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Golden Gate Assembly is an efficient and rapid cloning method but requires dedicated vectors. Here, we modified Golden Gate to expand its compatibility to a broader range of destination vectors while maintaining its strengths. Our Expanded Golden Gate (ExGG) assembly adds to the insert(s) type IIS restriction sites that generate protruding ends compatible with traditional type IIP sites on the recipient vector. The ligated product cannot be cleaved again, owing to a single-base change near the junction. This allows the reaction to proceed in a single tube without an intermediate purification step. ExGG can be used to introduce multiple fragments into a vector simultaneously, including shorter fragments (<100 bp) and fragments with shared sequences, which can be difficult to assemble with other fast cloning strategies. Thus, ExGG extends the convenience of Golden Gate to a much larger space of pre-existing vectors designed for conventional cloning., Competing Interests: The authors have no conflict of interest to declare., (© 2023 The Authors.)

We present RBPNet, a novel deep learning method, which predicts CLIP-seq crosslink count distribution from RNA sequence at single-nucleotide resolution. By training on up to a million regions, RBPNet achieves high generalization on eCLIP, iCLIP and miCLIP assays, outperforming state-of-the-art classifiers. RBPNet performs bias correction by modeling the raw signal as a mixture of the protein-specific and background signal. Through model interrogation via Integrated Gradients, RBPNet identifies predictive sub-sequences that correspond to known and novel binding motifs and enables variant-impact scoring via in silico mutagenesis. Together, RBPNet improves imputation of protein-RNA interactions, as well as mechanistic interpretation of predictions., (© 2023. BioMed Central Ltd., part of Springer Nature.)

One nucleotide substitution in codon 140 of HLA-DRB1*04:05:01:01 results in a novel allele, HLA-DRB1*04:358., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The study on nucleic acid retention in ion-pair reversed-phase high-performance liquid chromatography mainly focuses on size-dependence, however, other factors influencing retention behaviors have not been comprehensively clarified up to date. In this present work, the retention behaviors of oligonucleotides and double-stranded DNAs were investigated on silica-based C 18 stationary phase by ion-pair reversed-phase high-performance liquid chromatography. It is found that the retention of oligonucleotides was influenced by base composition and base sequence as well as size, and oligonucleotides prone to self-dimerization have weaker retention than those not prone to self-dimerization but with the same base composition. However, homo-oligonucleotides are suitable for the size-dependent separation as a special case of oligonucleotides. For double-stranded DNAs, the retention is also influenced by base composition and base sequence, as well as size. This may be attributed to the interaction of exposed bases in major or minor grooves with the hydrophobic alky chains of stationary phase. In addition, no specific influence of guanine and cytosine content was confirmed on retention of double-stranded DNAs. Notably, the space effect resulted from the stereostructure of nucleic acids also influences the retention behavior in ion-pair reversed-phase high-performance liquid chromatography., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Guanine is the most strongly oxidized base in DNA; generation of a guanine radical cation as an intermediate in an oxidation reaction leads to migration through a resulting cationic hole in the DNA π-stack until it is trapped by irreversible reaction with water or other free radicals. In the case of normal sequences, the primary position of Guanine oxidations by one-electron oxidants such as carbonate radical anions, BPT(7,8,9,10-tetrahydroxytetrahydrobenzo[a]pyrene), and riboflavin are 5'-G in GG doublets and the central G in a GGG triplet. According to results, the properties of guanine oxidation on abasic site containing sequences are independent from the position of AP(apurinic/apyrimidinic) site in the presence of carbonate radical anions under a short irradiation time, although this radical is exposed to solvent by the existence of an abasic site. The lack of abasic site effect on guanine oxidative damage by the carbonate radical may be due to a sequence-independent property of the initial electron transfer rate in the hole injection step, or may relate to an electron transfer mechanism with large reorganization energy dependency. Consequently, the carbonate radical anions may easily migrate to another single G in the charge re-distribution step. Meanwhile, there is a strong dependency on the presence of an AP(apurinic/apyrimidinic) site in the cleavage patterns of guanine oxidations by physically large oxidizing agents, such as BPT(7,8,9,10-tetrahydroxytetrahydrobenzo[a]pyrene) and riboflavin. These radicals show strong AP(apurinic/apyrimidinic) site dependency and clear G-site selectivity.Communicated by Ramaswamy H. Sarma.

Stimuli responsivity has been extensively pursued in dynamic DNA nanotechnology, due to its incredible application potentials. Among diverse dynamic systems, redox-responsive DNA assembly holds great promise for broad applications, especially considering that redox processes widely exist in various physiological environments. However, only a few studies have been reported on redox-sensitive dynamic DNA assembly. Albeit ingenious, most of these studies are either dependent on the DNA sequence or involve chemical modification. Herein, a facile and universal mechanism to realize redox-responsive self-assembly of DNA nanocages (tetrahedron and cube) driven by the interconversion between cystamine and cysteamine toward dynamic DNA nanotechnology is reported., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Genomic rearrangements, encompassing mutational changes in the genome such as insertions, deletions or inversions, are essential for genetic diversity. These rearrangements are typically orchestrated by enzymes that are involved in fundamental DNA repair processes, such as homologous recombination, or in the transposition of foreign genetic material by viruses and mobile genetic elements1,2. Here we report that IS110 insertion sequences, a family of minimal and autonomous mobile genetic elements, express a structured non-coding RNA that binds specifically to their encoded recombinase. This bridge RNA contains two internal loops encoding nucleotide stretches that base-pair with the target DNA and the donor DNA, which is the IS110 element itself. We demonstrate that the target-binding and donor-binding loops can be independently reprogrammed to direct sequence-specific recombination between two DNA molecules. This modularity enables the insertion of DNA into genomic target sites, as well as programmable DNA excision and inversion. The IS110 bridge recombination system expands the diversity of nucleic-acid-guided systems beyond CRISPR and RNA interference, offering a unified mechanism for the three fundamental DNA rearrangements-insertion, excision and inversion-that are required for genome design.

Populations and species are threatened by human pressure, but their fate is variable. Some depleted populations, such as that of the northern elephant seal (Mirounga angustirostris), recover rapidly even when the surviving population was small. The northern elephant seal was hunted extensively and taken by collectors between the early 1800s and 1892, suffering an extreme population bottleneck as a consequence. Recovery was rapid and now there are over 200,000 individuals. We sequenced 260 modern and 8 historical northern elephant seal nuclear genomes to assess the impact of the population bottleneck on individual northern elephant seals and to better understand their recovery. Here we show that inbreeding, an increase in the frequency of alleles compromised by lost function, and allele frequency distortion, reduced the fitness of breeding males and females, as well as the performance of adult females on foraging migrations. We provide a detailed investigation of the impact of a severe bottleneck on fitness at the genomic level and report on the role of specific gene systems.

Fundoscopy is the standard method for diagnosis and follow-up of intraocular retinoblastoma, but it is sometimes insufficient to discern whether tumors are inactivated following treatments. In this work, we hypothesized that the amount of conserved nuclear DNA sequences in the cell-free DNA (cfDNA) fraction of the aqueous humor (AH) might complement fundoscopy for retinoblastoma follow-up. To address our hypothesis, we developed highly sensitive droplet digital polymerase chain reaction (ddPCR) methods to quantify highly conserved DNA sequences of nucleus-encoded genes (GAPDH and B4GALNT1) and of a mitochondrial gene, MT-ATP6. We obtained AH samples during intravitreal treatments. We analyzed 42 AH samples from 25 patients with intraocular retinoblastoma and 11 AH from controls (non-cancer patients). According to clinical criteria, we grouped patients as having progression-free or progressive retinoblastoma. cfDNA concentration in the AH was similar in both retinoblastoma groups. Copy counts for nucleus-derived sequences of GAPDH and B4GALNT1 were significantly higher in the AH from patients with progressive disease, compared to the AH from progression-free patients and control non-cancer patients. The presence of mitochondrial DNA in the AH explained that both retinoblastoma groups had similar cfDNA concentration in AH. The optimal cut-off point for discriminating between progressive and progression-free retinoblastomas was 108 GAPDH copies per reaction. Among patients having serial AH samples analyzed during their intravitreal chemotherapy, GAPDH copies were high and decreased below the cut-off point in those patients responding to chemotherapy. In contrast, one non-responder patient remained with values above the cut-off during follow-up, until enucleation. We conclude that the measurement of conserved nuclear gene sequences in AH allows follow-up of intraocular retinoblastoma during intravitreal treatment. The method is applicable to all patients and could be relevant for those in which fundoscopy evaluation is inconclusive., (© 2022 The Authors. The Journal of Pathology: Clinical Research published by The Pathological Society of Great Britain and Ireland and John Wiley & Sons Ltd.)

The obligate intracellular bacterium Chlamydia has a unique developmental cycle that alternates between two contrasting cell types. With a hardy envelope and highly condensed genome, the small elementary body (EB) maintains limited metabolic activities yet survives in extracellular environments and is infectious. After entering host cells, EBs differentiate into larger and proliferating reticulate bodies (RBs). Progeny EBs are derived from RBs in late developmental stages and eventually exit host cells. How expression of the chlamydial genome consisting of nearly 1,000 genes governs the chlamydial developmental cycle is unclear. A previous microarray study identified only 29 Chlamydia trachomatis immediate early genes, defined as genes with increased expression during the first hour postinoculation in cultured cells. In this study, we performed more sensitive RNA sequencing (RNA-Seq) analysis for C. trachomatis cultures with high multiplicities of infection. Remarkably, we observed well over 700 C. trachomatis genes that underwent 2- to 900-fold activation within 1 hour postinoculation. Quantitative reverse transcription real-time PCR analysis was further used to validate the activated expression of a large subset of the genes identified by RNA-Seq. Importantly, our results demonstrate that the immediate early transcriptome is over 20 times more extensive than previously realized. Gene ontology analysis indicates that the activated expression spans all functional categories. We conclude that over 70% of C. trachomatis genes are activated in EBs almost immediately upon entry into host cells, thus implicating their importance in initiating rapid differentiation into RBs and establishing an intracellular niche conducive with chlamydial development and growth.

The genus Acacia is a large group of woody legumes containing an enormous amount of morphological diversity in leaf shape. This diversity is at least in part the result of an innovation in leaf development where many Acacia species are capable of developing leaves of both bifacial and unifacial morphologies. While not unique in the plant kingdom, unifaciality is most commonly associated with monocots, and its developmental genetic mechanisms have yet to be explored beyond this group. In this study, we identify an accession of Acacia crassicarpa with high regeneration rates and isolate a clone for genome sequencing. We generate a chromosome-level assembly of this readily transformable clone, and using comparative analyses, confirm a whole-genome duplication unique to Caesalpinoid legumes. This resource will be important for future work examining genome evolution in legumes and the unique developmental genetic mechanisms underlying unifacial morphogenesis in Acacia.

Pathogen lineage nomenclature systems are a key component of effective communication and collaboration for researchers and public health workers. Since February 2021, the Pango dynamic lineage nomenclature for SARS-CoV-2 has been sustained by crowdsourced lineage proposals as new isolates were sequenced. This approach is vulnerable to time-critical delays as well as regional and personal bias. Here we developed a simple heuristic approach for dividing phylogenetic trees into lineages, including the prioritization of key mutations or genes. Our implementation is efficient on extremely large phylogenetic trees consisting of millions of sequences and produces similar results to existing manually curated lineage designations when applied to SARS-CoV-2 and other viruses including chikungunya virus, Venezuelan equine encephalitis virus complex and Zika virus. This method offers a simple, automated and consistent approach to pathogen nomenclature that can assist researchers in developing and maintaining phylogeny-based classifications in the face of ever-increasing genomic datasets.

4-Aminobiphenyl (ABP) and its structure analog 2-aminofluorene (AF) are well-known carcinogens. In the present work, an unusual sequence effect in the 5'-CTTCTG1G2TCCTCATTC-3' DNA duplex is reported for ABP- and AF-modified G. Specifically, the ABP modification at G1 resulted in a mixture of 67% major groove B-type (B) and 33% stacked (S) conformers, while at the ABP modification at G2 exclusively resulted in the B-conformer. The AF modification at G1 and G2 lead to 25%:75% and 83%:17% B:S population ratios, respectively. These differences in preferred conformation are due to an interplay between stabilizing (hydrogen bonding and stacking that is enhanced by lesion planarity) and destabilizing (solvent exposure) forces at the lesion site. Furthermore, while the B-conformer is a thermodynamic stabilizer and the S-conformer is a destabilizer in duplex settings, the situation is reversed at the single strands/double strands (ss/ds) junction. Specifically, the twisted biphenyl is a better stacker at the ss/ds junction than the coplanar AF. Therefore, the ABP modification leads to a stronger strand binding affinity of the ss/ds junction than the AF modification. Overall, the current work provides conformational insights into the role of sequence and lesion effects in modulating DNA replication.

Background: MicroRNA isoforms (isomiRs), tRNA-derived fragments (tRFs), and rRNA-derived fragments (rRFs) represent most of the small non-coding RNAs (sncRNAs) found in cells. Members of these three classes modulate messenger RNA (mRNA) and protein abundance and are dysregulated in diseases. Experimental studies to date have assumed that the subcellular distribution of these molecules is well-understood, independent of cell type, and the same for all isoforms of a sncRNA., Results: We tested these assumptions by investigating the subcellular distribution of isomiRs, tRFs, and rRFs in biological replicates from three cell lines from the same tissue and same-sex donors that model the same cancer subtype. In each cell line, we profiled the isomiRs, tRFs, and rRFs in the nucleus, cytoplasm, whole mitochondrion (MT), mitoplast (MP), and whole cell. Using a rigorous mathematical model we developed, we accounted for cross-fraction contamination and technical errors and adjusted the measured abundances accordingly. Analyses of the adjusted abundances show that isomiRs, tRFs, and rRFs exhibit complex patterns of subcellular distributions. These patterns depend on each sncRNA's exact sequence and the cell type. Even in the same cell line, isoforms of the same sncRNA whose sequences differ by a few nucleotides (nts) can have different subcellular distributions., Conclusions: SncRNAs with similar sequences have different subcellular distributions within and across cell lines, suggesting that each isoform could have a different function. Future computational and experimental studies of isomiRs, tRFs, and rRFs will need to distinguish among each molecule's various isoforms and account for differences in each isoform's subcellular distribution in the cell line at hand. While the findings add to a growing body of evidence that isomiRs, tRFs, rRFs, tRNAs, and rRNAs follow complex intracellular trafficking rules, further investigation is needed to exclude alternative explanations for the observed subcellular distribution of sncRNAs., (© 2024. The Author(s).)

Complete genome sequencing of a virus from a white snakeroot plant (Ageratina altissima (L.) King & H. Rob.) collected in the Great Smoky Mountains National Park, USA, revealed a quadricistronic organization resembling that of umbraviruses. ORFs 1 and 2 are putatively translated via a -1 ribosomal frameshift mechanism as a single polypeptide with a role in viral replication, whereas the 3'-proximal and extensively overlapping ORFs 3 and 4 code for proteins involved in long distance trafficing and cell-to-cell movement within the host. Sequence comparisons and phylogenetic analysis strongly suggested that this virus is a previously undescribed member of the genus Umbravirus (family Tombusviridae), for which the name "white snakeroot virus A" (WSVA) is proposed. In addition, we identified and initiated characterization of its possible helper virus, a putative new member of the genus Luteovirus., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Objective: To explore the genetic etiology of a child with Primary hypertrophic osteoarthropathy., Methods: A child who was admitted to Zhongnan Hospital of Wuhan University on July 27, 2021 was selected as the study subject. Genomic DNA was extracted from peripheral blood samples of the child and his parents and subjected to whole exome sequencing. Suspected splicing variant was verified by Sanger sequencing of family members. In vitro function was validated through a minigene assay, whilst the suspected exonic deletion was validated by long-fragment PCR., Results: Whole exome sequencing revealed that the child has harbored compound heterozygous variants of HPGD gene, including a heterozygous deletion (exon 3 del) derived from his father and a splicing variant (c.421+1G>T) derived from his mother. Long-fragment PCR verified that the child and his father had both harbored a 7 565 bp heterozygous deletion (c.218-1304&#95;324+6156del), whilst the minigene assay proved that the splicing variant has resulted in skipping of exon 4., Conclusion: The heterozygous c.218-1304&#95;324+6156del deletion and the c.421+1G>T splicing variant of the HPGD gene probably underlay the pathogenesis in this child. Above finding has enriched the mutational spectrum of the HPGD gene and provided a basis for genetic counseling and prenatal diagnosis for this family.

A novel grapevine viroid was discovered in an asymptomatic grapevine of Indian rootstocks. The whole genome sequence of the viroid (370 nt) was determined by high-throughput sequencing as well as RT-PCR followed by cloning and Sanger sequencing. The terminal conserved region (TCR), central conserved region (CCR) upper strand, and CCR lower strand are conserved regions found in the viroid that are unique to the members of the genus Apscaviroid. Based on our findings and the demarcation criteria for viroids, the novel viroid, which we have tentatively named "grapevine yellow speckle viroid 3" is a putative new member of the genus Apscaviroid., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Precisely modulating the kinetics of toehold-mediated DNA strand displacements (TMSD) is essential for its application in DNA nanotechnology. The sequence in the toehold region significantly influences the kinetics of TMSD. However, due to the large sample space resulting from various arrangements of base sequences and the resulted complex secondary structures, such a correlation is not intuitive. Herein, machine learning was employed to reveal the relationship between the kinetics of TMSD and the toehold sequence as well as the correlated secondary structure of invader strands. Key factors that influence the rate constant of TMSD were identified, such as the number of free hydrogen bonding sites in the invader, the number of free bases in the toehold, and the number of hydrogen bonds in intermediates. Moreover, a predictive model was constructed, which successfully achieved semi-quantitative prediction of rate constants of TMSD even with subtle distinctions in toehold sequence., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

We report the design of a single RNA sequence capable of adopting one of two ribozyme folds and catalyzing the cleavage and/or ligation of the respective substrates. The RNA is able to change its conformation in response to its environment, hence it is called chameleon ribozyme (CHR). Efficient RNA cleavage of two different substrates as well as RNA ligation by CHR is demonstrated in separate experiments and in a one pot reaction. Our study shows that sequence variants of the hairpin ribozyme intersect with the hammerhead ribozyme and that rather short RNA molecules can have comprehensive conformational flexibility, which is an important feature for the emergence of new functional folds in early evolution., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Chromatin interactions create spatial proximity between distal regulatory elements and target genes in the genome, which has an important impact on gene expression, transcriptional regulation, and phenotypic traits. To date, several methods have been developed for predicting gene expression. However, existing methods do not take into consideration the effect of chromatin interactions on target gene expression, thus potentially reducing the accuracy of gene expression prediction and mining of important regulatory elements. In this study, we developed a highly accurate deep learning-based gene expression prediction model (DeepCBA) based on maize chromatin interaction data. Compared with existing models, DeepCBA exhibits higher accuracy in expression classification and expression value prediction. The average Pearson correlation coefficients (PCCs) for predicting gene expression using gene promoter proximal interactions, proximal-distal interactions, and both proximal and distal interactions were 0.818, 0.625, and 0.929, respectively, representing an increase of 0.357, 0.16, and 0.469 over the PCCs obtained with traditional methods that use only gene proximal sequences. Some important motifs were identified through DeepCBA; they were enriched in open chromatin regions and expression quantitative trait loci and showed clear tissue specificity. Importantly, experimental results for the maize flowering-related gene ZmRap2.7 and the tillering-related gene ZmTb1 demonstrated the feasibility of DeepCBA for exploration of regulatory elements that affect gene expression. Moreover, promoter editing and verification of two reported genes (ZmCLE7 and ZmVTE4) demonstrated the utility of DeepCBA for the precise design of gene expression and even for future intelligent breeding. DeepCBA is available at http://www.deepcba.com/ or http://124.220.197.196/., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Pathological cardiac hypertrophy (CH) may lead to heart failure and sudden death. MicroRNAs (miRNAs) have been documented to play crucial parts in CH. The objective of this research was to discuss the potential along with molecule mechanism of miR-495-3p in CH. In vivo CH model was induced by aortic banding (AB) in rats. Cellular hypertrophy in H9c2 rat cardiomyocytes was stimulated by angiotensin II (Ang II) treatment. Haematoxylin and eosin (HE), echocardiography and immunofluorescence staining were used to examine the alterations in cardiac function. The outcomes showed that miR-495-3p expression was high in rat model as well as in Ang II-stimulated cardiomyocytes. Besides, silenced miR-495-3p attenuated CH both in vitro and in vivo. Mechanically, miR-495-3p bound to pumilio RNA binding family member 2 (Pum2) 3'UTR and silenced its expression. Rescue assays further notarized that Pum2 silence abrogated the inhibitory impacts of miR-495-3p inhibitor on CH. In a word, the present research uncovered that miR-495-3p promoted CH by targeting Pum2. Therefore, miR-495-3p may be a novel therapeutic molecule for this disease.

The ability to transform matter between numerous physical states or shapes without wires or external devices is a major challenge for robotics and materials design. Organisms can transform their shapes using biomolecules carrying specific information and localize at sites where transitions occur. Here, we introduce gel automata, which likewise can transform between a large number of prescribed shapes in response to a combinatorial library of biomolecular instructions. Gel automata are centimeter-scale materials consisting of multiple micro-segments. A library of DNA activator sequences can each reversibly grow or shrink different micro-segments by polymerizing or depolymerizing within them. We develop DNA activator designs that maximize the extent of growth and shrinking, and a photolithography process for precisely fabricating gel automata with elaborate segmentation patterns. Guided by simulations of shape change and neural networks that evaluate gel automata designs, we create gel automata that reversibly transform between multiple, wholly distinct shapes: four different letters and every even or every odd numeral. The sequential and repeated metamorphosis of gel automata demonstrates how soft materials and robots can be digitally programmed and reprogrammed with information-bearing chemical signals., (© 2024. The Author(s).)

Deoxyribonucleic acid triplexes have potential roles in a range of biological processes involving gene and transcriptional regulation. A major challenge in exploiting the formation of these higher-order structures to target genes in vivo is their low stability, which is dependent on many factors including the length and composition of bases in the sequence. Here, different DNA base modifications have been explored, primarily using native mass spectrometry, in efforts to enable stronger binding between the triplex forming oligonucleotide (TFO) and duplex target sites. These modifications can also be used to overcome pyrimidine interruptions in the duplex sequence in promoter regions of genomes, to expand triplex target sequences for antigene therapies. Using model sequences with a single pyrimidine interruption, triplex forming oligonucleotides containing locked nucleic acid base modifications were shown to have a higher triplex binding propensity than DNA-only and dSpacer-containing TFOs. However, the triplex forming ability of these systems was limited by the competitive formation of multiple higher order assemblies. Triplex forming sequences that correspond to specific gene targets from the Pseudomonas aeruginosa genome were also investigated, with LNA-containing TFOs the only variant able to form triplex using these sequences. This work indicates the advantages of utilizing synthetically modified TFOs to form triplex assemblies in vivo for potential therapeutic applications and highlights the advantages of native mass spectrometry for the study of their formation.

Fast and accurate 3D RNA structure prediction remains a major challenge in structural biology, mostly due to the size and flexibility of RNA molecules, as well as the lack of diverse experimentally determined structures of RNA molecules. Unlike DNA structure, RNA structure is far less constrained by basepair hydrogen bonding, resulting in an explosion of potential stable states. Here, we propose a convolutional neural network that predicts all pairwise distances between residues in an RNA, using a recently described smooth parametrization of Euclidean distance matrices. We achieve high-accuracy predictions on RNAs up to 100 nt in length in fractions of a second, a factor of 10 7 faster than existing molecular dynamics-based methods. We also convert our coarse-grained machine learning output into an all-atom model using discrete molecular dynamics with constraints. Our proposed computational pipeline predicts all-atom RNA models solely from the nucleotide sequence. However, this method suffers from the same limitation as nucleic acid molecular dynamics: the scarcity of available RNA crystal structures for training., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)