Nucleic Acid Calipers for High-Throughput Macromolecular Structure Determination
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Solution Overview
Problem
Current methods for determining macromolecular structures, such as X-ray crystallography and solution NMR, face challenges with conformational heterogeneity and require extensive cysteine engineering for single-molecule FRET, lacking low-cost, high-throughput solutions for collecting long-range distance restraints.
Innovation Solution
The use of single-stranded nucleic acid calipers and splints to measure long-range distances between points on macromolecules via force spectroscopy, allowing for high-throughput structure determination and characterization of structural intermediates stabilized by tension, without the need for extensive cysteine engineering.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If single-molecule FRET is used to obtain long-range distance restraints, then structural information can be obtained, but extensive cysteine engineering and complex instrumentation are required
Solution Approach 1:
The patent introduces a nucleic acid caliper as an intermediary molecular ruler that bridges two points on the target macromolecule. This caliper comprises a reference length and measurement domains that hybridize to the target, allowing distance measurement without requiring complex FRET instrumentation or extensive protein engineering. The caliper acts as a physical mediator that directly reports distance through its structural configuration.
Solution Approach 2:
The patent replaces the optical FRET measurement system with a mechanical molecular ruler approach. Instead of using fluorescent markers and optical detection, the invention uses a nucleic acid caliper with defined structural dimensions that physically span the distance to be measured. The distance information is encoded in the mechanical structure of the caliper itself, which can be detected through its effect on the overall molecular assembly.
2Reliability
If conventional structure determination methods are used, then mature techniques are available, but they cannot handle conformational heterogeneity and require bulk averaging
Solution Approach 1:
The patent applies segmentation by dividing the measurement approach into individual single-molecule measurements rather than bulk averaging. Each nucleic acid caliper independently measures distances on individual target molecules, allowing the preservation of conformational information that would otherwise be lost in ensemble averaging. This segmentation enables the characterization of heterogeneous populations by analyzing each molecule's unique structural state.
Solution Approach 2:
The nucleic acid caliper system is self-service in that it automatically reports distance information through its hybridization to the target and its structural configuration. The caliper's measurement domains selectively bind to specific sites on the target, and the resulting complex structure inherently encodes the distance measurement without requiring external intervention or complex data processing to resolve conformational states.
3Loss of information
If long-range distance restraints are collected using existing methods, then structural information is obtained, but throughput is low and cost is high
Solution Approach 1:
The nucleic acid caliper design incorporates universal features that enable high-throughput application. The caliper uses standard nucleic acid hybridization principles that can be applied to any target with appropriate binding sites, making the method universally applicable across different macromolecules. The modular design with reference and measurement domains allows the same basic structure to be used for various distance measurements by simply changing the hybridization targets, enabling scalable high-throughput operation.
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 high-resolution, high-throughput measurement of long-range distance restraints, facilitating the determination of macromolecular structures with reduced experimental complexity and cost, and the ability to observe fleeting structural intermediates.
Implementation Method 1
measuring long-range distances between randomly selected points on the target of interest, for example via force spectroscopy
Implementation Method 2
a target splint that is a single-stranded oligonucleotide having partial sequence complementarity to the single-stranded nucleic acid caliper, wherein the reference and target splints bind to separate distinct sequences of the single-stranded nucleic acid caliper
Data Source
AI summary
Provided herein are compositions and methods for determining the structure of individual targets using by determining long-range distances within such targets.