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Long non-coding RNAs (lncRNAs) work together with diverse RNA-binding proteins (RBPs) to fulfill key regulations in important cellular functions. Here, we present a protocol to detect lncRNA-RBP interactions in vitrousing a tRNA scaffold containing a streptavidin aptamer pull-down assay. We describe steps for preparing both protein and lncRNA transcripts, lncRNA-protein interaction detection with an in vitrobinding assay, and western blot analysis. This protocol is applicable to screen for RNA-interacting proteins using cell lysates followed by mass spectrometry analysis.

Analyses of long non-coding RNA (lncRNA)-protein interactions provide key clues for understanding the molecular basis of lncRNA-modulated biological processes. Here, we detail a yeast three-hybrid assay to identify the lncRNA-interacting protein. We describe steps for lncRNA bait preparation, screening an RNA-binding proteins (RBPs) cDNA library, and validation of the lncRNA-RBP interaction. The assay can also be further applied to delineate the region of RBP that mediates the RNA-protein interaction.

The CRISPR/Cas9 system has been widely applied for plant genome editing. The commonly used SpCas9 has been shown to rely on the protospacer adjacent motif (PAM) sequences in the canonical form NGG and non-canonical NAG. Although these PAM sequences are extensively distributed across plant genomes, a broader scope of PAM sequence is required to expand the range of genome editing. Here we report the adoption of three variant enzymes, xCas9, SpCas9-NG and XNG-Cas9, to produce targeted mutation in soybean. Sequencing results determined that xCas9 with the NGG and KGA (contains TGA and GGA) PAMs successfully induces genome editing in soybean genome. SpCas9-NG could recognize NGD (contains NGG, NGA and NGT), RGC (contains AGC and GGC), GAA and GAT PAM sites. In addition, XNG-Cas9 was observed to cleave soybean genomic regions with NGG, GAA and AGY (contains AGC and AGT) PAM. Moreover, off-target analyses on soybean editing events induced by SpCas9 and xCas9 indicated that two high-fidelity Cas9 variants including eSpCas9 (enhanced specificity SpCas9) and exCas9 (enhanced specificity xCas9) could improve the specificity of the GGA PAM sequence without reducing on-target editing efficiency. These findings significantly expand the scope of Cas9-mediated genome editing in soybean.

Under shock loading, the spall strength of nanocrystals exhibits intricate grain-size effects due to the presence of abundant grain boundary and dislocation activities. However, the influence of size on spall toughness and void evolution has been largely overlooked. This study employs molecular dynamics simulations to investigate the damage accumulation characteristics of nanocrystalline aluminum across various grain sizes. Unlike the trade-off observed in quasi-static loading conditions, our study reveals a consistency in which grain size governs both nanovoid nucleation and coalescence, yielding a novel spall strength–toughness synergy. These insights highlight grain sizes that are particularly susceptible to spall fracture, offering a crucial understanding of nanocrystal failure mechanisms in extreme environments.

Simple sequence repeat (SSR) genetic markers, also referred to as microsatellites, function in map‐based cloning and for marker‐assisted selection in plant breeding. The objectives of this study were to determine the abundance of SSRs in the soybean genome and to develop and test soybean SSR markers to create a database of locus‐specific markers with a high likelihood of polymorphism. A total of 210,990 SSRs with di‐, tri‐, and tetranucleotide repeats of five or more were identified in the soybean whole genome sequence (WGS) which included 61,458 SSRs consisting of repeat units of di‐ (≥10), tri‐ (≥8), and tetranucleotide (≥7). Among the 61,458 SSRs, (AT)n, (ATT)n and (AAAT)n were the most abundant motifs among di‐, tri‐, and tetranucleotide SSRs, respectively. After screening for a number of factors including locus‐specificity using e‐PCR, a soybean SSR database (BARCSOYSSR_1.0) with the genome position and primer sequences for 33,065 SSRs was created. To examine the likelihood that primers in the database would function to amplify locus‐specific polymorphic products, 1034 primer sets were evaluated by amplifying DNAs of seven diverse Glycine max(L.) Merr. and one wild soybean (Glycine sojaSiebold & Zucc.) genotypes. A total of 978 (94.6%) of the primer sets amplified a single polymerase chain reaction (PCR) product and 798 (77.2%) amplified polymorphic amplicons as determined by 4.5% agarose gel electrophoresis. The BARCSOYSSR1.0 SSR markers can be found in SoyBase (http://soybase.org; verified 21 June 2010) the USDA‐ARS Soybean Genome Database.