3' Blocked AbSeq Oligonucleotides to Prevent Template Extension
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Solution Overview
Problem
Current methods struggle to quantitatively analyze protein expression in cells while simultaneously measuring gene expression, and existing protein-profiling techniques face issues with antibody oligonucleotides extending undesirably, necessitating compositions and methods to prevent or reduce this extension.
Innovation Solution
A protected cellular component-binding composition is generated by adding blocker nucleotides to the 3' end of cellular component-binding reagent specific oligonucleotides using enzymes like terminal transferase, creating a terminal blocked region that prevents extension by reverse transcriptase or polymerase, allowing specific binding and hybridization with barcoding oligonucleotides.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If antibody oligonucleotides are used as templates for barcoding reaction, then protein expression can be analyzed, but the oligonucleotides extend undesirably forming double-stranded nucleic acids
Solution Approach 1:
The patent applies preliminary anti-action by adding blocker nucleotides to the 3' end of the antibody oligonucleotide template before the barcoding reaction. These blocker nucleotides prevent the polymerase from extending the template further, thereby preventing the formation of unwanted double-stranded nucleic acids while allowing the desired barcoding to proceed accurately.
Solution Approach 2:
The patent uses an intermediary approach by introducing a specific enzyme (terminal transferase or polymerase) that mediates the addition of blocker nucleotides to the 3' end of the antibody oligonucleotide. This intermediary enzyme controls the extension process to prevent undesired reactions while maintaining the template's functionality for barcoding.
2Adaptability or versatility
If the 3' end of the oligonucleotide is extended, then more binding sites are available, but the extension creates unwanted double-stranded structures
Solution Approach 1:
The patent applies local quality by adding blocker nucleotides specifically at the 3' end of the oligonucleotide template. This localized modification prevents extension at the critical 3' end while leaving the rest of the oligonucleotide structure intact and functional for hybridization, thereby maintaining binding site availability without creating unwanted double-stranded structures.
3Reliability
If blocker nucleotides are added to prevent extension, then double-stranded nucleic acid formation is reduced, but additional enzymatic steps are required
Solution Approach 1:
The patent applies preliminary action by performing the addition of blocker nucleotides to the 3' end of the oligonucleotide template before the main barcoding reaction. This preliminary step prevents unwanted extension during the subsequent barcoding process, reducing double-stranded nucleic acid formation. The use of terminal transferase or polymerase in a single enzymatic step makes the process efficient and not significantly complex.
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 effectively prevents undesirable extension of antibody oligonucleotides, reducing the formation of double-stranded nucleic acids and enhancing the accuracy of protein and gene expression analysis by ensuring precise hybridization and ligation with barcoding oligonucleotides.
Implementation Method 1
contacting the cellular component-binding composition with one or more blocker nucleotides and an enzyme capable of catalyzing the addition said one or more blocker nucleotides to the 3' end of the cellular component-binding reagent specific oligonucleotide
Implementation Method 2
the enzyme is a terminal transferase. In some embodiments, said terminal transferase comprises a terminal deoxynucleotidyl transferase (TDT), a polyadenylate polymerase (PAP) a poly(U)polymerase (PUP), or a combination thereof
Implementation Method 3
allowing specific binding and hybridization with barcoding oligonucleotides
Implementation Method 4
the cellular component-binding reagent is capable of specifically binding to at least one of a plurality of cellular component targets of a cell
Data Source
AI summary
Disclosed herein include systems, methods, compositions, and kits for generating protected cellular component-binding compositions. There are provided, in some embodiments, protected cellular component-binding compositions comprising a cellular component-binding reagent associated with a protected cellular component-binding reagent specific oligonucleotide. A protected cellular component-binding reagent specific oligonucleotide can comprise a terminal blocked region. In some embodiments, the 3′ end of the protected cellular component-binding reagent specific oligonucleotide is incapable of being extended by a reverse transcriptase or a polymerase.