Fluorophore Modification Service

Introduction

Fluorophores are luminescent molecules that can absorb and re-emit light at specific wavelengths and change after binding with oligonucleotides. They can track in real time, conduct quantitative analysis, and perform high-resolution imaging of nucleic acids, which is crucial for FISH, live cell research, diagnostic analysis, and validation of target participation in treatment.

At Creative Biolabs, we offer end-to-end fluorophore-conjugated oligonucleotide solutions: customizing the attachment of various fluorophores (FAM, Cy3, Cy5, etc.) through proprietary chemical methods; High-purity synthesis by HPLC/MS, and probes tailored for imaging, diagnosis, or treatment monitoring. With over 20 years of professional expertise, our tools combine precision and reliability to accelerate your research.

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Fluorophores

Fig.1 The most common fluorophores summarized by structure and emission color.¹

Basic Properties

Fluorophores are light-emitting molecules covalently conjugated to oligonucleotides at specific positions, 5', 3', or internal bases, enabling real-time visualization of cellular uptake, trafficking, and target interactions. Common options include Cyanine dyes (Cy3, Cy5) and fluorescein derivatives, optimized to minimize interference with oligonucleotide function.

Key Advantages

Their key advantages include enhanced detection sensitivity (supporting sub-nanomolar tracking in live-cell imaging or in vivo biodistribution), dual functionality (combining therapeutic activity like gene silencing with diagnostic capabilities such as tumor targeting validation), and reduced off-target effects (site-specific labeling preserves base-pairing specificity and limits steric hindrance).

Design Considerations

For effective design, focus on labeling position (terminal modifications at 3'/5' optimize signal without disrupting hybridization), quenching mitigation (avoid placing fluorophores near G-quadruplex regions or nucleases to prevent signal loss), and linker chemistry (use non-polar, flexible linkers like C6 or PEG spacers to reduce conformational strain).

Workflow

1. Sequence Design & Fluorophore Selection

   Share your target sequence and preferred fluorophore (such as Cy5 for in vivo imaging needs), and our team of experts will step in to optimize labeling sites. This careful adjustment ensures a balance between the oligonucleotide's stability and the strength of the fluorescent signal, laying a solid foundation for subsequent experiments.

2. Synthesis & Conjugation

   We employ automated phosphoramidite chemistry to achieve precise incorporation of fluorophores during solid-phase synthesis, ensuring each modification is accurately positioned. After synthesis, post-synthesis HPLC purification is performed to thoroughly remove any unbound fluorophores, guaranteeing the purity of the conjugated oligonucleotides.

3. Quality Control

   Our rigorous quality control process covers two key aspects: purity verification, through IP-HPLC and MALDI-TOF analysis; and functionality validation, which includes testing fluorescence intensity and confirming the target binding affinity, ensuring the modified oligonucleotides perform as expected.

4. Delivery

   You will receive lyophilized oligonucleotides along with comprehensive reports, including HPLC traces and mass spectrometry data, within 4-8 weeks. This timely delivery, paired with detailed documentation, supports a smooth transition to your experimental workflow.

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Customer Reviews

"Creative Biolabs' fluorophore-modified oligos transformed our live-cell imaging. The Cy5 conjugation stayed bright for 72+ hours. Their team suggested a 3' placement that didn't disrupt target binding. HPLC reports confirmed 99% purity, and delivery hit the 5-week mark exactly."

"We needed dual-labeled FRET probes, and Creative Biolabs delivered perfectly. The Cy3-Cy5 pair had precise Förster distances, making our mRNA binding assays finally reproducible. Their scientists adjusted the linker to avoid G-quadruplex quenching, signal was stronger than our initial design. Fast turnaround and clear data sheets made this partnership seamless."

"Scaling up fluorophore-modified siRNAs for in vivo work? Creative Biolabs nailed it. From 10nmol to 1mmol, each batch had consistent fluorescence intensity. They suggested a modification that didn't alter liver uptake, critical for our therapy tracking. Their QC included serum stability data, saving us weeks of prep."

FAQs

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Reference

1. Gil-Rivas, Alba, et al. "New advances in the exploration of esterases with PET and fluorescent probes." Molecules 28.17 (2023): 6265. DOI: 10.3390/molecules28176265. Distributed under Open Access license CC BY 4.0, without modification.