Blocking Moiety Oligonucleotides for Strand-Specific RNA Sequencing
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Solution Overview
Problem
Conventional methods for preparing strand-specific RNA-sequencing libraries are prone to biased ligation and require additional enzymatic steps, such as UNG digestion, which are time-consuming and not 100% efficient, leading to residual second strand cDNA and false interpretation of RNA-sequencing data.
Innovation Solution
A method involving the use of oligonucleotide primers covalently coupled with a blocking moiety at their 5' terminal nucleotide to prevent ligation, allowing for the generation of strand-specific RNA-sequencing libraries without additional enzymatic steps, specifically through reverse transcription and DNA polymerase reactions, followed by adapter ligation and sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If dUTP is incorporated in the second strand DNA synthesis followed by UNG digestion, then strand-specificity is achieved, but the library construction process becomes more complicated and time-consuming
Solution Approach 1:
The invention extracts and removes the UNG digestion step from the library construction process by using blocking moieties during primer design. The blocking moiety prevents ligation of the second strand to adapters, eliminating the need for subsequent enzymatic removal of the second strand, thereby simplifying the workflow while maintaining strand-specificity
Solution Approach 2:
The blocking moiety is incorporated into the primer design at the beginning of the library construction process. This preliminary action prevents unwanted ligation events before they can occur, avoiding the need for corrective enzymatic steps later in the process
2Reliability
If UNG digestion is used to remove second strand cDNA, then strand-specificity is improved, but residual second strand cDNA remains causing false interpretation
Solution Approach 1:
Instead of relying on enzymatic digestion that may leave residuals, the invention completely removes the second strand from participating in ligation reactions through the blocking moiety. This ensures complete elimination of second strand cDNA interference without residual contamination
Solution Approach 2:
The blocking moiety acts as a disposable element that permanently prevents second strand ligation. The blocked second strand is rendered non-functional for the intended purpose from the outset, ensuring no false signals are generated
3Ease of manufacture
If conventional adapter ligation is used without blocking moieties, then library construction is simpler, but biased ligation occurs due to structural properties of RNA and adapters
Solution Approach 1:
The blocking moiety is placed locally at the 5' end of the primer, creating a localized modification that specifically prevents unwanted ligation events without affecting other aspects of the library construction process. This localized intervention maintains overall simplicity while eliminating bias
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 results in highly specific and efficient RNA sequencing that distinguishes sense from anti-sense transcripts, improving the accuracy of RNA quantification and reducing the complexity of the library construction process.
Implementation Method 1
generating (a) single-stranded first DNA strand(s) (cDNA), which is/are complementary to the RNA, by subjecting the RNA to reverse transcription by using a reverse transcriptase
Implementation Method 2
generating a second DNA strand by using a DNA polymerase
Data Source
AI summary
The invention refers to a novel method of preparing strand-specific RNA-sequencing libraries that can be used to identify DNA coding and non-coding strands that are transcribed to RNA. Such strand-specific RNA-sequencing libraries are especially useful in discovering anti-sense RNA and non-coding RNA. Random primer oligonucleotides, covalently coupled to a moiety, which blocks ligation, are used for RT reaction or the subsequent generation of the second DNA strand so that only one strand of the generated double-stranded DNA is ligated to sequencing adapters at the 5′ nucleotide and sequenced by paired-end sequencing.