Acridinium Ester Conjugates for Signal Localization in Sequencing
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Solution Overview
Problem
Current chemiluminescence molecules, such as luminol and hydrogen peroxide, suffer from signal diffusion due to the separation of the signaling molecule from the tri-phosphate reversible terminator after excitation, limiting their use in sequencing methods that require signal localization.
Innovation Solution
Development of novel acridinium-ester-containing compounds (AE compounds) that include a nucleotide moiety, allowing for signal localization through the use of acridinium ester-tri-phosphate reversible terminator conjugates in chemiluminescence-based one-color nucleic acid sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Illumination intensity
If currently available chemiluminescence molecules (luminol, hydrogen peroxide, dioxetanes, oxalate derivatives) are used in sequencing, then chemiluminescence signal can be generated, but signal diffusion occurs due to separation of the signaling molecule from the tri-phosphate reversible terminator after excitation
Solution Approach 1:
The patent merges the chemiluminescence signaling molecule with the tri-phosphate reversible terminator into a single conjugate structure. This is achieved by chemically linking the chemiluminescence molecule to the reversible terminator through a linker, ensuring that the signaling molecule remains physically associated with the incorporated nucleotide throughout the sequencing process, thereby preventing signal diffusion and enabling precise signal localization.
Solution Approach 2:
The patent introduces a linker as an intermediary component that connects the chemiluminescence molecule to the tri-phosphate reversible terminator. This linker acts as a bridge that maintains the spatial relationship between the signaling molecule and the incorporated nucleotide, allowing the chemiluminescence signal to remain localized at the site of nucleotide incorporation while still enabling the necessary chemical reactions.
2Productivity
If chemiluminescence molecules are separated from the tri-phosphate reversible terminator after excitation, then the chemiluminescence reaction can proceed, but signal localization is lost which limits sequencing application
Solution Approach 1:
The patent combines the chemiluminescence molecule and the tri-phosphate reversible terminator into a unified conjugate structure that is incorporated as a single unit during DNA synthesis. This merging ensures that the chemiluminescence signal remains precisely localized to the position of nucleotide incorporation, enabling both efficient sequencing and accurate base identification through spatially resolved detection.
Solution Approach 2:
The patent creates a composite molecule consisting of three functional components: the chemiluminescence molecule, the tri-phosphate reversible terminator, and a linker. This composite structure integrates the chemical reactivity of the terminator with the optical signaling capability of the chemiluminescence molecule, allowing simultaneous achievement of sequencing efficiency and measurement precision.
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
The AE compounds achieve effective signal localization, enabling the use of chemiluminescence in nucleic acid sequencing methods, which improves the accuracy and efficiency of base identification in template DNA strands.
Implementation Method 1
Chemiluminescence refers to a chemical reaction resulting in the production of light. When applied to a sequencing platform... Described herein are novel acridinium-ester-containing compounds (AE compounds)... in chemiluminescence-based one-color nucleic acid sequencing
Data Source
AI summary
Acridinium ester-containing compounds are provided herein. Also provided herein are chemiluminescence-based one-color sequencing methods and methods of using the acridinium ester-containing compounds in chemiluminescence-based one-color sequencing.