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DOE OSTI2022

Modification at the C2'-O-Position with 2-Methylbenzothiophene Induces Unique Structural Changes and Thermal Transitions on Duplexes of RNA and DNA

Oligonucleotides can be chemically modified for a variety of applications that include their use as biomaterials, in therapeutics, or as tools to understand biochemical processes, among others. This work focuses on the functionalization of oligonucleotides of RNA and DNA (12- or 14-nucleotides long) with methylbenzothiophene (BT), at the C2'-O-position, which led to unique structural features. Circular dichroism (CD) analyses showed that positioning the BT units on one strand led to significant thermal destabilization, while duplexes where each strand contained 4-BT rings formed a distinct arrangement with cooperativity/interactions among the modifications (evidenced from the appearance of a band with positive ellipticity at 235 nm). Interestingly, the structural arrays displayed increased duplex stabilization (>10 °C higher than the canonical analogue) as a function of [Na + ] with an unexpected structural rearrangement at temperatures above 50 °C. Density functional theory–polarizable continuum model (DFT-PCM) calculations were carried out, and the analyses were in agreement with induced structural changes as a function of salt content. A model was proposed where the hydrophobic surface allows for an internal nucleobase rearrangement into a more thermodynamically stable structure, before undergoing full denaturation, with increased heat. While this behavior is not common, B- to Z-form duplex transitions can occur and are dependent on parameters that were probed in this work, i.e., temperature, nature of modification, or ionic content. To take advantage of this phenomenon, we probed the ability of the modified duplexes to be recognized by Zα (an RNA binding protein that targets Z-form RNA) via electrophoretic analysis and CD. Interestingly, the protein did not bind to canonical duplexes of DNA or RNA; however, it recognized the modified duplexes, in a [monovalent/divalent salt] dependent manner. Overall, the findings describe methodology to attain unique structural motifs of modified duplexes of DNA or RNA, and control their behavior as a function of salt concentration. While their affinity to RNA binding proteins, and the corresponding mechanism of action, requires further exploration, the tunable properties can be of potential use to study this, and other, types of modifications. The novel arrays that formed, under the conditions described herein, provide a useful way to explore the structure and behavior of modified oligonucleotides, in general.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

NASA NTRSDate not supplied

MULTI-OMICS STUDY OF THE EFFECT OF REDOX-ACTIVE METALLOPORPHYRIN ON MURINE RETINA DURING SPACEFLIGHT

Astronauts returning from spaceflight have experienced eye problems, which may decrease retinal performance and lead to long-term effects on visual acuity. This study leverages the collected data from spaceflown murine retinas that were treated with redox-active metalloporphyrin (BuOE) to mitigate spaceflight-induced changes and respective ground controls. 10-week-old adult C57BL/6 male mice (n=5 in each of BuOE treated and saline control groups for spaceflown and ground control samples) were flown on Space-X 24 to the ISS national lab, kept in low earth orbit for 35 days and returned to Earth alive. Our multi-omics analysis of RNA-sequencing and reduced representation bisulfite sequencing (RRBS) data generated from subsequent murine retina tissues uncovered genes, pathways, and epigenetic modifications consistent with therapeutic potential of BuOE. From RNA-Seq analysis of spaceflown murine samples, the treatment group show differentially expressed genes relative to saline controls that reached significance (adjusted p-value < 0.05) and included genes Gpx3 and Crhbp, which are related to protection against cell oxidative damage and cellular response to organonitrogen compounds. Ranked fold-changes from the same contrast were used for gene set enrichment analysis, which showed biological processes reaching significance (adjusted p-value < 0.05) including glutathione metabolic processes and cellular response to xenobiotic stimulus. RRBS data of the spaceflown murine samples found 139 hyper or hypo differentially methylated sites spread across chromosomes 1-19 (20% promoters, 21% exons, 43% introns | 20 CpG islands, 7 CpG shores) with a 10% methylation difference (q-value < 0.05).The findings from this investigation have the potential to provide valuable insights into the molecular mechanisms underlying conditions like spaceflight associated neuro-ocular syndrome and assess the effectiveness of BuOE as a countermeasure for astronauts experiencing neuro-ophthalmic abnormalities, which can lead to long-term effects on visual acuity.

Biostatistics↗

NASA NTRS2024

Multi-Omics Study of the Effect of Redox-Active Metalloporphyrin on Murine Retina During Spaceflight

Biostatistics↗

DOE OSTI2021

Diagnostic Testing for COVID-19 Bridging Study for QIAamp Viral RNA Extraction vs Beckman RNAdvance vs Thermofisher MagMAX

This report describes testing that was performed by LANL’s Biological Agent Testing Lab (BATL) to validate modifications to the CDC EUA 2019-Novel Coronavirus (2019- nCoV) Real-Time RT-PCR Diagnostic Panel (EUA-CDC-nCoV-IFU). BATL intends to implement the modifications to increase thoughput for daily testing . BATL validated the viral RNA extraction process, using the orignial component, QIAamp Viral RNA Mini Kit (Cat # 52906) and the new components, Beckman Coulter magnetic 96-well plate RNAdvance Viral kit (Cat # C63510), Thermofisher MagMAX Viral/Pathogen Nucleic Acid Isolation Kit (Cat # A48310). Equivalency was demonstrated between the original component and the Beckman Coulter magnetic 96-well plate RNAdvance Viral kit (Cat # C63510). Equivalency was also demonstrated between the original component and the Thermofisher MagMAX Viral/Pathogen Nucleic Acid Isolation Kit (Cat # A48310). Subsequently, substitution of the original component with either of these kits for viral RNA extraction increased BATL’s extraction capability from 100 samples per day to 279 samples per day.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Putting Humpty Dumpty Back Together Again: What Does Protein Quantification Mean in Bottom-Up Proteomics?

Bottom-up proteomics provides peptide measurements and has been invaluable for moving proteomics into large-scale analyses. Commonly, a single quantitative value is reported for each protein-coding gene by aggregating peptide quantities into protein groups following protein inference or parsimony. However, given the complexity of both RNA splicing and post-translational protein modification, it is overly simplistic to assume that all peptides that map to a singular protein-coding gene will demonstrate the same quantitative response. Here, by assuming that all peptides from a protein-coding sequence are representative of the same protein, we may miss the discovery of important biological differences. To capture the contributions of existing proteoforms, we need to reconsider the practice of aggregating protein values to a single quantity per protein-coding gene.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Derivatization of Mirror-Image L -Nucleic Acids with 2'-OMe Modification for Thermal and Structural Stabilization

To further expand the functionality and enhance the stability of mirror-image nucleic acids as advanced agents for basic research and therapeutic design, we have synthesized 2'-deoxy-2'-methoxy- L -uridine phosphoramidite and incorporated it into L -DNA and L -RNA by solid-phase synthesis quantitatively. We found that the thermostability of L -nucleic acids is dramatically improved after introducing the modifications. Moreover, we successfully crystallized both L -DNA and L -RNA duplexes containing the 2'-OMe modifications and sharing identical sequences. Crystal structure determination and analysis revealed the overall structures of the mirror-image nucleic acids, and for the first time it was possible to interpret the structural deviations caused by 2'-OMe and 2'-OH groups in the oligonucleotides, which are very similar. In conclusion, this novel chemical nucleic acid modification has the potential to be used to design nucleic acid-based therapeutics and materials in the future.

2'-OMe-modifications↗

DOE OSTI2025

A semidominant point mutation of Mediator tail subunit MED5b in Arabidopsis leads to altered enrichment of H3K27me3 and reduced expression of targets of MYC2

Abstract The Mediator complex coordinates regulatory input for transcription driven by RNA polymerase II in eukaryotes. reduced epidermal fluorescence4-3 (ref4-3) is a semidominant mutation that results in a single amino acid substitution in the Mediator tail subunit Med5b. Previous characterization of ref4-3 revealed altered expression of a variety of loci in Arabidopsis, including those contributing to phenylpropanoid biosynthesis. Examination of existing RNA-seq data indicated that loci enriched for the transcriptionally repressive chromatin modification H3K27me3 are overrepresented among genes that are misregulated in ref4-3. We used ChIP-seq and RNA-seq to examine the possibility that perturbation of H3K27me3 homeostasis in ref4-3 plants contributed to altered transcript levels. We observed that ref4-3 results in a modest global reduction of H3K27me3 at enriched loci and that this reduction is not dependent on gene expression; however, altered H3K27me3 was not strongly predictive of altered expression in ref4-3 plants. Instead, our analyses revealed a substantial enrichment of targets of the MYC2 transcriptional regulator among genes that exhibit decreased expression in ref4-3. Consistent with previous characterization of ref4-3, we observed that ref4-3-dependent decreased expression of MYC2 targets can be suppressed by loss of another Mediator tail subunit, MED25. This observation is consistent with previous biochemical characterization of MYC2. Our data highlight the diverse and distinct impacts that a single amino acid change in the tail subunit of Mediator can have on transcriptional circuits and raise the prospect that Mediator directly contributes to H3K27me3 homeostasis in plants.

Long, Jiaxin (ORCID:0000000335150706)↗

DOE OSTI2022

Genome-wide fetalization of enhancer architecture in heart disease

Heart disease is associated with re-expression of key transcription factors normally active only during prenatal development of the heart. However, the impact of this reactivation on the regulatory landscape in heart disease is unclear. Here, we use RNA-seq and ChIP-seq targeting a histone modification associated with active transcriptional enhancers to generate genome-wide enhancer maps from left ventricle tissue from up to 26 healthy controls, 18 individuals with idiopathic dilated cardiomyopathy (DCM), and five fetal hearts. Healthy individuals have a highly reproducible epigenomic landscape, consisting of more than 33,000 predicted heart enhancers. In contrast, we observe reproducible disease-associated changes in activity at 6,850 predicted heart enhancers. Combined analysis of adult and fetal samples reveals that the heart disease epigenome and transcriptome both acquire fetal-like characteristics, with 3,400 individual enhancers sharing fetal regulatory properties. We also provide a comprehensive data resource (http://heart.lbl.gov) for the mechanistic exploration of DCM etiology.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2025

Viral delivery of an RNA-guided genome editor for transgene-free germline editing in Arabidopsis

Genome editing is transforming plant biology by enabling precise DNA modifications. However, delivery of editing systems into plants remains challenging, often requiring slow, genotype-specific methods such as tissue culture or transformation1. Plant viruses, which naturally infect and spread to most tissues, present a promising delivery system for editing reagents. However, many viruses have limited cargo capacities, restricting their ability to carry large CRISPR-Cas systems. Here we engineered tobacco rattle virus (TRV) to carry the compact RNA-guided TnpB enzyme ISYmu1 and its guide RNA. This innovation allowed transgene-free editing of Arabidopsis thaliana in a single step, with edits inherited in the subsequent generation. By overcoming traditional reagent delivery barriers, this approach offers a novel platform for genome editing, which can greatly accelerate plant biotechnology and basic research.

Weiss, Trevor↗

DOE OSTI2022

Spacer2PAM: A computational framework to guide experimental determination of functional CRISPR-Cas system PAM sequences

Abstract RNA-guided nucleases from CRISPR-Cas systems expand opportunities for precise, targeted genome modification. Endogenous CRISPR-Cas systems in many prokaryotes are attractive to circumvent expression, functionality, and unintended activity hurdles posed by heterologous CRISPR-Cas effectors. However, each CRISPR-Cas system recognizes a unique set of protospacer adjacent motifs (PAMs), which requires identification by extensive screening of randomized DNA libraries. This challenge hinders development of endogenous CRISPR-Cas systems, especially those based on multi-protein effectors and in organisms that are slow-growing or have transformation idiosyncrasies. To address this challenge, we present Spacer2PAM, an easy-to-use, easy-to-interpret R package built to predict and guide experimental determination of functional PAM sequences for any CRISPR-Cas system given its corresponding CRISPR array as input. Spacer2PAM can be used in a ‘Quick’ method to generate a single PAM prediction or in a ‘Comprehensive’ method to inform targeted PAM libraries small enough to screen in difficult to transform organisms. We demonstrate Spacer2PAM by predicting PAM sequences for industrially relevant organisms and experimentally identifying seven PAM sequences that mediate interference from the Spacer2PAM-informed PAM library for the type I-B CRISPR-Cas system from Clostridium autoethanogenum. We anticipate that Spacer2PAM will facilitate the use of endogenous CRISPR-Cas systems for industrial biotechnology and synthetic biology.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRSDate not supplied

Genomics Study of Effect of Redox-Active Metalloporphyrin on Murine Retina During Spaceflight

Astronauts returning from spaceflight have experienced eye problems, which may decrease retinal performance and lead to long-term effects on visual acuity. This study leverages the collected data from spaceflown murine retinas that were treated with redox-active metalloporphyrin (BuOE) to mitigate spaceflight-induced changes. 10-week-old adult C57BL/6 male mice (n=5 in each of BuOE treated and saline control groups) were flown on Space-X 24 to the ISS national lab, kept in low earth orbit for 35 days and returned to Earth alive. Our analysis of RNA-sequencing data generated from subsequent murine retina tissues uncovered genes, pathways, and epigenetic modifications consistent with therapeutic potential of BuOE. For spaceflown murine samples, the treatment group show differentially expressed genes relative to saline controls that reached significance (adjusted p-value < 0.05) and included genes Gpx3 and Crhbp, which are related to protection against cell oxidative damage and cellular response to organonitrogen compounds. Ranked fold-changes from the same contrast were used for gene set enrichment analysis, which showed biological processes reaching significance (adjusted p-value < 0.05) including glutathione metabolic processes and cellular response to xenobiotic stimulus. The findings from this investigation have the potential to provide valuable insights into the molecular mechanisms underlying conditions like spaceflight associated neuro-ocular syndrome and assess the effectiveness of BuOE as a countermeasure for astronauts experiencing neuro-ophthalmic abnormalities, which can lead to long-term effects on visual acuity.

Machine Learning↗

NASA NTRSDate not supplied

Genomics Study of Effect of Redox-Active Metalloporphyrin on Murine Retina During Spaceflight

Astronauts returning from spaceflight have experienced eye problems, which may decrease retinal performance and lead to long-term effects on visual acuity. This study leverages the collected data from spaceflown murine retinas that were treated with redox-active metalloporphyrin (BuOE) to mitigate spaceflight-induced changes. 10-week-old adult C57BL/6 male mice (n=5 in each of BuOE treated and saline control groups) were flown on Space-X 24 to the ISS national lab, kept in low earth orbit for 35 days and returned to Earth alive. Our analysis of RNA-sequencing data generated from subsequent murine retina tissues uncovered genes, pathways, and epigenetic modifications consistent with therapeutic potential of BuOE. For spaceflown murine samples, the treatment group show differentially expressed genes relative to saline controls that reached significance (adjusted p-value < 0.05) and included genes Gpx3 and Crhbp, which are related to protection against cell oxidative damage and cellular response to organonitrogen compounds. Ranked fold-changes from the same contrast were used for gene set enrichment analysis, which showed biological processes reaching significance (adjusted p-value < 0.05) including glutathione metabolic processes and cellular response to xenobiotic stimulus. The findings from this investigation have the potential to provide valuable insights into the molecular mechanisms underlying conditions like spaceflight associated neuro-ocular syndrome and assess the effectiveness of BuOE as a countermeasure for astronauts experiencing neuro-ophthalmic abnormalities, which can lead to long-term effects on visual acuity.

Machine Learning↗

DOE OSTI2024

Symmetry breaking of fluorophore binding to a G-quadruplex generates an RNA aptamer with picomolar K D

Fluorogenic RNA aptamer tags with high affinity enable RNA purification and imaging. The G-quadruplex (G4) based Mango (M) series of aptamers were selected to bind a thiazole orange based (TO1-Biotin) ligand. Using a chemical biology and reselection approach, we have produced a MII.2 aptamer–ligand complex with a remarkable set of properties: Its unprecedented K D of 45 pM, formaldehyde resistance (8% v/v), temperature stability and ligand photo-recycling properties are all unusual to find simultaneously within a small RNA tag. Crystal structures demonstrate how MII.2, which differs from MII by a single A23U mutation, and modification of the TO1-Biotin ligand to TO1-6A-Biotin achieves these results. MII binds TO1-Biotin heterogeneously via a G4 surface that is surrounded by a stadium of five adenosines. Breaking this pseudo-rotational symmetry results in a highly cooperative and homogeneous ligand binding pocket: A22 of the G4 stadium stacks on the G4 binding surface while the TO1-6A-Biotin ligand completely fills the remaining three quadrants of the G4 ligand binding face. Similar optimization attempts with MIII.1, which already binds TO1-Biotin in a homogeneous manner, did not produce such marked improvements. We use the novel features of the MII.2 complex to demonstrate a powerful optically-based RNA purification system.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

RNA oligomers at atomic resolution containing 1‐methylpseudouridine, an essential building block of mRNA vaccines

Abstract All widely used mRNA vaccines against COVID‐19 contain in their sequence 1‐methylpseudouridine ( m1Ψ ) instead of uridine. In this publication, we report two high resolution crystal structures (at up to 1.01 and 1.32 Å, respectively) of one such double‐stranded 12‐mer RNA sequence crystallized in two crystal forms. The structures are compared with similar structures which do not contain this modification. Additionally, the X‐ray structure of 1‐methyl‐pseudouridine itself was determined.

Nievergelt, Philipp↗

NASA NTRS1999

Left-handed Z-DNA: structure and function

Z-DNA is a high energy conformer of B-DNA that forms in vivo during transcription as a result of torsional strain generated by a moving polymerase. An understanding of the biological role of Z-DNA has advanced with the discovery that the RNA editing enzyme double-stranded RNA adenosine deaminase type I (ADAR1) has motifs specific for the Z-DNA conformation. Editing by ADAR1 requires a double-stranded RNA substrate. In the cases known, the substrate is formed by folding an intron back onto the exon that is targeted for modification. The use of introns to direct processing of exons requires that editing occurs before splicing. Recognition of Z-DNA by ADAR1 may allow editing of nascent transcripts to be initiated immediately after transcription, ensuring that editing and splicing are performed in the correct sequence. Structural characterization of the Z-DNA binding domain indicates that it belongs to the winged helix-turn-helix class of proteins and is similar to the globular domain of histone-H5.

Review↗

DOE OSTI2021

2'-O methylation of RNA cap in SARS-CoV-2 captured by serial crystallography

The genome of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus has a capping modification at the 5'-untranslated region (UTR) to prevent its degradation by host nucleases. These modifications are performed by the Nsp10/14 and Nsp10/16 heterodimers using S-adenosylmethionine as the methyl donor. Nsp10/16 heterodimer is responsible for the methylation at the ribose 2'-O position of the first nucleotide. To investigate the conformational changes of the complex during 2'-O methyltransferase activity, we used a fixed-target serial synchrotron crystallography method at room temperature. We determined crystal structures of Nsp10/16 with substrates and products that revealed the states before and after methylation, occurring within the crystals during the experiments. Here we report the crystal structure of Nsp10/16 in complex with Cap-1 analog ( m7 GpppA m2'-O ). Inhibition of Nsp16 activity may reduce viral proliferation, making this protein an attractive drug target.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Shade triggers posttranscriptional PHYTOCHROME-INTERACTING FACTOR-dependent increases in H3K4 trimethylation

The phytochrome (phy)-PHYTOCHROME-INTERACTING FACTOR (PIF) sensory module perceives and transduces light signals to direct target genes (DTGs), which then drive the adaptational responses in plant growth and development appropriate to the prevailing environment. These signals include the first exposure of etiolated seedlings to sunlight upon emergence from subterranean darkness and the change in color of the light that is filtered through, or reflected from, neighboring vegetation (“shade”). Previously, we identified three broad categories of rapidly signal-responsive genes: those repressed by light and conversely induced by shade; those repressed by light, but subsequently unresponsive to shade; and those responsive to shade only. Here, we investigate the potential role of epigenetic chromatin modifications in regulating these contrasting patterns of phy-PIF module-induced expression of DTGs in Arabidopsis (Arabidopsis thaliana). Using RNA-seq and ChIP-seq to determine time-resolved profiling of transcript and histone 3 lysine 4 trimethylation (H3K4me3) levels, respectively, we show that, whereas the initial dark-to-light transition triggers a rapid, apparently temporally coincident decline of both parameters, the light-to-shade transition induces similarly rapid increases in transcript levels that precede increases in H3K4me3 levels. Together with other recent findings, these data raise the possibility that, rather than being causal in the shade-induced expression changes, H3K4me3 may function to buffer the rapidly fluctuating shade/light switching that is intrinsic to vegetational canopies under natural sunlight conditions.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Editorial: Transcriptional and epigenetic landscapes of abiotic stress response in plants

In nature, plants constantly face various biotic and abiotic stresses that impact their growth, development, and productivity. Among these, abiotic stresses often have a more severe impact than biotic stresses. For instance, drought has been reported to cause greater yield losses than the combined impact of all plant pathogens (Gupta et al., 2020). Abiotic stresses are the immediate outcome of climate change, and the magnitude of these stresses has gradually increased every year with the rise in global temperatures. Thus, it has become imperative to study the impact of these stresses on plants and how plants respond to them at different levels to show resilient traits. This includes analysing the plants at morpho-physiological, biochemical, and molecular levels. Researchers often compare stressed plants to control (non-stressed) plants or evaluate contrasting genotypes, such as tolerant and sensitive lines, to elucidate the mechanisms underlying stress responses. While these studies have provided some insights, a comprehensive understanding of the intricate mechanisms governing plant responses to abiotic stress remains largely unknown. Recent advances in next-generation tools and technologies have enabled researchers to dissect the molecular basis of plant stress responses at genomic, transcriptomic, proteomic, metabolomic, epigenetic and epigenomic levels. Among these, knowledge of the transcriptional/epigenomic landscape of the trait-associated variations is limited. Given the importance of transcriptional changes and histone modifications in abiotic stress responses, this Research Topic was edited to collage the knowledge available on transcriptional and epigenetic landscapes of abiotic stress response in plants. The Research Topic features eight original research articles and one review, covering various aspects of transcriptome and epigenetic reprogramming in plants during abiotic stresses. Four of the research articles employ transcriptomics integrated with other omics approaches to explore transcriptome reprogramming, candidate gene identification, and the role of long non-coding RNA during different stresses. Two articles focus on the functional characterization of specific candidate genes involved in stress response, while another provides a genome-wide analysis of a stress-responsive gene family. Additionally, one study investigates genome-wide histone modifications, specifically H3K4me3 and H3K27me3, in response to abiotic stresses.

59 BASIC BIOLOGICAL SCIENCES↗