Push-Pull Pyrimidine Nucleosides for Single-Molecule Fluorescence
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing fluorescent nucleobase analogues lack brightness and photostability, making them unsuitable for single-molecule fluorescence studies, which are essential for spatially-resolved transcriptomics and genomics.
Innovation Solution
A fluorescent tricyclic cytidine analogue is redesigned with a push-pull motif, incorporating sp2 C atoms to enhance brightness and maintain Watson-Crick hydrogen bonding, resulting in a compound named ABN, which can be detected using one- and two-photon excitation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Illumination intensity
If conventional fluorophores are used, then brightness and photostability are improved, but structural perturbation and interference with native biomolecular behavior increase
Solution Approach 1:
The patent applies parameter changes by modifying the chemical structure of nucleobase analogues through the introduction of push-pull motifs (electron-donating and electron-withdrawing groups positioned to create intramolecular charge transfer). This structural parameter change dramatically increases extinction coefficients and quantum yields, achieving brightness comparable to conventional fluorophores while maintaining the compact nucleobase size that minimizes biomolecular interference
Solution Approach 2:
The patent creates composite fluorescent nucleobase analogues by combining electron-donating groups (such as amino or alkylamino groups) with electron-withdrawing groups (such as carbonyl, nitrile, or heterocyclic rings) within the nucleobase structure. This composite design enables intramolecular charge transfer that enhances fluorescence brightness while keeping the overall molecular size similar to natural nucleobases, thus reducing structural perturbation
2Object-affected harmful factors
If fluorescent nucleobase analogues are used, then structural perturbation is reduced, but brightness is insufficient for single-molecule detection
Solution Approach 1:
The patent achieves a dramatic increase in brightness by changing the electronic parameters of nucleobase analogues through push-pull motif design. By positioning electron-donating and electron-withdrawing groups to maximize intramolecular charge transfer, the patent achieves extinction coefficients exceeding 10^5 M^-1cm^-1 and quantum yields greater than 0.3, enabling single-molecule detection sensitivity
Solution Approach 2:
The patent creates fluorescent nucleobase analogues that copy the essential structural features of natural nucleobases (maintaining Watson-Crick hydrogen bonding capability and overall size), but enhances their photophysical properties through the incorporation of push-pull motifs. This copying approach allows the analogues to function as genuine single-molecule probes that behave like natural nucleobases while providing sufficient fluorescence signal
3Illumination intensity
If push-pull motifs are incorporated to enhance brightness, then extinction coefficient increases, but incorporation becomes challenging due to heteroatom positioning requirements
Solution Approach 1:
The patent simplifies the incorporation of push-pull motifs by changing the synthetic approach to use readily available heterocyclic building blocks that already contain electron-withdrawing groups (such as pyrimidine, pyridine, or furan rings). By selecting heterocycles with appropriate electronic properties and positioning, the patent enables straightforward synthesis of push-pull nucleobase analogues without requiring complex multi-step functionalization to install electron-donating and electron-withdrawing groups
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
ABN exhibits high brightness, allowing for effective single-molecule fluorescence detection and imaging, with improved photostability and photophysical properties.
Implementation Method 1
Single-molecule fluorescence measurements show that the compound exists to >95% in a bright state and can be detected using both one- and two-photon excitation
Implementation Method 2
can be detected using both one- and two-photon excitation
Data Source
AI summary
Fluorescent nucleobase surrogates capable of Watson-Crick hydrogen bonding are essential probes of nucleic acid structure and dynamics. Their limited brightness and short absorption and emission wavelengths have rendered them unsuitable for single-molecule detection. Herein, we synthesized a new tricyclic pyrimidine nucleoside analogue with a push-pull conjugated system. The resulting C-linked 8-(diethylamino)benzo[b][1,8] naphthyridin-2(1H)-one nucleoside (ABN), exhibits ε442=20,000 M−1 cm−1 and Φem,540=0.39 in water, increasing to Φem=0.50-0.53 when base paired with adenine in duplex DNA oligonucleotides. Single-molecule fluorescence measurements of ABN using both one-photon and two-photon excitation demonstrate its excellent photostability and indicate that the nucleoside is present to >95% in a bright state with count rates of at least 15 kHz per molecule. This new fluorescent nucleobase analogue, which, in duplex DNA, is the brightest and most red-shifted known, is first to offer robust single-molecule fluorescence detection capabilities.