Modified Template Switching Oligos for Specific cDNA Synthesis
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing RNA sequencing methods, particularly those using template switching for single-cell applications, suffer from non-specific reverse transcription and template switching oligo concatemerization, leading to biased and inefficient cDNA synthesis.
Innovation Solution
The use of template switching oligonucleotides (TSOs) with specific 3′ and 5′ end modifications, such as 3′ ddT and 5′ abasic sites, along with optimized reverse transcriptases and reaction buffers, to reduce concatemerization and enhance specificity and yield of cDNA synthesis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If template switching oligonucleotides are used for cDNA synthesis in single-cell RNA sequencing, then full-length cDNA enrichment and adapter addition are achieved, but non-specific reverse transcription and oligo concatemerization dominate the sequencing libraries
Solution Approach 1:
The patent applies local quality by introducing specific modifications at the 5' and 3' ends of the template switching oligonucleotide. The 5' end contains a modified nucleotide (such as dG or dA) that prevents concatemerization, while the 3' end maintains the template switching capability. This localized modification strategy resolves the contradiction by preserving the useful template switching function while eliminating the harmful concatemerization effect at specific locations.
Solution Approach 2:
The patent converts the harmful effect of non-specific reverse transcription into a beneficial outcome by designing the modified TSO to specifically anneal to non-templated nucleotides added by reverse transcriptase. The modification allows the TSO to distinguish between specific and non-specific reverse transcription products, thereby enriching full-length cDNA while preventing concatemerization of the oligo itself.
2Ease of operation
If standard template switching oligonucleotides are used, then the reaction is simple to perform, but concatemerization of the TSO occurs leading to biased cDNA synthesis
Solution Approach 1:
The patent applies parameter changes by modifying the chemical structure of the template switching oligonucleotide at specific positions. The introduction of modified nucleotides (such as 5'-dG or 5'-dA) changes the physical and chemical properties of the oligo, preventing self-annihilation and concatemerization. This structural parameter change maintains the ease of use in the protocol while significantly improving the accuracy and representativeness of the cDNA synthesis.
3Reliability
If ligation-based approaches are used for adapter addition, then the process is well-established, but they have many shortcomings including inefficiency and bias
Solution Approach 1:
The patent replaces the mechanical ligation-based approach with a template switching mechanism that occurs naturally during reverse transcription. Instead of using T4 RNA ligase to join adapters to cDNA ends, the modified TSO enables automated adapter addition through the reverse transcriptase enzyme's template switching activity. This substitution eliminates the inefficiencies and biases of ligation-based methods while maintaining procedural simplicity.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach significantly reduces non-specific reverse transcription and concatemerization, improving the yield and specificity of cDNA synthesis, especially in low-input RNA samples, thereby enhancing the accuracy of RNA sequencing.
Implementation Method 1
the TSO can anneal by base-pairing to non-templated nucleotides that have been added to the 5'-end of a target nucleic acid molecule
Data Source
AI summary
The present invention provides optimized template switching oligonucleotides, methods, and kits for performing reverse transcription. The optimized template switching oligonucleotides include modifications of 5′ and 3′ ends to prevent the formation of concatemers and to enhance the specificity of reverse transcription.