mRNA Quantification via Nucleotide Digestion and SCC Correction
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Solution Overview
Problem
Current methods for determining mRNA concentration rely on absorbance spectra assuming single-stranded RNA and a universal standard coefficient, which fail to account for secondary structures and chemically modified nucleotides, leading to inaccurate results.
Innovation Solution
A method involving digestion of mRNA into nucleotide monophosphates to derive sequence-corrected molar extinction coefficients (SCC) for accurate concentration determination, using a base to break down mRNA into nucleotide monophosphates and applying a sequence-corrected coefficient (SCC) to absorbance measurements.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If a universal standard coefficient is used for mRNA quantification, then the measurement process is simple and fast, but the accuracy is poor due to ignoring secondary structures and chemically modified nucleotides
Solution Approach 1:
The mRNA molecule is segmented into individual nucleotide monophosphates through base digestion. This segmentation eliminates the confounding effects of secondary structures and nearest-neighbor interactions that plague intact mRNA measurements. By measuring the absorbance of individual nucleotide components rather than the intact molecule, the method achieves accurate concentration determination without being influenced by structural variations or chemical modifications.
Solution Approach 2:
The method changes the physical-chemical state of the mRNA from intact folded structure to digested nucleotide monophosphates. This parameter change (from structured polymer to individual monomers) fundamentally alters the absorbance characteristics, eliminating the variability caused by secondary structures and enabling the use of a universal correction factor based on nucleotide composition rather than requiring sequence-specific calibration for each mRNA variant.
2Measurement precision
If absorbance measurement of intact mRNA is performed, then the procedure is quick and straightforward, but the results are inaccurate due to convoluted absorbance spectra from secondary structures and matrix effects
Solution Approach 1:
The method performs preliminary base digestion of the mRNA before absorbance measurement. This preliminary action converts the complex intact mRNA into simpler nucleotide monophosphates, eliminating secondary structures and matrix effects that would otherwise interfere with accurate measurement. The digestion step, while adding time, enables subsequent rapid and accurate quantification using a universal correction factor.
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
Provides precise mRNA concentration measurements by accounting for secondary structures and chemically modified nucleotides, enhancing accuracy and reliability.
Implementation Method 1
digesting a mRNA mixture by adding a base to a solution of mRNA and incubating the mixture to digest the mRNA into nucleotide monophosphates
Implementation Method 2
measuring an absorbance of a nucleotide solution comprising a plurality of nucleotides
Data Source
AI summary
Provided herein are methods for determining a concentration of RNA (e.g. mRNA) in a solution. Certain aspects of the disclosure relate to digesting the target RNA polymer by adding a base to a solution of RNA, optionally comprising at least one chemically modified nucleotide, and incubating the mixture to digest the RNA into a plurality of nucleotides. Certain other aspects of the disclosure relate to measuring the absorbance of the digested RNA mixture at 260 nm; while other aspects relate to using the absorbance measurement with a value of a relative percent of each nucleotide in the digestion mixture, and a mass-corrected coefficient of each nucleotide in the mixture to determine a concentration of the RNA. Other aspects of the disclosure relate to measuring the concentration of RNA in a solution using computer-implemented techniques.