RCA Product with Multiple Free Ends
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Solution Overview
Problem
Rolling circle amplification (RCA) products are limited due to having only one 3′ end and one 5′ end, which restricts opportunities for hybridization, ligation, or copying sequences from other molecules.
Innovation Solution
A complex comprising multiple nucleic acid molecules hybridized together, each with a specific sequence structure including complementary sequences, spacer sequences, and end sequences, allowing for hybridization and extension of free ends to facilitate various assays.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If traditional RCA products are used, then amplification of circular nucleic acid is achieved, but the number of free ends is limited to one 3′ end and one 5′ end
Solution Approach 1:
The RCA product is segmented into multiple individual nucleic acid molecules, each containing separate 3′ and 5′ ends. This segmentation transforms the single continuous RCA product into discrete units with independent ends, enabling multiple hybridization and ligation reactions to occur simultaneously across different molecules.
Solution Approach 2:
The invention transitions from a one-dimensional linear RCA product to a multi-dimensional structure where multiple molecules are spatially arranged to provide numerous accessible ends. By creating a complex comprising multiple molecules with exposed 3′ and 5′ ends, the system gains additional functional dimensions for parallel biochemical operations.
2Adaptability or versatility
If traditional RCA products are used, then sequence amplification is achieved, but opportunities for hybridization and ligation are restricted
Solution Approach 1:
The nucleic acid molecules are pre-configured with exposed 3′ and 5′ ends before hybridization and ligation reactions. This preliminary arrangement of reactive ends enables immediate and efficient hybridization with target sequences and subsequent ligation reactions without requiring additional processing steps to create free ends.
Solution Approach 2:
The complex of multiple nucleic acid molecules serves as an intermediary structure that facilitates hybridization and ligation reactions. The multiple exposed ends act as intermediaries that can simultaneously interact with multiple target sequences, enabling parallel processing and increasing overall reaction efficiency.
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
Enables efficient hybridization with target polynucleotides, extension of nucleic acid molecules, and sequencing, overcoming the limitations of traditional RCA products by providing multiple hybridizable ends.
Implementation Method 1
the first complementary sequence of one molecule is directly or indirectly hybridized with the second complementary sequence of another molecule in the complex
Implementation Method 2
digesting the product of step (a) with the nicking endonuclease, to nick the double-stranded region close to the loop and release a free end that comprises the end sequence
Data Source
AI summary
Described herein is a complex comprising multiple nucleic acid molecules that are hybridized together, each comprising, from 5′ to 3′, a first complementary sequence, a spacer sequence and a second complementary sequence, and either/or a 5′ end sequence that is 5′ of the first complementary sequence and terminates in a 5′ phosphate and a 3′ end sequence that is 3′ of the second complementary sequence and terminates in a 3′ hydroxyl. In this complex: the first complementary sequence of one molecule is directly or indirectly hybridized with the second complementary sequence of another molecule in the complex and the spacer sequence and the 3′ and/or 5′ end sequences are single-stranded. Methods of making and using the complex are also provided.