Single Base Pair Research Articles

Decoding the Complex Functional Landscape of the ykkC Riboswitches.

The ykkC class is the most diverse riboswitch class to date, recognizing structurally and chemically diverse ligands using only minor changes in sequence and structure. Structural studies have demonstrated how sequence changes correspond to altered specificity; however, they are insufficient to define the requirements for functional riboswitch specificity. Here, we report an extensive mutational analysis of the ppGpp riboswitch to investigate the functional role in transcriptional control for this variant riboswitch. Disruption of the terminator hairpin at a single base pair is sufficient to abolish nearly all function, highlighting the fine-tuning of the terminator hairpin to its corresponding aptamer domain. This fine-tuning has been observed in other riboswitches, suggesting that high levels of tunability may be a common feature of riboswitches. Additionally, mutational analysis shows that the previously reported binding site position, G93, does not necessarily correspond to PRPP-driven function as expected. Phylogenetic analysis of natural riboswitches that contain G93 revealed an additional ykkC subclass that binds to both XMP and GMP. This variant subclass is associated with genes for de novo GMP synthesis. Identification of this variant class provides further evidence for small sequence changes corresponding to altered ligand specificity.

Novel Primer Design for Significantly Reducing Fluorescent Interferences in the Synthesis of DNA-Templated Copper Nanoclusters for the Detection of the HLA-B*5801 Gene.

The optimal sequence for synthesizing copper nanoclusters is a promising research area. Initially, random dsDNA sequences yielded low fluorescence intensity, which constrained visual detection under UV light. Poly-AT dsDNA sequences later produced visible fluorescence, but it caused significant interference in negative samples when combined with gene amplification techniques. This interference occurs because the single-stranded poly-AT primer can self-anneal into a double-stranded AT sequence, efficiently synthesizing copper nanoclusters. To mitigate this, we designed a poly-AAT sequence at the primer's 5' end, creating a single base pair mismatch every three nucleotides during self-annealing. This adjustment reduced synthesis efficiency of copper nanoclusters in negative samples, improving the visual distinction between negative and positive results. We applied this method to identify the HLA-B*5801 gene, thereby demonstrating its efficacy even within a GC-rich region of human genomic DNA. Our method showed 100% agreement with a commercial qPCR kit, with results distinguishable under UV light. We conclude that the poly-AAT sequence is more suitable for integrating copper nanoclusters synthesis with nucleic acid amplification detection techniques, with potential applications in microelectronics, biosensing, and catalysis.

DNA calorimetric force spectroscopy at single base pair resolution

DNA hybridization is a fundamental molecular reaction with wide-ranging applications in biotechnology. The knowledge of the temperature dependence of the thermodynamic parameters of duplex formation is crucial for quantitative predictions throughout the DNA stability range. It is commonly assumed that enthalpies and entropies are temperature independent, and heat capacity changes ΔCp equal zero. However, it has been known that this assumption is a poor approximation for a long time. Here, we combine single-DNA mechanical unzipping experiments using a temperature jump optical trap with a tailored statistical analysis to derive the ten heat-capacity change parameters of the nearest-neighbor model. Calorimetric force spectroscopy establishes a groundbreaking approach to studying nucleic acids that can be further extended to chemically modified DNA, RNA, and DNA/RNA hybrid structures.

Nucleosome Engagement Regulates RORγt Structure and Dynamics.

The retinoic acid-related orphan receptor gamma (RORγt) acts as the major transcriptional activator in Th17 cell development and function to mediate adaptive immune defenses against pathogenic infection. RORγt engages accessible DNA response elements in the genome and interplays with coactivator proteins and accessory transcription factors to drive gene expression. However, how the chromatin environment mediates RORγt structure, dynamics, and function remains unclear. Here, we profile how the nucleosome promotes or restricts access to the main RORγt DNA response elements found in native enhancers and promoters, revealing preferential binding in regions of free DNA and nucleosomal entry/exit sites, with single base-pair resolution. Solution phase measurements using hydrogen deuterium exchange coupled to mass spectrometry identify novel allosteric effects that influence RORγt binding and mediate chromatin dynamics. A high-resolution structure of RORγt bound to the nucleosome reveals how structured elements assemble to confer binding specificity and avidity to chromatin substrates. The observations suggest an activation model where RORγt binding to chromatinized DNA promotes coregulator recruitment and chromatin decompaction.

Identifying the interactions conferring functional mechanical rigidity on RNase-resistant RNA from Zika virus

Some viruses counter host-cell efforts to digest invading viral RNA by using special structures resistant to host RNases, known as exoribonuclease-resistant RNAs (xrRNAs). xrRNAs typically form an unusual fold with the 5'-end threaded through a ring consisting of a multihelix junction closed by a pseudoknot. By using single-molecule force spectroscopy (SMFS), we previously showed that a Zika virus xrRNA is extremely rigid mechanically, withstanding very high forces, and that this mechanical resistance-not simply the knot-like fold topology-is essential for RNase resistance. Here, we have determined which interactions are most important for generating mechanical rigidity in the Zika virus xrRNA, by systematically mutating tertiary contacts. We found that removing any of the tertiary contacts involving the threaded 5' end was sufficient to abrogate mechanical resistance. In contrast, breaking a single pseudoknot base pair was not sufficient to do so: Two broken pairs were needed. This hierarchy of interaction importance for mechanical rigidity was supported by simulations mapping how mechanical tension was distributed within the xrRNA. For all mutants, RNase resistance varied in lock-step with mechanical resistance, confirming the primary role of mechanical rigidity in xrRNA function. This work reveals which interactions are most important for Zika xrRNA function, with implications for targeting the xrRNA therapeutically.

Mis-splicing drives loss of function of p53E224D point mutation.

The tumor suppressor p53 (Trp53), also known as p53, is the most commonly mutated gene in cancer. Canonical p53 DNA damage response pathways are well characterized and classically thought to underlie the tumor suppressive effect of p53. Challenging this dogma, mouse models have revealed that p53-driven apoptosis and cell cycle arrest are dispensable for tumor suppression. Here, we investigated the inverse context of a p53 mutation predicted to drive the expression of canonical targets but is detected in human cancer. We established a novel mouse model with a single base pair mutation (GAG>GAT, p53E221D) in the DNA-Binding domain that has wild-type function in screening assays, but is paradoxically found in human cancer in Li-Fraumeni syndrome. Using mouse p53E221D and the analogous human p53E224D mutants, we evaluated expression, transcriptional activation, and tumor suppression in vitro and in vivo. Expression of human p53E224D from cDNA translated to a fully functional p53 protein. However, p53E221D/E221D RNA transcribed from the endogenous locus is mis-spliced resulting in nonsense-mediated decay. Moreover, fibroblasts derived from p53E221D/E221D mice do not express a detectable protein product. Mice homozygous for p53E221D exhibited increased tumor penetrance and decreased life expectancy compared to p53WT/WT animals. Mouse p53E221D and human p53E224D mutations lead to splice variation and a biologically relevant p53 loss of function in vitro and in vivo.

CtDNA as an Objective Marker for Postoperative Residual Disease in Primary Advanced High-Grade Serous Ovarian Cancer.

The surgeon's subjective intraoperative evaluation is the standard of care to assess postoperative residual disease (RD) in advanced epithelial ovarian cancer (EOC). We investigated the feasibility of ctDNA as an objective marker for postoperative RD. This prospective study included 27 patients with advanced ovarian cancer (FIGO IIIA1-IVB) who underwent primary surgery between July 2021 and July 2022. Blood samples were analyzed preoperatively and on days 2 (d2) and 10 (d10) postoperatively. Low-coverage whole genome sequencing (WGS) was used to identify structural variants (SVs) at single-base pair resolution, single nucleotide variants (SNVs), and indels in tumor tissue to develop personalized, tumor-informed digital polymerase chain reaction (dPCR) fingerprint assays for each patient. dPCR fingerprint assays were successfully developed for all patients by identifying one to eight SVs/SNVs per patient. ctDNA was detected in 96% (n = 26/27) of patients preoperatively and in 81% (n = 22/27) of patients at d10. Median ctDNA levels at d10 were significantly higher in patients with postoperative RD (median 367.38 copies (cps)/mL, 2.84% variant allele frequency; VAF) than in patients without postoperative RD (median 0.92 cps/mL, 0.017% VAF, p < 0.001). In patients with postoperative RD, ctDNA levels increased from the preoperative stage to d10 in seven out of eight patients (p = 0.016). In patients with complete tumor resection, ctDNA levels decreased from the preoperative stage to d10 in 17/19 patients (p < 0.001). A tumor-informed personalized ctDNA approach demonstrated feasibility, providing extremely high detection rates pre- and postoperatively. These results indicate that this approach could potentially be used for postoperative RD assessment in patients with primary advanced EOC.

Mechanism of forced-copy-choice RNA recombination by enteroviral RNA-dependent RNA polymerases.

Forced-copy-choice recombination occurs at the end of a template, differing from copy-choice recombination, which happens at internal positions. This mechanism may produce full-length genomes from fragments created by host antiviral responses. Previous studies from our laboratory demonstrated that poliovirus (PV) RNA-dependent RNA polymerase (RdRp) switches to an "acceptor" template in vitro when initiated on a heteropolymeric RNA-primed "donor" template. Surprisingly, recombinants showed template switching from the 3'-end of the donor template. We have developed a primed-template system to study PV RdRp-catalyzed forced-copy-choice RNA recombination. PV RdRp adds a single, non-templated nucleotide to the 3'-end of a blunt-ended, double-stranded RNA product, forming a "plus-one" intermediate essential for template switching. Non-templated addition of CMP was favored over AMP and GMP (80:20:1); UMP addition was negligible. A single basepair between the plus-one intermediate and the 3'-end of the acceptor template was necessary and sufficient for template switching, which could occur without RdRp dissociation. Formation of the plus-one intermediate was rate limiting for template switching. PV RdRp also utilized synthetic, preformed intermediates, including those with UMP 3'-overhangs. Reactions showed up to five consecutive template-switching events, consistent with a repair function for this form of recombination. PV RdRp may exclude UMP during forced-copy-choice RNA recombination to preclude creation of nonsense mutations during RNA fragment assembly. Several other picornaviral RdRps were evaluated, and all were capable of RNA fragment assembly to some extent. Lastly, we propose a structure-based hypothesis for the PV RdRp-plus-one intermediate complex based on an elongating PV RdRp structure.

Plant Productivity and Leaf Starch During Grain Fill Is Linked to QTL Containing Flowering Locus T1 (FT1) in Wheat (Triticum aestivum L.).

Shifts in the environment due to climate change necessitate breeding efforts aimed at adapting wheat to longer, warmer growing seasons. In this study, 21 modern wheat (Triticum aestivum L.) cultivars and 29 landraces were screened for flag leaf starch levels, with the goal of identifying a genetic marker for targeted breeding. The landrace PI 61693 was identified as having exceptionally high flag leaf starch values. Yield trials were carried out in a Berkut × PI 61693 recombinant inbred line (RIL) population and a negative correlation was observed between leaf starch, flowering time, and yield. Genetic mapping identified a Quantitative Trait Loci (QTL) explaining 22-34% variation for leaf starch, flowering time, biomass, and seed yield. The starch synthase TraesCS7D02G117800 (wSsI-1) is located in this region, which possibly accounts for leaf starch variation in this population; also within this QTL is TraesCS7D02G111600 (FT-D1). Sequencing of FT-D1 identified a single base pair deletion in the 3rd exon of the Berkut allele. This indel has recently been shown to significantly impact flowering time and productivity, and likely led to significant variation in flowering date and yield in this population. Here, we illustrate how allelic selection of FT-D1 within breeding programs may aid in adapting wheat to changing environments.

Canonical prolactin signaling and global mRNA expression in the skin of Holstein heifers carrying the SLICK1 allele of the prolactin receptor gene.

Canonical prolactin signaling and global mRNA expression in the skin of Holstein heifers carrying the SLICK1 allele of the prolactin receptor gene.

An extreme mutational hotspot in nlpD depends on transcriptional induction of rpoS.

Mutation rate varies within and between genomes. Within genomes, tracts of nucleotides, including short sequence repeats and palindromes, can cause localised elevation of mutation rate. Additional mechanisms remain poorly understood. Here we report an instance of extreme mutational bias in Pseudomonas fluorescens SBW25 associated with a single base-pair change in nlpD. These mutants frequently evolve in static microcosms, and have a cell-chaining (CC) phenotype. Analysis of 153 replicate populations revealed 137 independent instances of a C565T loss-of-function mutation at codon 189 (CAG to TAG (Q189*)). Fitness measures of alternative nlpD mutants did not explain the deterministic evolution of C565T mutants. Recognising that transcription can be mutagenic, and that codon 189 overlaps with a predicted promoter (rpoSp) for the adjacent stationary phase sigma factor, rpoS, transcription across this promoter region was measured. This confirmed rpoSp is induced in stationary phase and that C565T mutation caused significant elevation of transcription. The latter provided opportunity to determine the C565T mutation rate using a reporter-gene fused to rpoSp. Fluctuation assays estimate the C565T mutation rate to be ~5,000-fold higher than expected. In Pseudomonas, transcription of rpoS requires the positive activator PsrA, which we show also holds for SBW25. Fluctuation assays performed in a ∆psrA background showed a ~60-fold reduction in mutation rate confirming that the elevated rate of mutation at C565T mutation rate is dependent on induction of transcription. This hotspot suggests a generalisable phenomenon where the induction of transcription causes elevated mutation rates within defining regions of promoters.

High-throughput method characterizes hundreds of previously unknown antibiotic resistance mutations

A fundamental obstacle to tackling the antimicrobial resistance crisis is identifying mutations that lead to resistance in a given genomic background and environment. We present a high-throughput technique – Quantitative Mutational Scan sequencing (QMS-seq) – that enables quantitative comparison of which genes are under antibiotic selection and captures how genetic background influences resistance evolution. We compare four E. coli strains exposed to ciprofloxacin, cycloserine, or nitrofurantoin and identify 812 resistance mutations, many in genes and regulatory regions not previously associated with resistance. We find that multi-drug and antibiotic-specific resistance are acquired through categorically different types of mutations, and that minor genotypic differences significantly influence evolutionary routes to resistance. By quantifying mutation frequency with single base pair resolution, QMS-seq informs about the underlying mechanisms of resistance and identifies mutational hotspots within genes. Our method provides a way to rapidly screen for resistance mutations while assessing the impact of multiple confounding factors.

Identification of Streptomycin-Resistant Erwinia amylovora in Iowa.

Erwinia amylovora is a bacterial pathogen that causes fire blight, an important disease in apples and pears. Applying the antibiotic streptomycin during the phenological bloom stage is considered the most effective management tactic for fire blight. Although streptomycin-resistant (SmR) E. amylovora populations have emerged in major U.S. apple-producing regions, antibiotic resistance data for medium- to small-sized apple-producing regions such as the Midwest are still lacking. This Short Communication article collected symptomatic fire blight samples from Iowa apple orchards during 2022 and 2023, where recent fire blight outbreaks persisted despite streptomycin use. Among E. amylovora isolates from seven counties in Central and Eastern Iowa, approximately 90% of them were SmR. All SmR isolates exhibited a single base pair mutation in codon 43 of the rpsL gene, conferring resistance to streptomycin levels exceeding 1,000 μg/ml. Through clustered regularly interspaced short palindromic repeat (CRISPR) analysis, we characterized two E. amylovora genotypes unique to our region. Whole genome sequencing of one representative SmR isolate, IA01, confirmed its CRISPR genotype, and subsequent phylogenetic analysis suggested that IA01 is genetically similar to Michigan isolates and distinct from those in eastern and western regions of North America. Furthermore, the disease-causing ability of IA01 was comparable to that of the highly virulent Ea110 strain, a streptomycin-sensitive strain isolated from Michigan, in immature pears. Overall, this study underscores the urgent need for regional strategies to address antibiotic resistance and provides insights into its genetic basis and geographic distribution, which are crucial for sustainable orchard management.

Solid-State Nanopore Real-Time Assay for Monitoring Cas9 Endonuclease Reactivity.

The field of nanopore sensing is now moving beyond nucleic acid sequencing. An exciting avenue is the use of nanopore platforms for the monitoring of biochemical reactions. Biological nanopores have been used for this application, but solid-state nanopore approaches have lagged. This is due to the necessity of using higher salt conditions (e.g., 4 M LiCl) to improve the signal-to-noise ratio which completely abolish the activities of many biochemical reactions. We pioneered a polymer electrolyte solid-state nanopore approach that maintains a high signal-to-noise ratio even at a physiologically relevant salt concentration. Here, we report the monitoring of the restriction enzyme SwaI and CRISPR-Cas9 endonuclease activities under physiological salt conditions and in real time. We investigated the dsDNA cleavage activity of these enzymes in a range of digestion buffers and elucidated the off-target activity of CRISPR-Cas9 ribonucleoprotein endonuclease in the presence of single base pair mismatches. This approach enables the application of solid-state nanopores for the dynamic monitoring of biochemical reactions under physiological salt conditions.

The topography of nullomer-emerging mutations and their relevance to human disease.

The topography of nullomer-emerging mutations and their relevance to human disease.

Recognition of mixed-sequence double-stranded DNA regions using chimeric Invader/LNA probes.

Development of robust oligonucleotide-based probe technologies, capable of recognizing specific regions of double-stranded DNA (dsDNA) targets, continues to attract considerable attention due to the promise of tools for modulation of gene expression, diagnostic agents, and new modalities against genetic diseases. Our laboratory pursues the development of various strand-invading probes. These include Invader probes, i.e., double-stranded oligonucleotide probes with one or more +1 interstrand zipper arrangements of intercalator-functionalized nucleotides like 2'-O-(pyren-1-yl)methyl-RNA monomers, and chimeric Invader/γPNA probes, i.e., heteroduplex probes between individual Invader strands and complementary γPNA strands. Here we report on the biophysical properties and dsDNA-recognition characteristics of a new class of chimeric probes-chimeric Invader/LNA probes-which are comprised of densely modified Invader strands and fully modified complementary LNA strands. The chimeric Invader/LNA probes form labile and distorted heteroduplexes, due to an apparent incompatibility between intercalating pyrene moieties and LNA strands. In contrast, the individual Invader and LNA strands form very stable duplexes with complementary DNA, which provides the driving force for near-stoichiometric recognition of model double-stranded DNA targets with single base-pair accuracy. The distinctive properties of chimeric Invader/LNA probes unlock exciting possibilities in molecular biology, and diagnostic and therapeutic fields.

Nucleosome Spacing Can Fine-Tune Higher Order Chromatin Assembly.

Cellular chromatin displays heterogeneous structure and dynamics, properties that control diverse nuclear processes. Models invoke phase separation of conformational ensembles of chromatin fibers as a mechanism regulating chromatin organization in vivo. Here we combine biochemistry and molecular dynamics simulations to examine, at single base-pair resolution, how nucleosome spacing controls chromatin phase separation. We show that as DNA linkers extend from 25 bp to 30 bp, as examplars of 10N+5 and 10N (integer N) bp lengths, chromatin condensates become less thermodynamically stable and nucleosome mobility increases. Simulations reveal that this is due to trade-offs between inter- and intramolecular nucleosome stacking, favored by rigid 10N+5 and 10N bp linkers, respectively. A remodeler can induce or inhibit phase separation by moving nucleosomes, changing the balance between intra- and intermolecular stacking. The intrinsic phase separation capacity of chromatin enables fine tuning of compaction and dynamics, likely contributing to heterogeneous chromatin organization in vivo.

Predictive Modeling of Gene Expression and Localization of DNA Binding Site Using Deep Convolutional Neural Networks.

Despite the sequencing revolution, large swaths of the genomes sequenced to date lack any information about the arrangement of transcription factor binding sites on regulatory DNA. Massively Parallel Reporter Assays (MPRAs) have the potential to dramatically accelerate our genomic annotations by making it possible to measure the gene expression levels driven by thousands of mutational variants of a regulatory region. However, the interpretation of such data often assumes that each base pair in a regulatory sequence contributes independently to gene expression. To enable the analysis of this data in a manner that accounts for possible correlations between distant bases along a regulatory sequence, we developed the Deep learning Adaptable Regulatory Sequence Identifier (DARSI). This convolutional neural network leverages MPRA data to predict gene expression levels directly from raw regulatory DNA sequences. By harnessing this predictive capacity, DARSI systematically identifies transcription factor binding sites within regulatory regions at single-base pair resolution. To validate its predictions, we benchmarked DARSI against curated databases, confirming its accuracy in predicting transcription factor binding sites. Additionally, DARSI predicted novel unmapped binding sites, paving the way for future experimental efforts to confirm the existence of these binding sites and to identify the transcription factors that target those sites. Thus, by automating and improving the annotation of regulatory regions, DARSI generates experimentally actionable predictions that can feed iterations of the theory-experiment cycle aimed at reaching a predictive understanding of transcriptional control.

WITHDRAWN: Mechanism of genome editing tools and their application on genetic inheritance disorders

WITHDRAWN: Mechanism of genome editing tools and their application on genetic inheritance disorders

Selection and Characterization of Antibodies Recognizing Unnatural Base Pairs

Background: Introducing unnatural base pairs into a natural, double-stranded DNA construct is a powerful tool within synthetic biology. Accordingly, the ability to detect these unnatural base pairs has many applications, including the study and detection of semisynthetic organisms. Objective and Methods: The work described here aimed to select human antibodies for the specific recognition of Hirao’s base pair dDs–dPn in various natural DNA contexts by using a combination of phage and yeast display technologies. Results: We selected a total of six antibodies in yeast-displayed scFv format, and further tested three of these antibodies in soluble form as minibodies and IgGs. We also describe an assay that can be used to detect plasmids containing dDs–dPn pair. Conclusions: Our antibodies did not afford the desired specificity or sensitivity for detection of a single unnatural base pair among thousands of natural. However, our data indicate not only that such detection is possible but also that these antibodies may be candidates for further affinity and specificity maturation.