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RNA sequences encode structures that impact protein production and other cellular processes. Misfolded RNAs can also potentiate disease, but a complete picture is lacking. To establish more comprehensive and accurate RNA structure-function relationships, new methods are needed to interrogate RNA in native environments. Existing tools rely primarily on electrophiles that are constitutively on or triggered by UV light, often resulting in high background. Here we describe an alternative, chemically triggered approach to cross-link RNAs using bioorthogonal cyclopropenones (CpOs). These reagents selectively react with phosphines to provide ketenes─electrophiles that can trap neighboring nucleophiles to forge covalent cross-links. As a proof-of-concept, we conjugated a CpO motif to thiazole orange (TO-1). TO-1-CpO bound selectively to a model RNA aptamer (Mango) with nanomolar affinity, as confirmed by fluorescence turn-on. After phosphine administration, covalent cross-links were formed between the CpO and RNA. Cross-linking was both time and dose dependent. We further applied the chemically triggered tools to model RNAs under biologically relevant conditions. Collectively, this work expands the toolkit of probes for studying RNA and its native conformations.

A complete understanding of RNA biology requires methods for tracking transcripts in vivo. Common strategies rely on fluorogenic probes that are limited in sensitivity, dynamic range, and depth of interrogation, owing to their need for excitation light and tissue autofluorescence. To overcome these challenges, we report a bioluminescent platform for serial imaging of RNAs. The RNA tags are engineered to recruit light-emitting luciferase fragments (termed RNA lanterns) upon transcription. Robust photon production is observed for RNA targets both in cells and in live animals. Importantly, only a single copy of the tag is necessary for sensitive detection, in sharp contrast to fluorescent platforms requiring multiple repeats. Overall, this work provides a foundational platform for visualizing RNA dynamics from the micro to the macro scale.

OBJECTIVE: Currently, diagnosis of cerebrospinal fluid (CSF) rhinorrhea relies on a multimodal approach, increasing costs and ultimately delaying diagnosis. In the United States and internationally, the crux of such a diagnosis relies on confirmation testing (via biomarkers) and localization (e.g., imaging). Biomarker testing may require analysis at an outside facility, resulting in delays diagnosis and treatment. In addition, specialized imaging may be nonspecific and often requires an active leak for diagnosis. There remains a clear need for innovative new technology. METHODS: A comprehensive review was conducted on both foundational and innovative scholarly articles regarding current and emerging diagnosis modalities for CSF. RESULTS: Current modalities in CSF rhinorrhea diagnosis and localization include laboratory tests (namely, B2T immunofixation), imaging (CT and/or MRI) with or without intrathecal administration, and surgical exploration. Each of these modalities carry flaws, risks, and benefits, ultimately contributing to delays in diagnosis and morbidity. Promising emerging technologies include lateral flow immunoassays (LFI) and biologically functionalized field-effect transistors (BioFET). Nevertheless, these carry some drawbacks of their own, and require further validation. CONCLUSION: CSF rhinorrhea remains a challenging diagnosis, requiring a multimodal approach to differentiate from nonpathologic causes of rhinorrhea. Current methods in diagnosis are imperfect, as the ideal test would be a readily accessible, inexpensive, rapid, highly accurate point-of-care test without the need for excess fluid or specialized processing. Critical work is being done to develop promising, new, improved tests, though a clear successor has not yet emerged. LEVEL OF EVIDENCE: N/A.

Abstract Objective Currently, diagnosis of cerebrospinal fluid (CSF) rhinorrhea relies on a multimodal approach, increasing costs and ultimately delaying diagnosis. In the United States and internationally, the crux of such a diagnosis relies on confirmation testing (via biomarkers) and localization (e.g., imaging). Biomarker testing may require analysis at an outside facility, resulting in delays diagnosis and treatment. In addition, specialized imaging may be nonspecific and often requires an active leak for diagnosis. There remains a clear need for innovative new technology. Methods A comprehensive review was conducted on both foundational and innovative scholarly articles regarding current and emerging diagnosis modalities for CSF. Results Current modalities in CSF rhinorrhea diagnosis and localization include laboratory tests (namely, B2T immunofixation), imaging (CT and/or MRI) with or without intrathecal administration, and surgical exploration. Each of these modalities carry flaws, risks, and benefits, ultimately contributing to delays in diagnosis and morbidity. Promising emerging technologies include lateral flow immunoassays (LFI) and biologically functionalized field‐effect transistors (BioFET). Nevertheless, these carry some drawbacks of their own, and require further validation. Conclusion CSF rhinorrhea remains a challenging diagnosis, requiring a multimodal approach to differentiate from nonpathologic causes of rhinorrhea. Current methods in diagnosis are imperfect, as the ideal test would be a readily accessible, inexpensive, rapid, highly accurate point‐of‐care test without the need for excess fluid or specialized processing. Critical work is being done to develop promising, new, improved tests, though a clear successor has not yet emerged. Level of Evidence N/A

The SARS-CoV-2 pandemic has brought to light the need for expedient diagnostic testing. Cost and availability of large-scale testing capacity has led to a lag in turnaround time and hindered contact tracing efforts, resulting in a further spread of SARS-CoV-2. To increase the speed and frequency of testing, we developed a cost-effective single-tube approach for collection, denaturation, and analysis of clinical samples. The approach utilizes 1 µL microbiological inoculation loops to collect saliva, sodium dodecyl sulfate (SDS) to inactivate and release viral genomic RNA, and a diagnostic reaction mix containing polysorbate 80 (Tween 80). In the same tube, the SDS-denatured clinical samples are introduced to the mixtures containing all components for nucleic acids detection and Tween 80 micelles to absorb the SDS and allow enzymatic reactions to proceed, obviating the need for further handling of the samples. The samples can be collected by the tested individuals, further decreasing the need for trained personnel to administer the test. We validated this single-tube sample-to-assay method with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) and discovered little-to-no difference between Tween- and SDS-containing reaction mixtures, compared to control reactions. This approach reduces the logistical burden of traditional large-scale testing and provides a method of deployable point-of-care diagnostics to increase testing frequency.

Various self-cleaving ribozymes appearing in nature catalyze the sequence-specific intramolecular cleavage of RNA and can be engineered to catalyze cleavage of appropriate substrates in an intermolecular fashion, thus acting as true catalysts. The mechanisms of the small, self-cleaving ribozymes have been extensively studied and reviewed previously. Self-cleaving ribozymes can possess high catalytic activity and high substrate specificity; however, substrate specificity is also engineerable within the constraints of the ribozyme structure. While these ribozymes share a common fundamental catalytic mechanism, each ribozyme family has a unique overall architecture and active site organization, indicating that several distinct structures yield this chemical activity. The multitude of catalytic structures, combined with some flexibility in substrate specificity within each family, suggests that such catalytic RNAs, taken together, could access a wide variety of substrates. Here, we give an overview of 10 classes of self-cleaving ribozymes and capture what is understood about their substrate specificity and synthetic applications. Evolution of these ribozymes in an RNA world might be characterized by the emergence of a new ribozyme family followed by rapid adaptation or diversification for specific substrates.

Self-cleaving ribozymes are RNA molecules that catalyze a site-specific self-scission reaction. Analysis of self-cleavage is a crucial aspect of the biochemical study and understanding of these molecules. Here we describe a co-transcriptional assay that allows the analysis of self-cleaving ribozymes in different reaction conditions and in the presence of desired ligands and/or cofactors. Utilizing a standard T7 RNA polymerase in vitro transcription system under limiting Mg2+ concentration, followed by a 25-fold dilution of the reaction in desired conditions of self-cleavage (buffer, ions, ligands, pH, temperature, etc.) to halt the synthesis of new RNA molecules, allows the study of self-scission of these molecules without the need for purification or additional preparation steps, such as refolding procedures. Furthermore, because the transcripts are not denatured, this assay likely yields RNAs in conformations relevant to co-transcriptionally folded species in vivo.

RNA molecules can be conveniently synthesized in vitro by the T7 RNA polymerase (T7 RNAP). In some experiments, such as cotranscriptional biochemical analyses, continuous synthesis of RNA is not desired. Here, we propose a method for a single-pass transcription that yields a single transcript per template DNA molecule using the T7 RNAP system. We hypothesized that stalling the polymerase downstream from the promoter region and subsequent cleavage of the promoter by a restriction enzyme (to prevent promoter binding by another polymerase) would allow synchronized production of a single transcript per template. The single-pass transcription was verified in two different scenarios: a short self-cleaving ribozyme and a long mRNA. The results show that a controlled single-pass transcription using T7 RNAP allows precise measurement of cotranscriptional ribozyme activity, and this approach will facilitate the study of other kinetic events.

Laboratory evolution of functional RNAs has applications in many areas of chemical and synthetic biology. In vitro selections critically depend on the presence of functional molecules, such as aptamers and ribozymes, in the starting sequence pools. For selection of novel functions the pools are typically transcribed from random-sequence DNA templates, yielding a highly diverse set of RNAs that contain a multitude of folds and biochemical activities. The phenotypic potential, the frequency of functional RNAs, is very low, requiring large complexity of starting pools, surpassing 1015 different sequences, to identify highly active isolates. Furthermore, the majority of random sequences is not structured and has a high propensity for aggregation; the in vitro selection process thus involves not just enrichment of functional RNAs, but also their purification from aggregation-prone "free-riders". We reasoned that purification of the nonaggregating, monomeric subpopulation of a random-sequence RNA pool will yield pools of folded, functional RNAs. We performed six rounds of selection for monomeric sequences and show that the enriched population is compactly folded. In vitro selections originating from various mixtures of the compact pool and a fully random pool showed that sequences from the compact pool always dominate the population once a biochemical activity is detectable. A head-to-head competition of the two pools starting from a low (5 × 1012) sequence diversity revealed that the phenotypic potential of the compact pool is about 1000-times higher than the fully random pool. A selection for folded and monomeric RNA pools thus greatly increases the frequency of functional RNAs from that seen in random-sequence pools, providing a facile experimental approach to isolation of highly active functional RNAs from low-diversity populations.

An international consortium of radiocarbon laboratories has established the origin of the Church of St. Margaret of Antioch in Kopčany (Slovakia), because its age was not well known from previous investigations. In total, 13 samples of charcoal, wood, mortar, and plaster were analyzed. The 14C results obtained from the different laboratories, as well as between the different sample types, were in good agreement. Resulting the final 14C calibrated age of the Church, based on dating a single piece of a wooden levelling rod is 774–884 AD (95.4% confidence level), which is in very good agreement with Bayesian modeling result based on dating of wood, charcoal and mortar samples (788–884 AD, 95.4% confidence level). The probability distribution from OxCal calibration shows that 79% of the probability distribution lies in the period before 863 AD, implying that the Church could have been constructed before the arrival of Constantine (St. Cyril) and St. Methodius to Great Moravia. If we take as the terminus post quem the documented date of consecration of the church in Nitrava (828 AD), the Bayesian modeling suggests the age of the Church in the range of 837–884 AD (95.4% confidence level). Although the 14C results have very good precision, the specific plateau shape of the calibration curve in this period caused a wide range of the calibrated age. The Church represents, together with the St. George's Rotunda in Nitrianska Blatnica, probably the oldest standing purpose-built Christian church in the eastern part of Central Europe. [ABSTRACT FROM AUTHOR]

This paper is focused on the permeability of cement composite samples with various admixtures. Permeability was examined by the rapid chloride permeability test due to the simple and relatively quick performance among the many methods. Permeability is one of the durability parameters considering the pore system of the composite structure. Ion diffusion provides information about inner pore structure by passing a charge through the sample. In real-life conditions, not only chlorides but also other ions can penetrate into the structure and cause corrosion. Various cement supplements were used as admixtures. The reference sample consisted of cement, fine sand, and water, while the rest of the samples consisted of 20% blast furnace slag, bypass dust, eggshells or recycled glass instead of the cement. The results showed lower permeability in samples containing blast furnace slag and eggshells and a higher charge passage in samples with recycled glass and bypass dust than for the reference sample.

Aptamers are small, functional nucleic acids that bind a variety of targets, often with high specificity and affinity. Genomic aptamers constitute the ligand-binding domains of riboswitches, whereas synthetic aptamers find applications as diagnostic and therapeutic tools, and as ligand-binding domains of regulatory RNAs in synthetic biology. Discovery and characterization of aptamers has been limited by a lack of high-throughput approaches that uncover the target-binding domains and the biochemical properties of individual sequences. With the advent of high-throughput sequencing, large-scale analysis of in vitro selected populations of aptamers (and catalytic nucleic acids, such as ribozymes and DNAzmes) became possible. In recent years the development of new experimental approaches and software tools has led to significant streamlining of the selection-pool analysis. This article provides an overview of post-selection data analysis and describes high-throughput methods that facilitate rapid discovery and biochemical characterization of aptamers.

Aptamers are small, functional nucleic acids that bind a variety of targets, often with high specificity and affinity. Genomic aptamers constitute the ligand-binding domains of riboswitches, whereas synthetic aptamers find applications as diagnostic and therapeutic tools, and as ligand-binding domains of regulatory RNAs in synthetic biology. Discovery and characterization of aptamers has been limited by a lack of high-throughput approaches that uncover the target-binding domains and the biochemical properties of individual sequences. With the advent of high-throughput sequencing, large-scale analysis of in vitro selected populations of aptamers (and catalytic nucleic acids, such as ribozymes and DNAzmes) became possible. In recent years the development of new experimental approaches and software tools has led to significant streamlining of the selection-pool analysis. This article provides an overview of post-selection data analysis and describes high-throughput methods that facilitate rapid discovery and biochemical characterization of aptamers.

Abstract The SARS-CoV-2 pandemic has brought to light the need for expedient diagnostic testing. Cost and availability of large-scale testing capacity has led to a lag in turnaround time and hindered contact tracing efforts, resulting in a further spread of SARS-CoV-2. To increase the speed and frequency of testing, we developed a cost-effective single-tube approach for collection, denaturation, and analysis of clinical samples. The approach utilizes 1 µL microbiological inoculation loops to collect saliva, sodium dodecyl sulfate (SDS) to inactivate and release viral genomic RNA, and a diagnostic reaction mix containing polysorbate 80 (Tween 80). In the same tube, the SDS-denatured clinical samples are introduced to the mixtures containing all components for nucleic acids detection and Tween 80 micelles to absorb the SDS and allow enzymatic reactions to proceed, obviating the need for further handling of the samples. The samples can be collected by the tested individuals, further decreasing the need for trained personnel to administer the test. We validated this single-tube sample-to-assay method with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) and discovered little-to-no difference between Tween- and SDS-containing reaction mixtures, compared to control reactions. This approach reduces the logistical burden of traditional large-scale testing and provides a method of deployable point-of-care diagnostics to increase testing frequency.

Hepatitis delta virus (HDV)-like ribozymes are self-cleaving catalytic RNAs with a widespread distribution in nature and biological roles ranging from self-scission during rolling-circle replication in viroids to co-transcriptional processing of eukaryotic retrotransposons, among others. The ribozymes fold into a double pseudoknot with a common catalytic core motif and highly variable peripheral domains. Like other self-cleaving ribozymes, HDV-like ribozymes can be converted into trans-acting catalytic RNAs by bisecting the self-cleaving variants at non-essential loops. Here we explore the trans-cleaving activity of ribozymes derived from the largest examples of the ribozymes (drz-Agam-2 family), which contain an extended domain between the substrate strand and the rest of the RNA. When this peripheral domain is bisected at its distal end, the substrate strand is recognized through two helices, rather than just one 7 bp helix common among the HDV ribozymes, resulting in stronger binding and increased sequence specificity. Kinetic characterization of the extended trans-cleaving ribozyme revealed an efficient trans-cleaving system with a surprisingly high KM', supporting a model that includes a recently proposed activation barrier related to the assembly of the catalytically competent ribozyme. The ribozymes also exhibit a very long koff for the products (∼2 weeks), resulting in a trade-off between sequence specificity and turnover. Finally, structure-based searches for the catalytic cores of these ribozymes in the genome of the mosquito Anopheles gambiae, combined with sequence searches for their putative substrates, revealed two potential ribozyme-substrate pairs that may represent examples of natural trans-cleaving ribozymes.

The activity of DNA polymerase underlies numerous biotechnologies, cell division, and therapeutics, yet the enzyme remains incompletely understood. We demonstrate that both thermostable and mesophilic DNA polymerases readily utilize deoxyribonucleoside diphosphates (dNDPs) for DNA synthesis and inorganic phosphate for the reverse reaction, that is, phosphorolysis of DNA. For Taq DNA polymerase, the KMs of the dNDP and phosphate substrates are ∼20 and 200 times higher than for dNTP and pyrophosphate, respectively. DNA synthesis from dNDPs is about 17 times slower than from dNTPs, and DNA phosphorolysis about 200 times less efficient than pyrophosphorolysis. Such parameters allow DNA replication without requiring coupled metabolism to sequester the phosphate products, which consequently do not pose a threat to genome stability. This mechanism contrasts with DNA synthesis from dNTPs, which yield high-energy pyrophosphates that have to be hydrolyzed to phosphates to prevent the reverse reaction. Because the last common ancestor was likely a thermophile, dNDPs are plausible substrates for genome replication on early Earth and may represent metabolic intermediates later replaced by the higher-energy triphosphates.

Self-cleaving ribozymes were discovered 30 years ago and have been found throughout nature, from bacteria to animals, but little is known about their biological functions and regulation, particularly how cofactors and metabolites alter their activity. A hepatitis delta virus-like self-cleaving ribozyme maps upstream of a phosphoglucosamine mutase (glmM) open reading frame in the genome of the human gut bacterium Faecalibacterium prausnitzii. The presence of a ribozyme in the untranslated region of glmM suggests a regulation mechanism of gene expression. In the bacterial hexosamine biosynthesis pathway, the enzyme glmM catalyzes the isomerization of glucosamine 6-phosphate into glucosamine 1-phosphate. In this study, we investigated the effect of these metabolites on the co-transcriptional self-cleavage rate of the ribozyme. Our results suggest that glucosamine 6-phosphate, but not glucosamine 1-phosphate, is an allosteric ligand that increases the self-cleavage rate of drz-Fpra-1, providing the first known example of allosteric modulation of a self-cleaving ribozyme by the substrate of the adjacent gene product. Given that the ribozyme is activated by the glmM substrate, but not the product, this allosteric modulation may represent a potential feed-forward mechanism of gene expression regulation in bacteria.

Laboratory-evolved RNAs bind a wide variety of targets and serve highly diverse functions, including as diagnostic and therapeutic aptamers. The majority of aptamers have been identified using in vitro selection (SELEX), a molecular evolution technique based on selecting target-binding RNAs from highly diverse pools through serial rounds of enrichment and amplification. In vitro selection typically yields multiple distinct motifs of highly variable abundance and target-binding affinities. The discovery of new aptamers is often limited by the difficulty of characterizing the selected motifs, because testing of individual sequences tends to be a tedious process. To facilitate the discovery of new aptamers within in vitro selected pools, we developed Apta-Seq, a multiplex analysis based on quantitative, ligand-dependent 2' acylation of solvent-accessible regions of the selected RNA pools, followed by reverse transcription (SHAPE) and deep sequencing. The method reveals, in a single sequencing experiment, the identity, structural features, and target dissociation constants for aptamers present in the selected pool. Application of Apta-Seq to a human genomic pool enriched for ATP-binding RNAs yielded three new aptamers, which together with previously identified human aptamers suggest that ligand-binding RNAs may be common in mammals.

BackgroundIn this paper, we study the problem of RNA motif search in long genomic sequences. This approach uses a combination of sequence and structure constraints to uncover new distant homologs of known functional RNAs. The problem is NP-hard and is traditionally solved by backtracking algorithms.ResultsWe have designed a new algorithm for RNA motif search and implemented a new motif search tool RNArobo. The tool enhances the RNAbob descriptor language, allowing insertions in helices, which enables better characterization of ribozymes and aptamers. A typical RNA motif consists of multiple elements and the running time of the algorithm is highly dependent on their ordering. By approaching the element ordering problem in a principled way, we demonstrate more than 100-fold speedup of the search for complex motifs compared to previously published tools.ConclusionsWe have developed a new method for RNA motif search that allows for a significant speedup of the search of complex motifs that include pseudoknots. Such speed improvements are crucial at a time when the rate of DNA sequencing outpaces growth in computing. RNArobo is available at http://compbio.fmph.uniba.sk/rnarobo .

A two-step templated, ribosomal biosynthesis/printing method for the fabrication of protein microarrays for surface plasmon resonance imaging (SPRI) measurements is demonstrated. In the first step, a sixteen component microarray of proteins is created in microwells by cell free on chip protein synthesis; each microwell contains both an in vitro transcription and translation (IVTT) solution and 350 femtomoles of a specific DNA template sequence that together are used to create approximately 40 picomoles of a specific hexahistidine-tagged protein. In the second step, the protein microwell array is used to contact print one or more protein microarrays onto nitrilotriacetic acid (NTA)-functionalized gold thin film SPRI chips for real-time SPRI surface bioaffinity adsorption measurements. Even though each microwell array element only contains approximately 40 picomoles of protein, the concentration is sufficiently high for the efficient bioaffinity adsorption and capture of the approximately 100 femtomoles of hexahistidine-tagged protein required to create each SPRI microarray element. As a first example, the protein biosynthesis process is verified with fluorescence imaging measurements of a microwell array containing His-tagged green fluorescent protein (GFP), yellow fluorescent protein (YFP) and mCherry (RFP), and then the fidelity of SPRI chips printed from this protein microwell array is ascertained by measuring the real-time adsorption of various antibodies specific to these three structurally related proteins. This greatly simplified two-step synthesis/printing fabrication methodology eliminates most of the handling, purification and processing steps normally required in the synthesis of multiple protein probes, and enables the rapid fabrication of SPRI protein microarrays from DNA templates for the study of protein-protein bioaffinity interactions.

As with any outcome of an evolutionary process, the success of in vitro selection experiments depends critically on the starting population. In vitro selections isolate functional nucleic acids that fold into specific structures and form unique binding and catalytic sites. The selection outcomes therefore depend on the molecular and structural diversity of the initial pools. In addition, the experiments are strongly influenced by the length of the starting pool. Longer randomized regions support the formation of more complex structures and presumably allow formation of more intricate tertiary interactions, but they also tend to misfold and aggregate, whereas shorter pools are sufficient to yield simpler motifs. Furthermore, introducing a sequence bias that promotes secondary structure formation appears to prejudice the population towards more functional macromolecules. We review the literature on the influence of the starting pools on the predicted and actual outcomes of laboratory evolution experiments.

Self-cleaving ribozymes fold into intricate structures, which orient active site groups into catalytically competent conformations. Most ribozyme families have distinct catalytic cores stabilized by tertiary interactions between domains peripheral to those cores. We show that large hepatitis delta virus (HDV)-like ribozymes are activated by peripheral domains that bring two helical segments, P1 and P2, into proximity - a "pinch" that results in rate acceleration by almost three orders of magnitude. Kinetic analysis of ribozymes with systematically altered length and stability of the peripheral domain revealed that about one third of its free energy of formation is used to lower an activation energy barrier, likely related to a rate-limiting conformational change leading to the pre-catalytic state. These findings provide a quantitative view of enzyme regulation by peripheral domains and may shed light on the energetics of allosteric regulation.

Many nucleic acids and proteins require divalent metal ions such as Mg(2+) and Ca(2+) for folding and function. The lipophilic alignment media frequently used as membrane mimetics also bind these divalent metals. Here we demonstrate the use of (31)P NMR spectrum of a metal ion chelator (deoxycytidine diphosphate) to measure the bound [Mg(2+)] and [Ca(2+)] in situ for several biological model systems at relatively high divalent ion concentrations (1-10 mM). This method represents a general approach to measuring divalent metal ion binding in NMR samples where the amount and type of metal ion added to the system is known.

Background: Several authors have investigated the relationship between obesity – assessed only by body mass index (BMI) – and overactive bladder (OAB) symptoms. Objectives: The objective of this study was to determine the relationship between body fat percentage (BFP) and the severity of OAB symptoms with an impact on quality of life. Design: Cross-sectional study. The sample consisted of 206 overweight women; they were university students (BMI = 25.8 ± 3.0) with an average age of 30.6 ± 2.4 years. Body composition analysis was used, including assessment of BFP, visceral fat area (cm2/level), and other parameters. OAB symptoms were evaluated with an OAB questionnaire (OAB-q), voiding diary, and quality of life scale (I-QoL). Results: Ninety women had a BFP >32% and 116 had a BFP 32% (p < 0.01). The Patient Perception of Intensity of Urgency Scale significantly correlated with BFP (r = 0.466, p < 0.001). Women with a BFP above 32% had a 1.95 times greater chance of developing OAB (odds ratio = 1.95, 95% CI = 1.09–3.52, p < 0.02). Conclusion: Young women with a BFP >32% were 95% more likely to have OAB than other young women with a BPF