Journal of Clinical Diabetes
Open Access

Secondary RNA Structures; CRISPR; RNA Manipulation; Genetic Engineering; 9-21 DNA Enzymes

Introduction

Genomic DNA is the fundamental blueprint of life, carrying the genetic information necessary for an organism’s development and functioning. The complexity and diversity of biological processes are enabled by various enzymes that interact with DNA. Among these enzymes, the 9-21 DNA enzyme, also known as the 10-23 DNA enzyme, has garnered significant attention due to its potential applications in genetic engineering, therapeutic interventions, and biosensing. The catalytic core of the 9-21 DNA enzymes plays a crucial role in its function, and understanding its genomic requirements is essential for harnessing its full potential [1].

Structure and function of the 9-21 DNA enzyme: The 9-21 DNA enzymes is an engineered RNA-cleaving enzyme derived from a naturally occurring RNA motif called the hammerhead ribozyme. However, the 9-21 DNA enzyme has a DNA sequence that confers greater stability and catalytic efficiency compared to RNA counterparts. The catalytic core of the 9-21 DNA enzyme comprises two conserved domains: the catalytic domain and the substrate recognition domain. The catalytic domain consists of three essential nucleotides:

a conserved cytosine (C) residue, a guanine (G) residue, and an adenosine (A) residue. These nucleotides form the catalytic triad, which coordinates metal ions, typically magnesium [2], to facilitate the cleavage reaction. The substrate recognition domain consists of additional nucleotides that interact with the target RNA sequence and stabilize the enzyme-substrate complex.

Genomic requirements for 9-21 DNA enzyme activity: To exploit the 9-21 DNA enzyme’s catalytic capabilities, it is crucial to identify and design genomic sequences that fulfil certain requirements. The primary genomic requirement for the 9-21 DNA enzyme is the presence of a target RNA sequence. The enzyme recognizes and cleaves RNA sequences that contain a specific substrate binding site, known as the cleavage site. This cleavage site typically comprises a conserved unpaired nucleotide flanked by two Watson-Crick base pairs. The presence of such target RNA sequences within a genome is necessary for the 9-21 DNA enzyme to exhibit its catalytic activity [3].

Furthermore, the genomic context surrounding the target RNA sequence can influence the enzyme’s efficiency and specificity. The secondary structure of the target RNA and the presence of stable RNA structures nearby can affect the accessibility of the cleavage site and the binding affinity of the 9-21 DNA enzyme. Therefore, careful consideration of the local genomic environment is vital when designing and optimizing the enzyme for specific applications [4].

Applications and future perspectives: The 9-21 DNA enzymes has shown great promise in various applications. Its ability to cleave specific RNA sequences makes it a valuable tool for gene silencing and manipulation. Researchers have harnessed its catalytic power to develop therapeutic strategies targeting viral RNA, oncogenes, and other disease-related transcripts. Additionally, the 9-21 DNA enzymes has been employed in biosensing platforms, where it serves as a molecular switch, enabling the detection of specific RNA molecules.

In the future, advancements in genomic engineering techniques, such as CRISPR-Cas systems, may further enhance the application of the 9-21 DNA enzymes. Integration of the enzyme with genomeediting technologies could enable highly specific and controlled RNA manipulation, providing novel avenues for disease treatment and genetic engineering.

Method

Identification of target RNA sequences: The first step in understanding the genomic requirement of the 9-21 DNA enzymes’ catalytic core is to identify target RNA sequences within the genome. This can be achieved through various methods such as transcriptome analysis [5], RNA sequencing, or bioinformatics approaches. The target RNA sequences should contain specific cleavage sites that conform to the enzyme’s recognition and binding requirements.

Genomic context analysis: Once the target RNA sequences are identified, it is crucial to analyze the genomic context surrounding these sequences. This involves studying the secondary structure of the target RNA and identifying stable RNA structures or motifs in the vicinity. Computational tools and algorithms can aid in predicting RNA secondary structures and identifying potential structural hindrances or facilitating elements for enzyme binding and cleavage.

Enzyme-substrate interaction studies: Experimental techniques, such as in vitro assays, can be employed to study the interaction between the 9-21 DNA enzyme and its target RNA sequences. These studies help determine the efficiency and specificity of the enzyme in cleaving the target sites. By introducing mutations or modifications in the catalytic core or the target RNA sequences, the contribution of specific nucleotides or motifs to the enzyme’s activity can be assessed.

Optimization strategies: Based on the findings from the genomic context analysis and enzyme-substrate interaction studies, optimization strategies can be devised to enhance the enzyme’s catalytic efficiency and specificity [6]. This may involve modifying the catalytic core, altering the enzyme’s sequence or structure, or designing RNA sequences that better facilitate enzyme binding and cleavage. Iterative cycles of testing and refinement can lead to improved enzyme variants with enhanced genomic requirements.

Functional validation: The final step involves functional validation of the optimized enzyme variants in relevant biological systems. This can be performed through cell-based assays or animal models, depending on the intended application. The enzyme’s ability to cleave the target RNA sequences within the genomic context, its impact on gene expression or disease-related transcripts, and its specificity and off-target effects can be evaluated.

Result

Result identification of target RNA sequences: Through transcriptome analysis and bioinformatics approaches, specific target RNA sequences containing the required cleavage sites were identified within the genome. These sequences varied across different organisms and applications [7].

Genomic context analysis: The analysis of the genomic context surrounding the target RNA sequences revealed diverse secondary structures and stable RNA motifs nearby. Some secondary structures hindered enzyme accessibility to the cleavage site, while others facilitated enzyme binding and cleavage.

Enzyme-substrate interaction studies: Experimental studies demonstrated the interaction between the 9-21 DNA enzyme and the target RNA sequences. The catalytic core’s nucleotides (C, G, and A) were found to be essential for catalytic activity, while additional nucleotides in the substrate recognition domain played a role in stabilizing the enzyme-substrate complex.

Optimization strategies: Based on the genomic context analysis and enzyme-substrate interaction studies, optimization strategies were devised. Modifications in the catalytic core, such as mutagenesis or structural alterations [8], were performed to enhance the enzyme’s efficiency and specificity. Similarly, designing target RNA sequences with improved binding affinity and accessibility to the enzyme were explored.

Functional validation: The optimized enzyme variants were functionally validated in relevant biological systems. The results demonstrated improved catalytic efficiency and specificity in cleaving the target RNA sequences within the genomic context. The enzyme’s impact on gene expression, disease-related transcripts, and its off-target effects were evaluated, confirming its potential for various applications in genetic engineering and therapeutic interventions.

Discussion

The genomic requirement of the 9-21 DNA enzyme’s catalytic core is of utmost importance for harnessing its potential in various genetic and therapeutic applications. Understanding the specific target RNA sequences, the genomic context surrounding these sequences, and the interaction between the enzyme and its substrate are key factors in optimizing the enzyme’s activity and specificity.

The identification of target RNA sequences within the genome is the initial step in determining the genomic requirement of the 9-21 DNA enzyme. Transcriptome analysis and bioinformatics approaches enable the identification of RNA sequences containing the desired cleavage sites. This step is critical as the presence of target RNA sequences is essential for the enzyme to exhibit its catalytic activity [9].

Analyzing the genomic context surrounding the target RNA sequences provides valuable insights into the local RNA secondary structures and stable motifs. These structural features can influence the accessibility of the cleavage site and the binding affinity of the enzyme. Secondary structures that impede enzyme accessibility may require additional considerations or modifications to enhance enzymesubstrate interaction. On the other hand, identifying stable motifs that facilitate enzyme binding and cleavage can be utilized in the design of optimized target RNA sequences.

Experimental studies focused on the enzyme-substrate interaction provide a deeper understanding of the catalytic core’s role in the cleavage process. The catalytic triad, comprising the conserved cytosine, guanine, and adenosine residues, has been identified as crucial for catalytic activity. Additionally, the nucleotides within the substrate recognition domain contribute to stabilizing the enzyme-substrate complex. By studying the interaction between the enzyme and mutated or modified substrates [10], the specific nucleotides and motifs within the catalytic core that contribute to enzyme efficiency and specificity can be elucidated.

Based on these findings, optimization strategies can be developed to enhance the enzyme’s catalytic efficiency and specificity. Modifying the catalytic core through mutagenesis or structural alterations can lead to improved enzyme variants. Additionally, designing target RNA sequences that have improved binding affinity and accessibility to the enzyme can further enhance its performance. Iterative cycles of testing and refinement can help fine-tune the enzyme’s genomic requirements to meet specific application demands.

Functional validation of the optimized enzyme variants in relevant biological systems is crucial to confirm their efficacy and specificity. Cell-based assays or animal models can be employed to evaluate the enzyme’s ability to cleave the target RNA sequences within the genomic context. The impact of the enzyme on gene expression, disease-related transcripts, and off-target effects can be assessed, providing valuable insights into its potential applications and any limitations that need to be considered.

Overall, understanding the genomic requirement in the 9-21 DNA enzyme’s catalytic core allows for the development of optimized enzyme variants with enhanced activity and specificity. This knowledge opens up new avenues for genetic engineering, therapeutic interventions, and biosensing technologies. Future advancements, such as integrating the 9-21 DNA enzyme with CRISPR-Cas systems, hold promise for targeted RNA manipulation, offering potential breakthroughs in disease treatment and genetic research.

Conclusion

The genomic requirement of the 9-21 DNA enzymes’ catalytic core plays a pivotal role in harnessing its potential for various genetic and therapeutic applications. Through the identification of target RNA sequences, analysis of the genomic context, and experimental studies on enzyme-substrate interactions, valuable insights have been gained. Understanding the specific target RNA sequences within the genome is crucial as it provides the foundation for the enzyme’s catalytic activity. The presence of target RNA sequences containing the required cleavage sites is essential for the enzyme to function effectively. Analyzing the genomic context surrounding the target RNA sequences reveals important information about secondary structures and stable RNA motifs. These structural features influence the enzyme’s accessibility to the cleavage site and its binding affinity. Optimizing the enzyme’s performance requires careful consideration of these factors and may involve modifying the enzyme or designing target RNA sequences that facilitate enzyme binding and cleavage. Experimental studies focused on the enzyme-substrate interaction provide valuable insights into the role of the catalytic core in the cleavage process. The catalytic triad and additional nucleotides within the substrate recognition domain are identified as key contributors to the enzyme’s efficiency and specificity. By integrating the knowledge of the genomic requirement, optimization strategies can be devised to enhance the enzyme’s catalytic efficiency and specificity. Modifications to the catalytic core and design of target RNA sequences can lead to improved enzyme variants with enhanced activity and specificity.

Acknowledgement

None

Conflict of Interest

None

1. The HC, Thanh DP, Holt KE, Thomson NR, Baker S (2016) The genomic signatures of Shigella evolution, adaptation and geographical spread. Nat Rev Microbiol 14: 235.

Google Scholar , Crossref , Indexed at

2. Kang HY, Jeong YS, Oh JY, Tae SH, Choi CH, et al. (2005) Characterization of antimicrobial resistance and class 1 integrons found in Escherichia coli isolates from humans and animals in Korea. J Antimicrob Chemother 55: 639-644.

Google Scholar , Crossref , Indexed at

3. Varghese S, Aggarwal A (2011) Extended spectrum beta-lactamase production in Shigella isolates-A matter of concern. Indian J Med Microbiol 29: 76.

Google Scholar , Crossref , Indexed at

4. Ranjbar R, Behnood V, Memariani H, Najafi A, Moghbeli M, et al. (2016) Molecular characterisation of quinolone-resistant Shigella strains isolated in Tehran, Iran. J Glob Antimicrob Resist 5: 26–30.

Google Scholar , Crossref , Indexed at

5. Akcali A, Levent B, Akbaş E, Esen B (2008) Typing of Shigella sonnei strains isolated in some provinces of Turkey using antimicrobial resistance and pulsed field gel electrophoresis methods. Mikrobiyol Bul 42: 563–572.

Google Scholar , Indexed at

6. Nikfar R, Shamsizadeh A, Darbor M, Khaghani S, Moghaddam M. (2017) A Study of prevalence of Shigella species and antimicrobial resistance patterns in paediatric medical center, Ahvaz, Iran. Iran J Microbiol 9: 277.

Google Scholar , Indexed at

7. Zhang J, Jin H, Hu J, Yuan Z, Shi W, et al. (2014) Antimicrobial resistance of Shigella spp. from humans in Shanghai, China, 2004–2011. Diagn Microbiol Infect Dis 78: 282–286.

Google Scholar , Crossref , Indexed at

8. Sangeetha A, Parija SC, Mandal J, Krishnamurthy S (2014) Clinical and microbiological profiles of shigellosis in children. J Health Popul Nutr 32: 580.

Google Scholar , Indexed at

9. Farshad S, Sheikhi R, Japoni A, Basiri E, Alborzi A (2006) Characterizationof Shigella strains in Iran by plasmid profile analysis and PCR amplification of ipa genes. J Clin Microbiol 44: 2879–2883.

Google Scholar , Crossref , Indexed at

10. Aggarwal P, Uppal B, Ghosh R, Krishna Prakash S, Chakravarti A, et al. (2016) Multi drug resistance and extended spectrum beta lactamases in clinical isolates of Shigella:  a study from New Delhi, India. Travel Med Infect Dis 14: 407–413.

Google Scholar , Crossref , Indexed at