Modified Oligonucleotide Probes for In Situ Hybridization Stability
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Solution Overview
Problem
Existing oligonucleotide probe-based assays for analyzing target nucleic acids face challenges in maintaining the stability of hybridization complexes and positional information, particularly under stringent wash conditions, and require customizable and affordable probes with modified nucleotides for effective analysis.
Innovation Solution
A method involving the modification of probes by attaching modified nucleotides to overhangs using a first oligonucleotide as a template or primer, which includes extending or ligating these nucleotides to enhance probe stability and positional stability within a sample, such as a hydrogel matrix, using polymerases like T4 or T7, and incorporating crosslinkable nucleotides for improved hybridization and detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If stringent wash conditions are used to reduce background noise, then signal purity is improved, but probe/target hybrid stability deteriorates
Solution Approach 1:
The patent modifies the chemical parameters of the probe by incorporating non-natural nucleotides with enhanced binding properties. These modified nucleotides change the thermodynamic parameters of hybridization, allowing the probe to maintain stable binding under stringent wash conditions that would otherwise cause dissociation. This resolves the contradiction by enabling high signal purity without sacrificing hybrid stability.
Solution Approach 2:
The probe is constructed as a composite molecule containing both natural and non-natural nucleotides. The non-natural nucleotides (such as those with expanded genetic alphabet bases) provide enhanced binding affinity and stability, while the natural nucleotides maintain sequence specificity. This composite structure allows the probe to withstand stringent wash conditions while maintaining target binding.
2Measurement precision
If positional stability of target analytes is enhanced to preserve spatial information, then spatial resolution is improved, but sample accessibility deteriorates
Solution Approach 1:
The probe is designed with pre-formed secondary structures including overhangs and stem-loop configurations that enable preliminary binding to target analytes. The overhang regions are engineered to facilitate initial attachment before full hybridization occurs, allowing the probe to secure positional information early in the binding process while maintaining accessibility during subsequent wash steps.
Solution Approach 2:
Different regions of the probe are assigned different functional properties: the hybridization region provides sequence-specific binding, the overhang regions provide structural stability and attachment points, and modified nucleotides at specific positions enhance local binding affinity. This local differentiation allows the probe to maintain spatial resolution while preserving sample accessibility through optimized regional functions.
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 approach enhances the stability and positional stability of probes within samples, allowing for more reliable and customizable analysis of target nucleic acids, including viral or cellular DNA/RNA, by maintaining hybridization and positional information, and enabling in situ sequencing and hybridization techniques.
Implementation Method 1
The polymerase can catalyze extension of the second overhang using the first oligonucleotide as a template, thereby attaching the one or more modified nucleotides to the second overhang
Implementation Method 2
the probe comprises (i) a hybridization region that hybridizes to the target nucleic acid in the sample
Implementation Method 3
affordable and easily customizable probes comprising modified nucleotides (e.g., crosslinkable nucleotides) for use in analysis of target nucleic acids
Data Source
AI summary
The present disclosure relates in some aspects to methods for analyzing a target nucleic acid in a biological sample. In some aspects, the methods involve the use of a set of oligonucleotides, for example a set of two or more oligonucleotides, wherein one or more oligonucleotides comprises modified nucleotides, for assessing target nucleic acids. In some aspects, the presence, amount, and/or identity of a target nucleic acid is analyzed in situ. Also provided are oligonucleotides, sets of oligonucleotides, compositions, and kits for use in accordance with the methods.