Compaction Oligonucleotide Folding for Dense Nucleic Acid Nanostructures
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Solution Overview
Problem
Existing polynucleotide sequencing technologies face limitations in throughput and signal-to-noise ratio, leading to increased costs due to barriers in surface chemistry and on-support polynucleotide amplification.
Innovation Solution
A method is developed for generating high-density nucleic acid nanostructures on a support using compaction oligonucleotides, involving hybridization with universal surface primers and rolling circle amplification, resulting in compact nanostructures with densities of 102-1015 per mm2, enhancing immobilization and amplification efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional surface chemistry and on-support polynucleotide amplification methods are used, then sequencing can be performed, but throughput is limited and signal-to-noise ratio is poor
Solution Approach 1:
The invention transitions from conventional 2D surface-bound polynucleotide amplification to 3D nucleic acid nanostructure formation. By using compaction oligonucleotides to fold linear concatemer templates into compact three-dimensional nanostructures (such as nanoballs), the patent achieves higher local concentration of template molecules and improved signal detection, directly addressing the signal-to-noise ratio limitation while increasing sequencing throughput
Solution Approach 2:
The invention implements a nested structure where linear concatemer templates are folded into compact nanostructures containing multiple copies of the target sequence. Each nanostructure nests multiple template copies in a condensed spatial arrangement, enabling parallel sequencing of multiple targets within a single detectable unit, thereby improving both throughput and signal-to-noise ratio
2Quantity of substance
If density of primers on support is increased, then amplification capacity improves, but manufacturing complexity increases
Solution Approach 1:
The invention performs preliminary action by pre-synthesizing linear concatemer templates with multiple copies of the target sequence before immobilization on the support. This pre-assembly approach allows standard primer densities to generate high effective template concentrations through the compact folding of pre-formed concatemers, avoiding the need for complex high-density surface chemistry while achieving enhanced amplification capacity
3Quantity of substance
If compaction oligonucleotides are used to form nanostructures, then nanostructure density increases to 102-1015 per mm2, but process complexity increases
Solution Approach 1:
The invention implements self-service through the autonomous self-assembly of compact nanostructures. Compaction oligonucleotides hybridize to complementary sequences on linear concatemer templates and spontaneously fold them into stable three-dimensional nanostructures without requiring external energy input or complex control mechanisms. This self-organizing process achieves high nanostructure densities while keeping the amplification process relatively simple
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 method improves sequencing efficiency by increasing nanostructure density and signal-to-noise ratio, thereby reducing costs and enhancing throughput.
Implementation Method 1
hybridizing a plurality of single stranded circular nucleic acid library molecules to the plurality of immobilized first universal surface primers
Implementation Method 2
conducting an on-support rolling circle amplification reaction with (i) a plurality of strand-displacing polymerases
Implementation Method 3
individual compaction oligonucleotides comprise a single-stranded linear oligonucleotide having a first binding region capable of hybridizing to a first portion of a concatemer molecule and a second binding region capable of hybridizing to a second portion of the concatemer molecule
Implementation Method 4
the plurality of immobilized concatemer molecules forms a compact nucleic acid nanostructure
Data Source
AI summary
The present disclosure provides compositions and related methods, e.g., for preparing immobilized nucleic acid nanostructures using compaction oligonucleotides. In some embodiments, rolling circle amplification reaction can be conducted with compaction oligonucleotides on-support or in-solution to generate concatemer molecules having multiple copies of a polynucleotide unit arranged in tandem. Each polynucleotide unit comprises a sequence-of-interest and at least one universal adaptor sequence that binds one end of a compaction oligonucleotide. The 5′ and 3′ regions of the compaction oligonucleotide can hybridize to the concatemer to pull together distal portions of the concatemer causing compaction of the concatemer to form a nanostructure. Nanostructures having tighter size and shape compared to concatemers generated in the absence of the compaction oligonucleotides. The compact and stable characteristics of the nucleic acid nanostructures improves sequencing accuracy by increasing signal intensity and they retain their shape and size during multiple sequencing cycles.