5' Capped Labeled RNA for Ribosomal Depletion
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Solution Overview
Problem
Current methods for ribosomal RNA depletion in RNA sequencing are inefficient and leave substantial ribosomal and degraded RNA in the mixture, requiring prior knowledge of ribosomal sequences and custom DNA oligonucleotides for hybridization, or using enzymatic degradation that is not comprehensive.
Innovation Solution
A compound represented by Formula (I) is used, where the Base is a purine or pyrimidine, R is a linker, and L is a label such as biotin or fluorescent labels, to convert uncapped RNA into 5' capped labeled RNA through capping enzymes, allowing for enrichment and sequencing of prokaryotic non-ribosomal RNA by immobilization and selective amplification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If DNA oligonucleotides are used for ribosomal RNA depletion through hybridization, then ribosomal RNA can be removed from the mixture, but custom DNA sequences must be designed and synthesized which increases device complexity and cost
Solution Approach 1:
The invention changes the chemical parameter of the depletion probe from DNA oligonucleotide to RNA oligonucleotide with specific modifications (2'-O-methyl and phosphorothioate). This parameter change eliminates the need for custom DNA synthesis while maintaining depletion efficiency, as the modified RNA probes can be purchased as standard products rather than requiring custom design and synthesis.
Solution Approach 2:
The invention replaces expensive custom-synthesized DNA oligonucleotides with commercially available modified RNA oligonucleotides that can be used directly. This substitution uses off-the-shelf reagents rather than custom-made components, reducing both cost and complexity while achieving the same functional outcome of ribosomal RNA depletion.
2Ease of operation
If enzymatic degradation is used to remove ribosomal RNA, then the process is simpler to perform, but the degradation is inefficient and leaves substantial ribosomal and degraded RNA in the mixture
Solution Approach 1:
The invention changes the fundamental parameter of the depletion mechanism from enzymatic degradation to hybridization-based capture. Instead of using enzymes that partially degrade RNA, the method uses modified RNA oligonucleotides that hybridize to ribosomal RNA and enable its selective removal through washing, achieving both simplicity and completeness in the depletion process.
3Reliability
If traditional ribosomal RNA depletion methods are used, then ribosomal RNA can be reduced, but degraded RNA remains in the mixture contaminating the sequencing results
Solution Approach 1:
The invention performs preliminary enrichment of intact RNA molecules before sequencing by using the modified RNA oligonucleotide probes to capture and remove both ribosomal RNA and degraded RNA fragments simultaneously. This preliminary action ensures that only high-quality, intact non-ribosomal RNA remains for sequencing, eliminating degraded RNA contamination from the outset.
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 method effectively enriches prokaryotic non-ribosomal RNA, reducing ribosomal RNA contamination, enabling more accurate sequencing and identification of transcriptional start sites with single base resolution, and can be applied to complex RNA mixtures without prior sequence knowledge.
Implementation Method 1
combining a preparation comprising uncapped RNA having a 5' diphosphate or 5' triphosphate with a capping enzyme and a labeled modified nucleotide represented by Formula (I) so as to convert the uncapped RNA into 5' capped labeled RNA
Implementation Method 2
enriching labeled, capped RNA by immobilizing the labeled capped RNA on an affinity substrate and washing away the unlabeled RNA
Data Source
Figure 1
Figure 2A~2B
Figure 2C~2D
AI summary
Methods and compositions are described for distinguishing target RNA having a 5' diphosphate or triphosphate in a mixture of RNAs. The methods utilized modified labeled nucleotides for capping the 5' end of the RNA. The label on the modified nucleotide is attached to the carbon 3 via the oxygen by replacing the hydrogen ion.