spostrzeżenie - Synthetic Biology - # Intracellular expression of DNA aptamers using bacterial retrons

Intracellular Expression and Characterization of a Fluorogenic DNA Aptamer Using the Bacterial Retron Eco2 System

Q: How could the retron-based expression system be further optimized to improve the yield and folding of intracellularly expressed DNA aptamers?

To enhance the yield and folding of intracellularly expressed DNA aptamers using the retron-based expression system, several optimization strategies can be implemented: Sequence Optimization: Careful selection of the insertion site within the retron msd region is crucial. Conducting thorough structural analyses to identify regions that allow for minimal interference with the native fold of the msd sequence can improve aptamer folding. Additionally, optimizing the aptamer sequence itself for stability and efficient folding can enhance its performance. Promoter Strength and Inducer Concentration: Tuning the promoter strength and inducer concentration can regulate the copy number of the aptamer construct, influencing its expression levels. Fine-tuning these parameters can help achieve optimal expression without overwhelming the cellular machinery. Plasmid Copy Number: Adjusting the plasmid copy number can impact the overall expression levels of the aptamer construct. Exploring different plasmid backbones or replication origins to modulate copy number can optimize the yield of functional aptamers. Cellular Environment: Understanding the intracellular factors that influence aptamer folding, such as chaperones, RNA-binding proteins, and ribosomal machinery, can guide the design of aptamer constructs that are compatible with the cellular environment. Modulating these factors through genetic engineering or co-expression of specific proteins can improve aptamer folding. Post-Transcriptional Modifications: Exploring post-transcriptional modifications, such as adding stabilizing elements or modifying the aptamer sequence for enhanced stability, can improve the overall folding and functionality of the aptamer within the cellular context. By implementing these optimization strategies, the retron-based expression system can be fine-tuned to improve the yield and folding of intracellularly expressed DNA aptamers, expanding their applications in cellular settings.

Q: What other types of functional DNA sequences, beyond aptamers, could be expressed using the retron platform, and how might they be applied in cellular contexts?

Beyond aptamers, the retron platform can be utilized to express a variety of functional DNA sequences with diverse applications in cellular contexts. Some examples include: Ribozymes and DNAzymes: Retrons can be used to express catalytic RNA or DNA molecules that can cleave specific RNA targets or catalyze chemical reactions within the cell. These molecules can be applied in gene regulation, biosensing, and therapeutic interventions. Riboswitches: DNA sequences that can regulate gene expression in response to specific ligands or environmental cues, known as riboswitches, can be expressed using retrons. By controlling gene expression at the transcriptional or translational level, riboswitches can be applied in metabolic engineering and synthetic biology applications. Antisense Oligonucleotides: Retrons can be harnessed to produce antisense oligonucleotides that bind to complementary mRNA sequences, modulating gene expression or inhibiting protein translation. This approach can be used for gene silencing, studying gene function, and developing therapeutic interventions. DNA Aptamer-Based Sensors: Apart from traditional aptamers, DNA sequences designed for sensing specific molecules or environmental conditions can be expressed using retrons. These aptamer-based sensors can be utilized for real-time monitoring of cellular processes, environmental pollutants, or disease biomarkers. DNA Origami Structures: Retrons can potentially be used to express DNA sequences that fold into intricate nanostructures, such as DNA origami. These structures can serve as scaffolds for drug delivery, molecular computing, or as platforms for organizing biomolecules within the cell. By leveraging the retron platform to express a diverse range of functional DNA sequences, researchers can explore novel applications in cellular contexts, ranging from gene regulation and sensing to nanotechnology and therapeutics.

Q: Given the observed differences in aptamer performance between in vitro and in vivo conditions, what insights could be gained by studying the intracellular factors and mechanisms that influence the folding and function of retron-expressed DNA constructs?

Studying the intracellular factors and mechanisms that influence the folding and function of retron-expressed DNA constructs can provide valuable insights into the following areas: Chaperone Interactions: Understanding how molecular chaperones interact with retron-expressed DNA constructs can elucidate their role in aptamer folding and stability within the cellular environment. Identifying specific chaperones that facilitate aptamer folding can guide the design of more robust constructs. Ribosomal Machinery: Investigating the interaction between retron-expressed DNA constructs and the ribosomal machinery can shed light on how translation efficiency and fidelity impact aptamer functionality. Optimizing the translation process can enhance the yield and quality of functional DNA aptamers. RNA Binding Proteins: Exploring the interactions between RNA binding proteins and retron-expressed DNA constructs can reveal potential regulatory mechanisms that influence aptamer folding and function. Modulating these interactions can fine-tune aptamer performance in vivo. Cellular Localization: Studying the subcellular localization of retron-expressed DNA constructs can provide insights into how compartmentalization affects aptamer folding and activity. Understanding the impact of different cellular environments on aptamer function can guide the design of constructs tailored for specific cellular compartments. Post-Transcriptional Modifications: Investigating post-transcriptional modifications that occur on retron-expressed DNA constructs can uncover additional layers of regulation that influence aptamer stability and function. Manipulating these modifications can enhance the performance of DNA constructs in vivo. By delving into the intracellular factors and mechanisms that impact the folding and function of retron-expressed DNA constructs, researchers can optimize the design and expression of functional DNA molecules for diverse applications in cellular contexts.

Główne pojęcia

The bacterial retron system Eco2 can be used to effectively express and characterize functional DNA aptamers, such as the fluorogenic Lettuce aptamer, within living cells.

Streszczenie

The study demonstrates the use of the bacterial retron system Eco2 to express a DNA light-up aptamer, Lettuce, within Escherichia coli cells. Key highlights:

Retrons are prokaryotic elements that can synthesize single-stranded DNA (ssDNA) in vivo, making them a promising platform for intracellular expression of DNA aptamers.
The authors used a structure-guided approach to insert the Lettuce aptamer domain into four different positions within the msd region of the Eco2 retron.
In vitro characterization showed that only the Lettuce variant inserted at position v4 could bind the DFHBI-1T ligand and induce fluorescence.
However, when expressed in E. coli cells, all four Lettuce variants showed increased fluorescence upon DFHBI-1T staining, suggesting improved folding in the intracellular environment compared to in vitro.
The authors observed a significant decrease in Eco2 RT-DNA abundance after integrating the Lettuce aptamer, but the remaining RT-DNA was still sufficient to produce detectable fluorescence.
The findings demonstrate the potential of retron systems to enable intracellular expression and characterization of functional DNA aptamers, which could expand their applications in cellular settings.

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biorxiv.org

Statystyki

The RT-DNA abundance of the Eco2-Lettuce variants was 30-70% of the endogenous Eco1 retron.
Cells expressing the 4LE-v4 variant showed a ~2.6-fold increase in fluorescence compared to the wild-type Eco2 control.
Cells expressing the 4LE-v1, v2, and v3 variants showed a 1.7 to 2-fold increase in fluorescence compared to the Eco2 control.

Cytaty

"Expanding the repertoire of retrons for the expression of DNA aptamers promises to significantly expand the repertoire of functional non-coding DNAs and enable the creation of new sensors and regulators with a wide range of intracellular applications."
"A major advantage of retron-based intracellular synthesis of ssDNA in vivo is its simplicity. The desired sequence can be cloned into the msd region of plasmid or genomically encoded retron Eco2. As such, the copy number of the aptamer can readily be tuned by inducer concentration, promoter strength and plasmid copy number."

Kluczowe wnioski z

Intracellular Expression of a Fluorogenic DNA Aptamer Using Retron Eco2

by Vibhute,M. A... o www.biorxiv.org 05-23-2024

https://www.biorxiv.org/content/10.1101/2024.05.21.595248v2

Głębsze pytania

How could the retron-based expression system be further optimized to improve the yield and folding of intracellularly expressed DNA aptamers?

To enhance the yield and folding of intracellularly expressed DNA aptamers using the retron-based expression system, several optimization strategies can be implemented:

Sequence Optimization: Careful selection of the insertion site within the retron msd region is crucial. Conducting thorough structural analyses to identify regions that allow for minimal interference with the native fold of the msd sequence can improve aptamer folding. Additionally, optimizing the aptamer sequence itself for stability and efficient folding can enhance its performance.

Promoter Strength and Inducer Concentration: Tuning the promoter strength and inducer concentration can regulate the copy number of the aptamer construct, influencing its expression levels. Fine-tuning these parameters can help achieve optimal expression without overwhelming the cellular machinery.

Plasmid Copy Number: Adjusting the plasmid copy number can impact the overall expression levels of the aptamer construct. Exploring different plasmid backbones or replication origins to modulate copy number can optimize the yield of functional aptamers.

Cellular Environment: Understanding the intracellular factors that influence aptamer folding, such as chaperones, RNA-binding proteins, and ribosomal machinery, can guide the design of aptamer constructs that are compatible with the cellular environment. Modulating these factors through genetic engineering or co-expression of specific proteins can improve aptamer folding.

Post-Transcriptional Modifications: Exploring post-transcriptional modifications, such as adding stabilizing elements or modifying the aptamer sequence for enhanced stability, can improve the overall folding and functionality of the aptamer within the cellular context.

By implementing these optimization strategies, the retron-based expression system can be fine-tuned to improve the yield and folding of intracellularly expressed DNA aptamers, expanding their applications in cellular settings.

What other types of functional DNA sequences, beyond aptamers, could be expressed using the retron platform, and how might they be applied in cellular contexts?

Beyond aptamers, the retron platform can be utilized to express a variety of functional DNA sequences with diverse applications in cellular contexts. Some examples include:

Ribozymes and DNAzymes: Retrons can be used to express catalytic RNA or DNA molecules that can cleave specific RNA targets or catalyze chemical reactions within the cell. These molecules can be applied in gene regulation, biosensing, and therapeutic interventions.

Riboswitches: DNA sequences that can regulate gene expression in response to specific ligands or environmental cues, known as riboswitches, can be expressed using retrons. By controlling gene expression at the transcriptional or translational level, riboswitches can be applied in metabolic engineering and synthetic biology applications.

Antisense Oligonucleotides: Retrons can be harnessed to produce antisense oligonucleotides that bind to complementary mRNA sequences, modulating gene expression or inhibiting protein translation. This approach can be used for gene silencing, studying gene function, and developing therapeutic interventions.

DNA Aptamer-Based Sensors: Apart from traditional aptamers, DNA sequences designed for sensing specific molecules or environmental conditions can be expressed using retrons. These aptamer-based sensors can be utilized for real-time monitoring of cellular processes, environmental pollutants, or disease biomarkers.

DNA Origami Structures: Retrons can potentially be used to express DNA sequences that fold into intricate nanostructures, such as DNA origami. These structures can serve as scaffolds for drug delivery, molecular computing, or as platforms for organizing biomolecules within the cell.

By leveraging the retron platform to express a diverse range of functional DNA sequences, researchers can explore novel applications in cellular contexts, ranging from gene regulation and sensing to nanotechnology and therapeutics.

Given the observed differences in aptamer performance between in vitro and in vivo conditions, what insights could be gained by studying the intracellular factors and mechanisms that influence the folding and function of retron-expressed DNA constructs?

Studying the intracellular factors and mechanisms that influence the folding and function of retron-expressed DNA constructs can provide valuable insights into the following areas:

Chaperone Interactions: Understanding how molecular chaperones interact with retron-expressed DNA constructs can elucidate their role in aptamer folding and stability within the cellular environment. Identifying specific chaperones that facilitate aptamer folding can guide the design of more robust constructs.

Ribosomal Machinery: Investigating the interaction between retron-expressed DNA constructs and the ribosomal machinery can shed light on how translation efficiency and fidelity impact aptamer functionality. Optimizing the translation process can enhance the yield and quality of functional DNA aptamers.

RNA Binding Proteins: Exploring the interactions between RNA binding proteins and retron-expressed DNA constructs can reveal potential regulatory mechanisms that influence aptamer folding and function. Modulating these interactions can fine-tune aptamer performance in vivo.

Cellular Localization: Studying the subcellular localization of retron-expressed DNA constructs can provide insights into how compartmentalization affects aptamer folding and activity. Understanding the impact of different cellular environments on aptamer function can guide the design of constructs tailored for specific cellular compartments.

Post-Transcriptional Modifications: Investigating post-transcriptional modifications that occur on retron-expressed DNA constructs can uncover additional layers of regulation that influence aptamer stability and function. Manipulating these modifications can enhance the performance of DNA constructs in vivo.

By delving into the intracellular factors and mechanisms that impact the folding and function of retron-expressed DNA constructs, researchers can optimize the design and expression of functional DNA molecules for diverse applications in cellular contexts.