Tethering Nucleic Acid Delivery for Nanoscale Reaction Sites
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Solution Overview
Problem
Current methods for loading molecules into nanoscale reaction sites, such as Zero Mode Waveguides (ZMWs), face challenges with entropic barriers, leading to inefficiencies in loading large reactant molecules, which limits the density and efficiency of molecular analysis in techniques like DNA sequencing and protein-nucleic acid complex detection.
Innovation Solution
The use of tethering nucleic acids with capture and binding moieties, where the tethering nucleic acid is immobilized and a molecule is attached via interaction with a binding moiety, followed by degradation of the second strand to collapse the first strand into a hairpin, facilitating the molecule's entry into the nanoscale site, enhancing loading efficiency and density.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If passive distribution methods are used to load molecules into nanoscale reaction sites, then the loading process is simple, but the loading efficiency and density are low due to entropic barriers
Solution Approach 1:
A tethering nucleic acid molecule acts as an intermediary carrier that is first loaded into the nanoscale reaction site, then captures the target molecule (template, primer, or enzyme) through binding moieties, and finally delivers it to the reaction site. This two-step intermediary process overcomes the entropic barriers that prevent direct loading of large molecules into confined nanoscale spaces.
Solution Approach 2:
The tethering nucleic acid is prepared in advance with specific binding moieties attached to it before being introduced to the reaction site. This preliminary preparation of the carrier molecule with its cargo-binding capability enables efficient capture and delivery of target molecules once inside the confined reaction volume.
2Quantity of substance
If large reactant molecules are loaded into nanoscale reaction sites, then the molecular analysis capability is enhanced, but entropic barriers significantly reduce loading efficiency
Solution Approach 1:
The tethering nucleic acid serves as a size-appropriate intermediary that can be efficiently loaded into the nanoscale site, then binds to and transports the larger target molecule (such as template-primer-enzyme complexes) into the confined space. This intermediary approach allows large molecules to be delivered despite entropic barriers that would prevent their direct loading.
Solution Approach 2:
The loading process is segmented into distinct steps: first the tethering nucleic acid is loaded, then it captures the target molecule through binding moieties, and finally delivers it to the reaction site. This segmentation of the loading process into manageable stages allows large molecules to be introduced efficiently without overwhelming entropic barriers.
3Productivity
If the density of molecules in reaction sites is increased, then the throughput of molecular analysis is improved, but the precision of single molecule analysis may be compromised
Solution Approach 1:
The tethering nucleic acid is designed with specific local properties including binding moieties at defined positions that enable selective capture of target molecules. This localized functional design ensures that even at higher densities, each reaction site maintains its ability to perform specific single-molecule analysis with high precision while contributing to overall increased throughput.
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 method significantly increases the loading efficiency and density of molecules into nanoscale sites, improving the performance of downstream reactions like sequencing by reducing the amount of input molecules needed and time required for loading, while minimizing background noise.
Implementation Method 1
the binding moiety and the capture moiety interact to attach the molecule to the tethering nucleic acid
Implementation Method 2
the first strand contains self-complementary regions that hybridize together to collapse the first strand into a hairpin
Implementation Method 3
degrading the second strand of the tethering nucleic acid, where the first strand contains self-complementary regions that hybridize together
Data Source
AI summary
The present invention provides methods, compositions, and systems for distributing molecules and complexes into reaction sites. In particular, the methods, compositions, and systems of the present invention result in an active loading of molecules and complexes into reaction sites with improved efficiency over loading by passive diffusion methods alone.