Recombinant Adiantum capillus-veneris Cytochrome b559 subunit alpha (psbE)

What is the structure and function of Cytochrome b559 subunit alpha (psbE) in photosystem II?

Cytochrome b559 is a key component of the photosystem II complex (PSII) that plays an essential role in its proper functioning and assembly. The alpha subunit of cytochrome b559, encoded by the psbE gene, forms part of the reaction center complex (RCII) alongside other protein subunits including D1 and D2 . Structurally, the full-length protein consists of 84 amino acids in some species, with a sequence that includes transmembrane domains allowing it to anchor in the thylakoid membrane . The protein contains a heme group with axial ligands that are critical for its function, as evidenced by site-directed mutants of the cyanobacterium Synechocystis sp. PCC6803 with mutated heme axial ligands being unable to grow photoautotrophically due to reduced PSII accumulation .

The primary functions of cytochrome b559 include:

• Stabilization of the PSII complex structure

• Protection against photoinhibition through cyclic electron flow

• Participation in the assembly process of PSII

• Potential role in water oxidation reactions

Research methods to study the structure typically involve isolation of PSII core complexes, often facilitated by using N-terminal His-tagged versions of the psbE protein .

How does RNA editing affect psbE expression in Adiantum species?

RNA editing represents a critical post-transcriptional modification process in Adiantum species that significantly impacts psbE expression. Comparative studies of RNA editing patterns across three Adiantum species (A. capillus-veneris, A. aleuticum, and A. shastense) have revealed striking variation in the number and location of RNA-editing sites . While A. capillus-veneris exhibits approximately 350 RNA-editing sites in its plastome, A. aleuticum and A. shastense display 505 and 509 sites, respectively .

The pattern of conservation in RNA-editing sites follows several principles:

• Reverse (U-to-C) editing sites show a higher degree of conservation than forward (C-to-U) sites

• Sites involving start and stop codons demonstrate high conservation across species

• Of the total 653 distinct RNA-editing sites found across the three Adiantum plastomes, only 234 were shared among all three species

These variations suggest that RNA-editing sites can be rapidly gained or lost throughout evolution, with different degrees of selective pressure maintaining certain edits. The conservation pattern hints at likely independent origins of both types of edits and suggests functional importance for editing sites that modify start or stop codons . This RNA editing process directly impacts the final protein sequence and potentially the structure and function of the expressed psbE protein.

What experimental models are suitable for studying recombinant Adiantum capillus-veneris psbE?

Selecting appropriate experimental models for studying recombinant A. capillus-veneris psbE requires consideration of both expression systems and analytical platforms. Based on current research approaches, the following models have proven effective:

Expression Systems:

• E. coli: Successfully used for expressing recombinant full-length cytochrome b559 subunit alpha with N-terminal His-tags, facilitating purification while maintaining protein functionality .

• Tobacco plants: Biolistic chloroplast transformation has been employed to replace wildtype psbE genes with His-tagged counterparts, allowing for in vivo studies of modified psbE proteins .

• Cyanobacteria (Synechocystis): Provides a prokaryotic photosynthetic system for studying mutations in psbE and observing their effects on PSII assembly and function .

Analytical Models:

• Oxygen evolution measurements: To assess PSII functionality in transformed plants or isolated complexes. Research shows His-tagged psbE variants can reduce oxygen evolution capacity by 10-30% compared to wildtype .

• Fluorescence analysis (Fv/Fm values): For evaluating photosynthetic efficiency, with studies showing only slight differences between wildtype and His-tagged psbE variants .

• 2D PAGE analysis: For assessing PSII complex assembly and stability, particularly useful for detecting changes in complex formation in mutant variants .

When selecting a model system, researchers should consider that modifications to psbE (such as His-tagging) may induce subtle functional changes, as evidenced by reduced oxygen evolution in transgenic plants despite minimal changes in Fv/Fm values .

What are the optimal methods for expressing and purifying recombinant Adiantum capillus-veneris psbE?

Expression and purification of recombinant A. capillus-veneris psbE requires carefully optimized protocols to ensure proper folding and functionality of this membrane protein. Based on current research practices, the following methodological approach is recommended:

Expression System Selection:

• E. coli: Provides high yield and established protocols for membrane protein expression . For psbE, expression constructs should include the full-length sequence (1-84 amino acids) with an N-terminal His-tag for purification.

• Storage conditions: After purification, the protein should be stored as a lyophilized powder or in Tris/PBS-based buffer with 6% trehalose at pH 8.0 .

Purification Protocol:

• Cell lysis under non-denaturing conditions

• Membrane fraction isolation via ultracentrifugation

• Solubilization using mild detergents (typically β-dodecyl maltoside)

• Immobilized metal affinity chromatography using the His-tag

• Size exclusion chromatography for further purification

Reconstitution Method:

• Brief centrifugation of the protein vial prior to opening

• Reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Addition of 5-50% glycerol (final concentration) for long-term storage at -20°C/-80°C

Quality Control Metrics:

• Purity assessment: >90% as determined by SDS-PAGE

• Functional assays: Spectroscopic analysis of heme incorporation

• Stability testing: Repeated freeze-thaw cycles should be avoided to maintain protein integrity

When expressed and purified correctly, the recombinant protein should exhibit the expected spectral characteristics of cytochrome b559 and maintain its ability to incorporate into PSII complexes in reconstitution experiments.

How can His-tagging affect the functionality of recombinant psbE proteins?

His-tagging of psbE proteins provides valuable purification advantages but can introduce functional modifications that researchers must account for in experimental design and data interpretation. Studies on tobacco plants with N-terminal His-tagged psbE have revealed several important considerations:

Functional Impacts:

• Oxygen evolution capacity in thylakoids prepared from plants with His-tagged psbE was reduced by 10-30% compared to wildtype plants, with the reduction correlating with the length of the His-tag .

• Fv/Fm values (a measure of photosynthetic efficiency) differed only slightly between wildtype and His-tagged variants, suggesting that basic photochemistry remains largely intact .

• No obvious phenotypic differences were observed in transgenic plants expressing His-tagged psbE, indicating that the modification is generally well-tolerated at the whole-plant level .

Structural Considerations:

• The His-tag location (N-terminal vs. C-terminal) is critical, as C-terminal tags may interfere with protein-protein interactions within the PSII complex.

• Tag length correlates with functional impact, with longer tags causing greater reduction in oxygen evolution capacity .

Purification Advantages:

• His-tagged psbE facilitates rapid, mild purification of higher plant PSII core complexes.

• The resulting purified complexes contain the main core subunits of PSII without detectable traces of LHC or PsaA/B polypeptides .

• Additional proteins such as Psb27 and PsbS can be co-purified, with PsbS appearing exclusively as a monomer in tobacco PSII core complexes, contrary to previous reports .

To minimize functional impacts while maximizing purification benefits, researchers should consider using the shortest effective His-tag sequence and validating the functional characteristics of the tagged protein against wildtype controls.

What analytical techniques are most effective for characterizing recombinant psbE structure and function?

Comprehensive characterization of recombinant psbE structure and function requires a multi-technique approach spanning biophysical, biochemical, and functional analyses. The following analytical techniques have proven most effective based on current research:

Structural Characterization:

• Mass Spectrometry

  • For confirming protein identity and post-translational modifications

  • Particularly useful for validating RNA editing events that alter amino acid sequences

• Circular Dichroism (CD) Spectroscopy

  • For analyzing secondary structure elements and conformational stability

  • Helps verify proper protein folding after recombinant expression

• Immunodetection Methods

  • Western blotting with specific antibodies to confirm protein identity

  • Used in conjunction with SDS-PAGE to verify purity and molecular weight

Functional Analysis:

• Oxygen Evolution Measurements

  • Quantifies PSII activity in reconstituted systems or thylakoid preparations

  • Sensitive to functional changes caused by protein modifications (e.g., His-tagging)

• Fluorescence Spectroscopy

  • Fv/Fm measurements assess photochemical efficiency

  • Can detect subtle functional changes in PSII complexes containing recombinant psbE

• Spectroscopic Analysis of Heme Properties

  • UV-Vis spectroscopy to confirm proper heme incorporation and redox properties

  • Critical for verifying functional integrity of cytochrome components

Interaction Studies:

• Blue Native PAGE

  • Analyzes incorporation of recombinant psbE into native-like PSII complexes

  • Helps assess impact of mutations or modifications on complex assembly

• Co-immunoprecipitation

  • Identifies interaction partners within the PSII complex

  • Useful for determining whether recombinant psbE maintains native protein-protein interactions

When applying these techniques, researchers should compare results from recombinant proteins with native controls to accurately interpret structural or functional differences introduced by the recombinant production process or protein modifications.

How can recombinant psbE be used to study photosystem II assembly mechanisms?

Recombinant psbE provides a powerful tool for dissecting the assembly mechanisms of photosystem II through both in vitro reconstitution and in vivo mutational approaches. These applications leverage the essential role of cytochrome b559 as an early assembly factor in PSII biogenesis.

In Vitro Reconstitution Studies:

• Purified His-tagged recombinant psbE can be used to reconstitute minimal PSII reaction center complexes, allowing researchers to study the sequential assembly process .

• Mixing recombinant psbE with other core PSII proteins helps identify the minimal components necessary for stable complex formation and initial photochemistry.

• This approach has revealed that properly folded cytochrome b559 (composed of psbE and psbF) likely forms a structural scaffold for subsequent incorporation of D1 and D2 proteins.

Mutational Analysis:

• Site-directed mutagenesis of heme axial ligands in recombinant psbE demonstrates the critical nature of these residues, as such mutations typically result in little PSII accumulation and inability to grow photoautotrophically in cyanobacteria .

• The observation that tandem gene amplification of the psbEFLJ operon can restore PSII accumulation in Cyt b559 mutants provides insight into dosage-compensation mechanisms .

• RNA-seq analysis of such mutants has shown greatly increased transcript levels of the psbEFLJ operon, suggesting that increased expression can overcome structural deficiencies in the mutant protein .

Interspecies Comparative Studies:

• The varying patterns of RNA editing in psbE across Adiantum species (A. capillus-veneris: 350 sites; A. aleuticum: 505 sites; A. shastense: 509 sites) provide a natural experiment for studying how post-transcriptional modifications impact protein function and PSII assembly .

• Comparison of edited versus non-edited psbE variants offers insights into the evolutionary pressures shaping photosystem II assembly and function.

A particularly insightful experimental approach involves combining His-tagged psbE with fluorescent protein fusions of other PSII components to track the assembly process in real-time using fluorescence microscopy or FRET techniques.

What insights can RNA editing studies of psbE provide about photosynthetic evolution?

RNA editing of psbE transcripts offers a unique window into photosynthetic evolution, particularly in ferns like Adiantum species where this process is extensive. Comparative analysis of RNA editing patterns provides several critical insights:

Evolutionary Rate and Conservation:

• Of 653 distinct RNA-editing sites found across three Adiantum plastomes, only 234 were shared among all three species, indicating rapid evolutionary change in editing patterns .

• The divergence between A. aleuticum/A. shastense and A. capillus-veneris occurred approximately 60 million years ago, while A. aleuticum and A. shastense diverged about 20 million years ago, providing a temporal framework for the evolution of RNA editing .

• This timeline suggests that RNA-editing sites can be rapidly gained or lost throughout evolution, potentially serving as adaptive mechanisms.

Conservation Patterns and Functional Significance:

• Reverse (U-to-C) editing sites show a higher degree of conservation than forward (C-to-U) sites, suggesting different selective pressures or mechanisms .

• Sites involving start and stop codons demonstrate particularly high conservation, indicating their critical functional importance .

• A. capillus-veneris exclusively shared only 22 editing sites with A. aleuticum and 24 with A. shastense, while 58 sites were unique to A. capillus-veneris .

Methodological Approaches for Evolutionary Studies:

• Transcriptome Analysis

  • RNA-seq data can be analyzed using read-mapping and SNP-calling software to identify RNA-editing sites

  • Comparison of editing sites across species requires generation of alignments for each plastid gene

• Comparative Analysis Framework

  • Edits can be categorized as shared among all species, shared between pairs of species, or unique to individual species

  • This categorization helps identify evolutionarily conserved edits that likely have critical functional roles

The varying degrees of conservation between both types of edits (C-to-U and U-to-C) and sites in start/stop codons versus other codons hint at likely independent origins of both types of edits and potentially different functional roles in photosynthetic adaptation .

How do modifications to psbE affect PSII activity and plant photosynthetic efficiency?

Impact of His-Tag Modifications:

• N-terminal His-tags added to psbE in tobacco plants reduced oxygen evolution capacity by 10-30%, with the reduction directly correlating with the length of the His-tag .

• Despite this reduction in oxygen evolution, Fv/Fm values (a measure of photochemical efficiency) differed only slightly between wildtype and His-tagged plants .

• Transgenic plants expressing His-tagged psbE did not exhibit obvious phenotypic differences, suggesting compensatory mechanisms at the whole-plant level .

Effects of Heme Axial Ligand Mutations:

• Site-directed mutants of the cyanobacterium Synechocystis with mutated heme axial ligands of Cyt b559 accumulated little PSII and were unable to grow photoautotrophically .

• This demonstrates the critical nature of the heme cofactor in psbE for PSII assembly and function.

Gene Dosage Effects and Compensation:

• In cyanobacteria, autotrophic transformants carrying mutations in Cyt b559 heme axial ligands were found to contain multiple tandem repeats (5-15 copies) of chromosomal segments containing the psbEFLJ operon .

• RNA-seq analysis showed greatly increased transcript levels of the psbEFLJ operon in these transformants .

• This tandem gene amplification restored PSII accumulation and photoautotrophic growth, demonstrating a dosage-compensation mechanism .

• Interestingly, the multiple copies were only maintained during autotrophic growth and gradually decreased under photoheterotrophic conditions, suggesting a selective pressure only when photosynthesis is essential .

Quantitative Analysis of PSII Efficiency:

• Two-dimensional PAGE analysis of membrane proteins revealed strong deficiency in PSII complexes in Cyt b559 mutants that was reversed in autotrophic transformants with amplified psbEFLJ genes .

• This provides a direct link between psbE expression levels and PSII complex formation and stability.

These findings suggest that while the structure and function of psbE are critical for photosynthetic efficiency, plants and cyanobacteria possess remarkable adaptive mechanisms to compensate for deficiencies through gene amplification or other regulatory adjustments.

How can researchers reconcile contradictory data about psbE function across different species?

Researchers frequently encounter seemingly contradictory data regarding psbE function when comparing results across different photosynthetic organisms. These apparent contradictions can emerge from genuine biological differences or methodological variations. The following systematic approach can help reconcile such discrepancies:

Sources of Apparent Contradictions:

• Evolutionary Divergence

  • The psbE gene shows considerable variation in RNA editing patterns even among closely related Adiantum species, with A. capillus-veneris exhibiting 350 RNA-editing sites compared to approximately 505-509 sites in related species .

  • These differences suggest that even within the same genus, the protein may have distinct functional characteristics.

• Post-transcriptional Modifications

  • The varying patterns of RNA editing can result in different protein sequences from identical gene sequences.

  • Particularly significant are the editing sites affecting start and stop codons, which show high conservation across species, suggesting critical functional importance .

• Experimental System Variations

  • His-tagged versions of psbE used for purification can reduce oxygen evolution capacity by 10-30% depending on tag length, while showing minimal impact on Fv/Fm values .

  • Different expression systems (E. coli vs. plant chloroplasts vs. cyanobacteria) may yield proteins with varying folding patterns and post-translational modifications.

Methodological Approaches for Reconciliation:

• Comparative Functional Analysis

  • Direct side-by-side testing of psbE from different species under identical experimental conditions

  • Standardized assays measuring oxygen evolution, fluorescence parameters, and spectroscopic properties

• Structural Comparison

  • Alignment of protein sequences after accounting for RNA editing events

  • Identification of conserved domains versus variable regions that might explain functional differences

• Heterologous Expression Studies

  • Expression of psbE from various species in a standard host system (e.g., cyanobacteria)

  • Complementation assays to determine functional equivalence across species

• Integration of Multiple Data Types

  • Combining biochemical, biophysical, and genetic data to build comprehensive models

  • Meta-analysis approaches to identify patterns across multiple studies

When addressing contradictory findings, it's essential to consider that apparent contradictions may actually reflect biological adaptations to different ecological niches or evolutionary histories, rather than experimental artifacts or errors.

What are the challenges in measuring the impact of RNA editing on psbE function?

Measuring the precise impact of RNA editing on psbE function presents several methodological and interpretational challenges that researchers must navigate carefully. These challenges span from molecular characterization to functional analysis:

Technical Challenges:

• Isolating Edited vs. Non-edited Forms

  • Natural systems contain mixtures of edited and non-edited transcripts

  • Creating systems with exclusively edited or non-edited forms requires sophisticated genetic engineering

• Temporal Dynamics of Editing

  • RNA editing patterns may change during development or in response to environmental conditions

  • Capturing these dynamics requires time-course sampling and analysis

• Site-Specific Effects

  • With hundreds of editing sites present in Adiantum species (350 in A. capillus-veneris, 505 in A. aleuticum, 509 in A. shastense) , determining the impact of individual sites requires systematic mutagenesis

  • Some edits may have synergistic or antagonistic effects when combined

Analytical Challenges:

• Distinguishing Direct from Indirect Effects

  • RNA editing may affect protein structure/function directly or influence regulatory processes like translation efficiency

  • Separating these mechanisms requires multi-level analysis from transcript to protein to function

• Quantitative Assessment

  • Determining the degree to which specific edits impact photosynthetic parameters requires sensitive and reproducible assays

  • Small functional changes may have significant physiological impacts over time

• Evolutionary Interpretation

  • Different patterns of conservation between C-to-U and U-to-C editing sites suggest distinct evolutionary origins and selective pressures

  • Understanding why certain edits are conserved while others vary requires integration of functional and phylogenetic data

Methodological Solutions:

• Site-Directed RNA Editing

  • Using CRISPR-based RNA editing tools to create transcripts with specific editing patterns

  • Allows direct comparison of defined variants

• Recombinant Protein Approaches

  • Expressing protein variants corresponding to different editing patterns

  • Enables direct biochemical and biophysical characterization

• Computational Prediction

  • Developing algorithms to predict functional impacts of RNA editing based on structural models

  • Helps prioritize sites for experimental validation

The fundamental challenge remains linking molecular changes at the RNA level to functional consequences at the protein and physiological levels. This requires integrating transcriptomic, proteomic, and physiological approaches to build a comprehensive understanding of how RNA editing shapes psbE function in photosynthetic organisms.

How should researchers design experiments to study the relationship between psbE and wound healing properties of Adiantum capillus-veneris?

Investigating the potential relationship between psbE and the wound healing properties of Adiantum capillus-veneris requires carefully designed experiments that bridge photosynthetic biochemistry and medicinal plant biology. While initial studies show that A. capillus-veneris extracts promote angiogenesis and protect fibroblasts from oxidative damage , the specific role of psbE in these effects remains unexplored.

Experimental Design Framework:

• Extract Fractionation and Component Analysis

  • Fractionate methanol extracts of A. capillus-veneris into hexane, ethyl acetate, n-butanol, and aqueous portions

  • Perform LC-MS/MS analysis to identify components in each fraction

  • Use protein precipitation and immunoblotting to detect psbE protein or fragments in medicinal extracts

• Recombinant psbE Testing

  • Express recombinant A. capillus-veneris psbE in E. coli with appropriate tags for purification

  • Test purified protein in angiogenesis and fibroblast protection assays

  • Compare activity of recombinant protein with plant extracts

• Structure-Activity Relationship Studies

  • Create truncated or mutated versions of recombinant psbE

  • Test these variants for wound healing properties

  • Identify active domains or residues responsible for biological activity

Assay Systems:

• Angiogenesis Assessment

  • Capillary-like tubular formation assays using human umbilical vein endothelial cells

  • Proliferation assays of endothelial cells

  • Chorioallantoic membrane (CAM) assay for in vivo angiogenesis assessment

• Fibroblast Protection

  • Tests for protection against damage to fibroblasts by oxygen free radicals

  • MTT assays on normal human dermal fibroblasts for cytotoxicity evaluation

  • Assessment of fibroblast migration and proliferation in wound models

• Molecular Mechanism Investigation

  • Gene expression analysis in treated cells (RNA-seq or qPCR)

  • Pathway analysis focusing on angiogenesis and anti-inflammatory markers

  • Binding partner identification through pull-down assays

Control Experiments:

• Comparative Analysis

  • Test extracts and recombinant proteins from other fern species with different psbE sequences

  • Compare A. capillus-veneris with A. aleuticum and A. shastense, which show different RNA editing patterns

  • Include plant species with high sequence similarity but no reported wound healing properties

• RNA Editing Impact

  • Express both edited and non-edited versions of psbE to determine if RNA editing affects bioactivity

  • Create chimeric proteins with domains from species with different healing properties

• Specificity Controls

  • Test other photosystem components to determine if the effects are specific to psbE

  • Assess other cytochromes to evaluate structural class effects versus sequence-specific effects

This experimental design allows researchers to systematically evaluate whether psbE contributes to the wound healing properties of A. capillus-veneris, potentially revealing novel therapeutic applications for this photosynthetic protein beyond its role in photosystem II.

What are the future research directions for Adiantum capillus-veneris psbE studies?

Research on Adiantum capillus-veneris psbE stands at the intersection of photosynthesis biochemistry, evolutionary biology, and potential therapeutic applications. Based on current knowledge, several promising research directions emerge:

Integration of RNA Editing and Protein Function:

• Comparative functional analysis of psbE proteins with different editing patterns from A. capillus-veneris (350 editing sites), A. aleuticum (505 sites), and A. shastense (509 sites)

• Development of computational models predicting how specific RNA edits affect protein structure and function

• Investigation of environmental factors that influence RNA editing patterns in psbE

Advanced Structural Biology Approaches:

• Cryo-EM or X-ray crystallography studies of PSII complexes containing native versus recombinant psbE

• Molecular dynamics simulations to understand how His-tags or mutations affect protein dynamics

• Structure-based design of psbE variants with enhanced stability or altered functions

Photosynthetic Engineering Applications:

• Exploration of the gene amplification mechanism observed in cyanobacteria as a potential strategy for enhancing photosynthetic efficiency

• Development of optimized His-tagged psbE variants that maintain full functionality while allowing easy purification

• Engineering of psbE to improve stress tolerance in crop plants

Medicinal Applications:

• Isolation and characterization of bioactive peptides derived from psbE that might contribute to the wound healing properties of A. capillus-veneris extracts

• Structure-activity relationship studies to identify the specific domains responsible for angiogenesis promotion or fibroblast protection

• Development of synthetic peptides based on psbE structure for therapeutic applications

Evolutionary Studies:

• Expanded phylogenetic analysis of psbE across fern species to understand the evolution of RNA editing patterns

• Investigation of the independent origins of C-to-U and U-to-C editing types suggested by different conservation patterns

• Exploration of the selective pressures that maintain editing sites in start and stop codons across species

These research directions would benefit from interdisciplinary approaches combining molecular biology, biochemistry, computational modeling, and medicinal chemistry to fully understand the multifaceted nature of psbE and its potential applications beyond photosynthesis research.

What methodological advances would enhance research on recombinant psbE proteins?

Advancing research on recombinant psbE proteins requires methodological innovations across multiple domains, from expression systems to analytical techniques. Based on current limitations and emerging technologies, the following methodological advances would significantly enhance this research area:

Expression System Optimization:

• Cell-Free Expression Systems

  • Development of specialized cell-free systems for membrane protein expression

  • Incorporation of nanodiscs or liposomes for proper folding of hydrophobic domains

  • Direct incorporation of heme cofactors during synthesis

• Chloroplast-Based Expression Platforms

  • Refinement of biolistic chloroplast transformation for expressing psbE variants with minimized functional impact

  • Development of inducible expression systems in chloroplasts for temporal control

  • Optimization of protein extraction protocols to maintain native-like structures

Purification and Structural Analysis:

• Advanced Tagging Strategies

  • Design of cleavable tags that allow tag removal after purification

  • Development of minimally disruptive tags that maintain full protein function

  • Site-specific labeling approaches for tracking psbE in complex formation

• Native Complex Isolation

  • Improved methods for isolating intact PSII complexes containing recombinant psbE

  • Development of gentle solubilization protocols that preserve protein-protein interactions

  • Adaptation of styrene-maleic acid lipid particle (SMALP) technology for PSII isolation

Functional Characterization:

• Single-Molecule Techniques

  • Application of single-molecule fluorescence to study assembly dynamics

  • Development of surface-enhanced Raman spectroscopy for heme environment characterization

  • Single-molecule force spectroscopy to assess protein stability and folding

• Time-Resolved Spectroscopy

  • Ultra-fast spectroscopy to capture electron transfer events involving psbE

  • Time-resolved crystallography to capture conformational changes during function

  • Development of spectroscopic fingerprints for different functional states

RNA Editing Analysis:

• Direct RNA Sequencing

  • Application of nanopore direct RNA sequencing for unbiased detection of RNA editing

  • Development of computational tools for automated editing site identification

  • Methods for quantifying editing efficiency at individual sites

• Site-Specific RNA Editing Tools

  • Adaptation of CRISPR-Cas13 systems for programmable RNA editing

  • Development of tools to create defined editing patterns in psbE transcripts

  • Methods for temporally controlled editing to study kinetics