Biology subjects

Barnes, S. A.

Publications and source records attributed to Barnes, S. A..

4 recordsLinked to original sources

bioRxiv2026

Fluorescently guided workflow with rationally engineered 5' ligation adapters for high-sensitivity and low-bias small RNA sequencing

MicroRNAs (miRNAs) act as key regulators of gene expression across diverse cellular processes, and their precise quantification can provide unique insight into disease pathogenesis. High-throughput sequencing allows for comprehensive small RNA profiling; however, standard commercial library preparation workflows are challenged by issues of low sensitivity and representational bias, limiting reliable profiling, especially in scenarios where samples are scarce. Several structural studies have shown that this bias primarily arises due to sequence and secondary structure variations between miRNAs and adapters during enzyme-catalyzed biochemical reactions. In this work, we propose a new approach to ligation adapter engineering using a bioinformatic analysis of the human miRNome to rationally design structure-forcing 5 adapters, that physically override localized, unpredictable structural variations during the intermediate ligation state. We show that this approach combined with a practical fluorescence-guided workflow, utilizing a fluorescently-labeled 3 adapter and novel Fluorescent Ligation Rulers (FLRs) to guide precise band excision, can minimize representational bias and increase the sensitivity of small RNA sequencing from low-input biological matrices. In comprehensive benchmarks using a synthetic panel, this method significantly reduced bias and outperformed alternative commercial protocols. Finally, we demonstrate that this workflow enhances biomarker detection and library quality in challenging clinical matrices, especially in cerebrospinal fluid. Overall, this protocol enables highly accurate miRNome characterization and is well-suited for biomarker discovery in challenging sample types.

molecular biology↗

bioRxiv2026

Cleavage specificity of E. coli YicC endoribonuclease

Escherichia coli YicC enzyme is the founding member of a family of endoribonucleases that is encoded in virtually all bacterial species. Previous structural studies revealed that this ribonuclease binds RNA by a novel mechanism in which the hexameric apoprotein presents an open channel that undergoes a large rotation upon RNA binding and clamps down on the RNA. The current study follows up on these findings by examining the cleavage of various oligonucleotide substrates designed to probe recognition elements required for YicC binding and cleavage. A 26-nucleotide RNA oligomer (oligo), with a KD in the low micromolar range, was the standard to which numerous oligos with altered sequence were compared. In vitro RNase assays and fluorescence anisotropy binding measurements indicated that the preferred substrates for YicC were relatively small RNAs that contain some secondary structure. Larger RNAs or highly structured RNAs were less-than-optimal substrates. Similarly, RyhB RNA, a [~]90-nucleotide, iron-responsive RNA of E. coli, which has been described as a target of YicC binding and/or cleavage, was a poor YicC substrate in our assays. These results suggest that the native substrates for YicC-family members are very small RNAs or RNA fragments derived from larger RNAs.

molecular biology↗

bioRxiv2025

Methylphenidate-induced reductions in choice impulsivity are associated with alterations in cortico-striatal beta oscillations.

Impulsive choice- preferring small, immediate rewards over larger, delayed reward- is a hallmark of many psychiatric disorders. Methylphenidate, a dopamine and noradrenaline transporter inhibitor, reduces impulsivity, but the neural mechanisms underlying this effect remain unclear. Identifying a neurophysiological signature of methylphenidates action could be used clinically to predict treatment response and guide drug development. To this end, we recorded local field potentials (LFP) from 32 electrodes spanning cortico-striatal circuits in rats performing a temporal discounting task, under saline or methylphenidate treatment conditions. Methylphenidate decreased beta frequency (15-30 Hz) power and connectivity for small, immediate rewards while simultaneously increasing beta power for large, delayed rewards. Reduced connectivity in cortico-striatal networks during small rewards predicted increased preference for delayed rewards. These findings suggest that methylphenidate influences discounting behavior by modulating reward-evoked beta oscillations in cortico-striatal networks. Thus, beta oscillations may serve as a translational biomarker of treatment responsivity to monoaminergic drugs, providing a circuit-level assay to measure and predict changes in impulsive behavior.

neuroscience↗

bioRxiv2024

Non-ionotropic signaling through the NMDA receptor GluN2B carboxy terminal domain drives morphological plasticity of dendritic spines and reverses fragile X phenotypes in mouse hippocampus

It is well known that activation of NMDA receptors can trigger long-term synaptic depression (LTD) and that a morphological correlate of this functional plasticity is spine retraction and elimination. Recent studies have led to the surprising conclusion that NMDA-induced spine shrinkage proceeds independently of ion flux and requires the initiation of de novo protein synthesis, highlighting an unappreciated contribution of mRNA translation to non-ionotropic NMDAR signaling. Here we used NMDA-induced spine shrinkage in slices of mouse hippocampus as a readout to investigate this novel modality of synaptic transmission. By using selective pharmacological and genetic tools, we find that structural plasticity is dependent on the ligand binding domain (LBD) of GluN2B-containing NMDA receptors and that metabotropic signaling occurs via the GluN2B carboxyterminal domain (CTD). Disruption of signaling by replacing the GluN2B CTD with the GluN2A CTD leads to increased spine density, dysregulated basal protein synthesis, and epileptiform activity in area CA3 reminiscent of phenotypes observed in the Fmr1-/y model of fragile X syndrome. By crossing the Fmr1-/y mice with animals in which the GluN2A CTD has been replaced with the GluN2B CTD, we observe a correction of these core fragile X phenotypes. These findings suggest that non-ionotropic NMDAR signaling through GluN2B may represent a novel therapeutic target for treatment of fragile X and related causes of intellectual disability and autism.

neuroscience↗