a-d' triad depends on the lateral a' contact within the local KIH motif. Structure-informed bioinformatic analyses of protein sequences reveal trends consistent with the thermodynamic data derived from our experimental model system in suggesting that heterotriads involving Leu and Ile are preferred over homotriads involving Leu and Ile.

Rejection Thresholds in Solid Chocolate‐Flavored Compound Coating

Classical detection thresholds do not predict liking, as they focus on the presence or absence of a sensation. Recently however, Prescott and colleagues described a new method, the rejection threshold, where a series of forced choice preference tasks are used to generate a dose-response function to determine hedonically acceptable concentrations. That is, how much is too much? To date, this approach has been used exclusively in liquid foods. Here, we determined group rejection thresholds in solid chocolate-flavored compound coating for bitterness. The influences of self-identified preferences for milk or dark chocolate, as well as eating style (chewers compared to melters) on rejection thresholds were investigated. Stimuli included milk chocolate-flavored compound coating spiked with increasing amounts of sucrose octaacetate, a bitter and generally recognized as safe additive. Paired preference tests (blank compared to spike) were used to determine the proportion of the group that preferred the blank. Across pairs, spiked samples were presented in ascending concentration. We were able to quantify and compare differences between 2 self-identified market segments. The rejection threshold for the dark chocolate preferring group was significantly higher than the milk chocolate preferring group (P= 0.01). Conversely, eating style did not affect group rejection thresholds (P= 0.14), although this may reflect the amount of chocolate given to participants. Additionally, there was no association between chocolate preference and eating style (P= 0.36). Present work supports the contention that this method can be used to examine preferences within specific market segments and potentially individual differences as they relate to ingestive behavior. This work makes use of the rejection threshold method to study market segmentation, extending its use to solid foods. We believe this method has broad applicability to the sensory specialist and product developer by providing a process to identify how much is too much when formulating products, even in the context of specific market segments. We illustrate this in solid chocolate-flavored compound coating, identifying substantial differences in the amount of acceptable bitterness in those who prefer milk chocolate compared to dark chocolate. This method provides a direct means to answer the question of how much is too much.

Curvature-driven effective attraction in multicomponent membranes

We study closed liquid membranes that segregate into three phases due to differences in the chemical and physical properties of its components. The shape and in-plane membrane arrangement of the phases are coupled through phase-specific bending energies and line tensions. We use simulated annealing Monte Carlo simulations to find low-energy structures, allowing both phase arrangement and membrane shape to relax. The three-phase system is the simplest one in which there are multiple interface pairs, allowing us to analyze interfacial preferences and pairwise distinct line tensions. We observe the system's preference for interface pairs that maximize differences in spontaneous curvature. From a pattern selection perspective, this acts as an effective attraction between phases of most disparate spontaneous curvature. We show that this effective attraction is robust enough to persist even when the interface between these phases is the most penalized by line tension. This effect is driven by geometry and not by any explicit component-component interaction.

Sensory Evaluation of Baked Chicken Wrapped with Antimicrobial Apple and Tomato Edible Films Formulated with Cinnamaldehyde and Carvacrol

The addition of plant essential oils to edible films and coatings has been shown to protect against bacterial pathogens and spoilage while also enhancing sensory properties of foods. This study evaluated the effect of adding 0.5 and 0.75% carvacrol (active ingredient of oregano oil) to apple- and tomato-based film-forming solutions and 0.5 and 0.75% cinnamaldehyde (active ingredient of cinnamon oil) to apple-based film-forming solutions on sensory properties of cooked chicken wrapped with these films. Paired preference tests indicated no difference between baked chicken wrapped with tomato and apple films containing 0.5% carvacrol and cinnamaldehyde compared to chicken wrapped with tomato or apple films without the plant antimicrobials. The taste panel indicated a higher preference for carvacrol-containing tomato-coated chicken over the corresponding apple coating. There was also a higher preference for cinnamaldehyde-containing apple films over corresponding carvacrol-containing wrapping. Films containing antibacterial active compounds derived from essential oils can be used to protect raw chicken pieces against bacterial contamination without adversely affecting preferences of wrapped chicken pieces after baking.

Germline VH/VL pairing in antibodies

Antibodies are key molecules of the adaptive immune response and are now a major class of biopharmaceuticals. Pairing of heavy and light chains is one of the ways of generating antibody diversity and, while little is known about mechanisms governing V(H)/V(L) pairing, previous studies have suggested that the germline source from which chains are paired is random. By selecting paired antibody protein sequences from human and mouse antibodies from the KabatMan database and mapping them onto their corresponding germline sequences, we find that pairing preferences do exist in the germline, but only for a small proportion of germline gene segments; others are much more promiscuous showing no preferences. The closest equivalent human and mouse gene families were identified and pairing preferences compared. This work may impact on the ability to generate more stable antibodies for use as biopharmaceuticals.

Simultaneous estimation of genetic linkage and preferential pairing factor for a triploid population with unphased markers

Triploids, recognized to occur more frequently in natural and experimental populations of many species than previously appreciated, display important economic and biological values. Despite this, however, linkage analysis for triploids has not been well explored. We develop a statistical model for estimating and testing the linkage between molecular markers in a triploid population derived from a tetraploid and diploid parent. The model incorporates one important meiotic feature of tetraploids by which more homologous chromosomes pair with a greater likelihood than less homologous chromosomes. By implementing the EM algorithm within the maximum likelihood framework, the model provides a procedure for simultaneous estimation of the linkage and preferential pairing factor. The model accommodates the segregating patterns of pseudotest markers and intercross markers with different amounts of informativeness. The utility of the model was validated through a real data analysis and simulation studies. The model provides a statistical tool for linkage analysis in a triploid population by taking into account meiotic behavior of tetraploids. Results from the model will help to shed light on the genetic diversity and origin of a polyploid population.

Contingent choice. Exploring the relationship between sweetened beverages and vegetable consumption

Adults and children are repeatedly exposed to the pairing of food and drink as found in meal deals and “combos”. There may arise from this indoctrination, a contingent relationship between drink context and food preference. Our multi-method research examines food and drink combining. A survey-based study examines the food and drink pairing preferences of adults (N=60), while a laboratory study with young children (N=75, aged three to five) examines the role of drink context on vegetable consumption. The adult survey finds strong food and drink combining preferences. The pairing of soft drinks with calorie dense foods is regarded favorably, while the pairing of soft drinks with vegetables is not. In child food trials, vegetable consumption is not influenced by the child’s fussiness but is influenced by the drink accompaniment. In limited contexts, these findings demonstrate the contingent relationship between drink context and food consumption. Both palate preference and associative learning may be mechanisms driving the effects of drink context on food consumption. The findings suggest simple consumer strategies that might be employed to change dietary patterns (e.g., drink water with meals), and hold straightforward policy implications (e.g., increase water as the default option in meal deals).

Antibody Repertoires in Humanized NOD-scid-IL2Rγnull Mice and Human B Cells Reveals Human-Like Diversification and Tolerance Checkpoints in the Mouse

Immunodeficient mice reconstituted with human hematopoietic stem cells enable the in vivo study of human hematopoiesis. In particular, NOD-scid-IL2Rγnull engrafted mice have been shown to have reasonable levels of T and B cell repopulation and can mount T-cell dependent responses; however, antigen-specific B-cell responses in this model are generally poor. We explored whether developmental defects in the immunoglobulin gene repertoire might be partly responsible for the low level of antibody responses in this model. Roche 454 sequencing was used to obtain over 685,000 reads from cDNA encoding immunoglobulin heavy (IGH) and light (IGK and IGL) genes isolated from immature, naïve, or total splenic B cells in engrafted NOD-scid-IL2Rγnull mice, and compared with over 940,000 reads from peripheral B cells of two healthy volunteers. We find that while naïve B-cell repertoires in humanized mice are chiefly indistinguishable from those in human blood B cells, and display highly correlated patterns of immunoglobulin gene segment use, the complementarity-determining region H3 (CDR-H3) repertoires are nevertheless extremely diverse and are specific for each individual. Despite this diversity, preferential DH-JH pairings repeatedly occur within the CDR-H3 interval that are strikingly similar across all repertoires examined, implying a genetic constraint imposed on repertoire generation. Moreover, CDR-H3 length, charged amino-acid content, and hydropathy are indistinguishable between humans and humanized mice, with no evidence of global autoimmune signatures. Importantly, however, a statistically greater usage of the inherently autoreactive IGHV4-34 and IGKV4-1 genes was observed in the newly formed immature B cells relative to naïve B or total splenic B cells in the humanized mice, a finding consistent with the deletion of autoreactive B cells in humans. Overall, our results provide evidence that key features of the primary repertoire are shaped by genetic factors intrinsic to human B cells and are principally unaltered by differences between mouse and human stromal microenvironments.

Preferences by Rhagoletis indifferens (Diptera, Tephritidae) for rectangles of various yellow colours and fluorescence

AbstractSticky rectangle traps of various yellow colours and fluorescence made of cardboard were field tested against western cherry fruit fly, Rhagoletis indifferens Curran, in paired trap preference experiments in Washington state. In a first experiment that involved comparisons of Alpha Scents (proprietary paint), Fluorescent Yellow (aerosol paint), Saffron Thread and Neon Light (semi‐gloss enamel paints) traps at two sites, the best performing trap was the Saffron Thread trap. In a second experiment comparing Citrus Splash (semi‐gloss enamel paint) with Alpha Scents and with Macaw and Lemon Zest (both semi‐gloss enamel paints) traps at a third site, the Citrus Splash trap outperformed the Alpha Scents trap. The Citrus Splash trap did not differ statistically from Saffron Thread and Lemon Zest traps, even though it caught 51% more flies than the Lemon Zest trap. The Alpha Scents trap caught significantly more non‐target Diptera than Fluorescent Yellow, Neon Light and Citrus Splash traps at two of three trap sites where non‐target Diptera populations were relatively high and overall it appeared less selective than the Citrus Splash trap. Results suggest that sticky rectangle traps painted semi‐gloss enamel Saffron Thread or Citrus Splash with low fluorescence could be highly useful for detecting R. indifferens.

Rejection thresholds in chocolate milk: Evidence for segmentation

Bitterness is generally considered a negative attribute in food, yet many individuals enjoy some bitterness in products like coffee or chocolate. In chocolate, bitterness arises from naturally occurring alkaloids and phenolics found in cacao. Fermentation and roasting help develop typical chocolate flavor and reduce the intense bitterness of raw cacao by modifying these bitter compounds. As it becomes increasingly common to fortify chocolate with ‘raw’ cacao to increase the amount of healthful phytonutrients, it is important to identify the point at which the concentration of bitter compounds becomes objectionable, even to those who enjoy some bitterness. Classical threshold methods focus on the presence or absence of a sensation rather than acceptability or hedonics. A new alternative, the rejection threshold, was recently described in the literature. Here, we sought to quantify and compare differences in rejection thresholds (RjT) and detection thresholds (DT) in chocolate milk spiked with a food safe bitterant (sucrose octaacetate). In experiment 1, a series of paired preference tests was used to estimate the RjT for bitterness in chocolate milk. In a new group of participants (experiment 2), we determined the RjT and DT using the forced choice ascending method of limits. In both studies, participants were segmented on the basis of self-declared preference for milk or dark solid chocolate. Based on sigmoid fits of the indifference–preference function, the RjT was ∼2.3 times higher for those preferring dark chocolate than the RjT for those preferring milk chocolate in both experiments. In contrast, the DT for both groups was functionally identical, suggesting that differential effects of bitterness on liking of chocolate products are not based on the ability to detect bitterness in these products.

A unifying framework for bivalent multilocus linkage analysis of allotetraploids

An allotetraploid has four paired sets of chromosomes derived from different diploid species, whose meiotic behavior is qualitatively different from the underlying diploids. According to a traditional view, meiotic pairing occurs only between homologous chromosomes, but new evidence indicates that homoeologous chromosomes may also pair to a lesser extent compared with homolog pairing. Here, we describe and assess a unifying analytical framework that incorporates differential chromosomal pairing into a multilocus linkage model. The preferential pairing factor is used to quantify the probability difference of pairing occurring between homologous chromosomes and homoeologous chromosomes. The unifying framework allows simultaneous estimation of the linkage, genetic interference and preferential pairing factor using commonly existing multiplex markers. We compared the unifying approach and traditional approaches assuming random chromosomal pairing by analyzing marker data collected in a full-sib family of tetraploid switchgrass, a bioenergy species whose diploid origins are undefined, but with subgenomes that are genetically well differentiated. The unifying framework provides a better tool for estimating the meiotic linkage and constructing a genetic map for allotetraploids.

Synonymous Codon Ordering: A Subtle but Prevalent Strategy of Bacteria to Improve Translational Efficiency

BackgroundIn yeast coding sequences, once a particular codon has been used, subsequent occurrence of the same amino acid tends to use codons sharing the same tRNA. Such a phenomenon of co-tRNA codons pairing bias (CTCPB) is also found in some other eukaryotes but it is not known whether it occurs in prokaryotes.Methodology/Principal FindingsIn this study, we focused on a total of 773 bacterial genomes to investigate their synonymous codon pairing preferences. After calculating the actual frequencies of synonymous codon pairs and comparing them with their expected values, we detected an obvious pairing bias towards identical codon pairs. This seems consistent with the previously reported CTCPB phenomenon, since identical codons are certainly read by the same tRNA. However, among co-tRNA but non-identical codon pairs, only 22 were often found overrepresented, suggesting that many co-tRNA codons actually do not preferentially pair together in prokaryotes. Therefore, the previously reported co-tRNA codons pairing rule needs to be more rigorously defined. The affinity differences between a tRNA anticodon and its readable codons should be taken into account. Moreover, both within-gene-shuffling tests and phylogenetic analyses support the idea that translational selection played an important role in shaping the observed synonymous codon pairing pattern in prokaryotes.ConclusionsOverall, a high level of synonymous codon pairing bias was detected in 73% investigated bacterial species, suggesting the synonymous codon ordering strategy has been prevalently adopted by prokaryotes to improve their translational efficiencies. The findings in this study also provide important clues to better understand the complex dynamics of translational process.

Novel isoxazole polycyclic aromatic hydrocarbons as DNA-intercalating agents

The third generation of isoxazole polycyclic aromatic hydrocarbons, acting as DNA-intercalator agents and possessing the binding constants in the range 104–105 M−1, in order to easily diffuse targeting remotely implanted tumors, has been synthesized in good yields according to the 1,3-dipolar cycloaddition methodology. The structure of the obtained cycloadducts has been determined by NOE experiments and supported by computational studies at PM3 level. All the obtained compounds have been tested for their in vitro cytotoxic activity and the most potent of them, (3RS,5SR)-2-benzyl-N,N-dimethyl-3-(pyren-1-yl)isoxazolidine-5-carboxamide (7d), showed an IC50 of 4 μM upon the human lung cancer (A-549) cells. Moreover, compound 7d showed binding constant for the intercalation with calf thymus DNA, poly-d(AT)2 and poly-d(GC)2 of 1.7 × 105 M−1, 1.6 × 105 M−1 and 0.3 × 105 M−1, respectively. Biological and docking studies showed that, in vitro, these compounds complex by intercalation between base pairs, approaching the DNA from its minor groove with a preference for the AT nucleobases pairs.

The d′--d--d′ Vertical Triad Is Less Discriminating Than the a′--a--a′ Vertical Triad in the Antiparallel Coiled-Coil Dimer Motif

Elucidating relationships between the amino-acid sequences of proteins and their three-dimensional structures, and uncovering non-covalent interactions that underlie polypeptide folding, are major goals in protein science. One approach toward these goals is to study interactions between selected residues, or among constellations of residues, in small folding motifs. The α-helical coiled coil has served as a platform for such studies because this folding unit is relatively simple in terms of both sequence and structure. Amino acid side chains at the helix-helix interface of a coiled coil participate in so-called "knobs-into-holes" (KIH) packing whereby a side chain (the knob) on one helix inserts into a space (the hole) generated by four side chains on a partner helix. The vast majority of sequence-stability studies on coiled-coil dimers have focused on lateral interactions within these KIH arrangements, for example, between an a position on one helix and an a' position of the partner in a parallel coiled-coil dimer, or between a--d' pairs in an antiparallel dimer. More recently, it has been shown that vertical triads (specifically, a'--a--a' triads) in antiparallel dimers exert a significant impact on pairing preferences. This observation provides impetus for analysis of other complex networks of side-chain interactions at the helix-helix interface. Here, we describe a combination of experimental and bioinformatics studies that show that d'--d--d' triads have much less impact on pairing preference than do a'--a--a' triads in a small, designed antiparallel coiled-coil dimer. However, the influence of the d'--d--d' triad depends on the lateral a'--d interaction. Taken together, these results strengthen the emerging understanding that simple pairwise interactions are not sufficient to describe side-chain interactions and overall stability in antiparallel coiled-coil dimers; higher-order interactions must be considered as well.

What the Numbers Say: A Digit-Based Test for Election Fraud

Is it possible to detect manipulation by looking only at electoral returns? Drawing on work in psychology, we exploit individuals' biases in generating numbers to highlight suspicious digit patterns in reported vote counts. First, we show that fair election procedures produce returns where last digits occur with equal frequency, but laboratory experiments indicate that individuals tend to favor some numerals over others, even when subjects have incentives to properly randomize. Second, individuals underestimate the likelihood of digit repetition in sequences of random integers, so we should observe relatively few instances of repeated numbers in manipulated vote tallies. Third, laboratory experiments demonstrate a preference for pairs of adjacent digits, which suggests that such pairs should be abundant on fraudulent return sheets. Fourth, subjects avoid pairs of distant numerals, so those should appear with lower frequency on tainted returns. We test for deviations in digit patterns using data from Sweden's 2002 parliamentary elections, Senegal's 2000 and 2007 presidential elections, and previously unavailable results from Nigeria's 2003 presidential election. In line with observers' expectations, we find substantial evidence that manipulation occurred in Nigeria as well as in Senegal in 2007.

A computational proposal for the experimentally observed discriminatory behavior of hypoxanthine, a weak universal nucleobase

A computational model composed of six nucleobases was used to investigate why hypoxanthine does not yield duplexes of equal stability when paired opposite each of the natural DNA nucleobases. The magnitudes of all nearest-neighbor interactions in a DNA helix were calculated, including hydrogen-bonding, intra- and interstrand stacking interactions, as well as 1-3 intrastrand stacking interactions. Although the stacking interactions in DNA relevant arrangements are significant and account for at least one third of the total stabilization energy in our nucleobase complexes, the trends in the magnitude of the stacking interactions cannot explain the relative experimental melting temperatures previously reported in the literature. Furthermore, although the total hydrogen-bonding interactions explain why hypoxanthine preferentially pairs with cytosine, the experimental trend for the remaining nucleobases (A, T, G) is not explained. In fact, the calculated pairing preference of hypoxanthine matches that determined experimentally only when the sum of all types of nearest-neighbor interactions is considered. This finding highlights a strong correlation between the relative magnitude of the total nucleobase-nucleobase interactions and measured melting temperatures for DNA strands containing hypoxanthine despite the potential role of other factors (including hydration, temperature, sugar-phosphate backbone). By considering a large range of sequence combinations, we reveal that the binding preference of hypoxanthine is strongly dependent on the nucleobase sequence, which may explain the varied ability of hypoxanthine to universally bind to the natural bases. As a result, we propose that future work should closely examine the interplay between the dominant nucleobase-nucleobase interactions and the overall strand stability to fully understand how sequence context affects the universal binding properties of modified bases and to aid the design of new molecules with ambiguous pairing properties.