ClickSeq

ClickSeq is a high-throughput sequencing method designed for the analysis of RNA structure. It combines selective 2'-hydroxyl acylation performed by transesterification using imidazole (SHAPE) chemistry with next-generation sequencing to provide nucleotide-resolution information about RNA flexibility and secondary structure.

Overview:

ClickSeq builds upon earlier SHAPE-Seq techniques. SHAPE reagents modify flexible nucleotides in RNA, often those that are unpaired or in dynamic regions. ClickSeq utilizes a modified SHAPE reagent with an alkyne tag. After modification, the RNA is fragmented, and the alkyne-modified nucleotides are tagged with a biotin azide using a click chemistry reaction. Biotinylated fragments are then enriched using streptavidin beads. Following enrichment, a sequencing library is prepared and sequenced using standard high-throughput sequencing platforms.

Principle:

The fundamental principle of ClickSeq relies on the differential reactivity of RNA nucleotides to SHAPE reagents based on their structural context. Flexible, unpaired nucleotides are more accessible and therefore more readily modified by the SHAPE reagent. The alkyne tag allows for selective enrichment of these modified fragments, making it possible to identify them via sequencing. By quantifying the frequency of modification at each nucleotide position, researchers can infer the underlying RNA secondary structure and dynamics.

Applications:

ClickSeq has applications in various areas of RNA research, including:

• RNA Structure Determination: Mapping RNA secondary and tertiary structures at nucleotide resolution.
• RNA Folding Studies: Investigating RNA folding pathways and conformational changes in response to various stimuli.
• RNA-Protein Interactions: Identifying regions of RNA that interact with proteins by observing changes in RNA structure upon protein binding.
• Drug Discovery: Screening for compounds that bind to RNA targets and alter their structure.
• Understanding RNA Function: Correlating RNA structure with its biological function.

Advantages:

ClickSeq offers several advantages over other RNA structure probing techniques, including:

• High Throughput: Allows for the analysis of large RNA molecules or complex RNA populations.
• Nucleotide Resolution: Provides information about RNA structure at the single-nucleotide level.
• Sensitivity: Capable of detecting subtle changes in RNA structure.
• Direct Measurement: Directly measures RNA modification, providing quantitative data on RNA flexibility.

Limitations:

While ClickSeq is a powerful technique, it has some limitations:

• SHAPE Reagent Bias: The choice of SHAPE reagent can influence the observed modification patterns.
• Library Preparation Artifacts: Biases introduced during library preparation can affect the accuracy of the results.
• Computational Analysis: Requires sophisticated computational tools for data analysis and interpretation.
• Cost: The use of next-generation sequencing can be expensive.