Quencher Modification Service
Introduction
Quenchers are key to molecular probes. They suppress fluorescence until the target binds, thereby enabling real-time hybridization monitoring and enhancing the accuracy of gene analysis and therapy. Oligonucleotides modified with quenchers exhibit enhanced anti-nuclease stability, improved specificity by reducing off-target interactions, and optimized pharmacokinetics through conjugation or sugar modifications. We offer customized services, including specific design, high-precision synthesis, and thorough QC/validation. Trust our professional expertise to accelerate your gene-silencing therapeutics or diagnostics with reliable and precise oligonucleotides.
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Quenchers
Quenchers are molecules used in oligonucleotide modifications to suppress the fluorescence of a nearby fluorophore, a key mechanism in fluorescent probes for nucleic acid detection. They work by energy transfer: when in close proximity to a fluorophore (e.g., FAM, Cy3), the quencher absorbs the fluorophore's emitted energy, preventing fluorescence. This allows precise monitoring of oligonucleotide-target interactions via changes in fluorescence signal.
Basics
Quenchers are non-fluorescent molecules that absorb fluorophore energy, suppressing background signals. When incorporated into oligonucleotides like molecular beacons, they enable real-time nucleic acid detection via fluorescence triggered by specific binding and enhance gene-silencing specificity by reducing non-target interactions. They fall into two main types:
- Non-fluorescent quenchers (NFQ), which dissipate energy as heat. These include broad-spectrum variants matching fluorophores like FAM or Cy5, and traditional aromatic ones better suited for shorter wavelengths.
- Fluorescent quenchers, which emit at longer wavelengths than paired fluorophores (e.g., ~580 nm with 520 nm-emitting fluorophores like FAM) to minimize crosstalk.
Key Advantages
- Enhanced Specificity: Quenchers reduce false positives by blocking unintended fluorophore activation, critical for diagnostics and in situ hybridization where distinguishing closely related sequences matters. Ensures reliable results even in complex samples.
- Improved Stability: Quenchers like Iowa Black resist enzymatic degradation, extending oligonucleotide half-life in serum-rich environments. Enables longer monitoring in vivo or cell cultures without performance loss.
- Dual Modality Flexibility: Compatible with phosphorothioate (PS) or 2'-O-methyl modifications, creating synergies to boost stability and targeting. Adaptable for research assays to therapeutic development.
Design Considerations
Positioning Terminal placement at the 3' or 5' end optimizes quenching efficiency without disrupting base pairing ensuring the oligonucleotide maintains its ability to bind to target sequences
Quencher Fluorophore Pairing Mismatched absorption or emission spectra can compromise performance so selecting pairs with overlapping spectra is key to maximizing signal to noise ratios
Backbone Compatibility It is important to avoid steric clashes with other modifications such as PS to maintain structural integrity ensuring the modified oligonucleotide functions as intended
Workflow
Design & Synthesis
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1 Sequence Optimization
Customize quencher type and placement using predictive algorithms that analyze target sequence secondary structure and binding dynamics to ensure optimal quenching efficiency and minimal off target interactions.
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2 Synthesis
Solid phase synthesis with high fidelity phosphoramidite chemistry ensures precise coupling of quencher modified nucleotides maintaining strict quality control at each step to prevent truncation or misincorporation.
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3 Purification & QC
HPLC/MS
Confirm sequence accuracy and quencher incorporation with over 99% purity using high resolution mass spectrometry and chromatographic separation techniques.
Fluorescence Validation
Verify quenching efficiency via Förster resonance energy transfer FRET assays measuring fluorescence quenching and dequenching upon target binding to ensure reliable performance.
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4 Timeline
6 to 10 weeks from design to delivery including analytical validation with expedited options available for urgent projects.
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5 Deliverables
Lyophilized oligonucleotides QC reports including UV Vis spectra and purity certificates and stability data documenting performance under various storage conditions to ensure long term reliability.
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What We Can Offer?
Customized Design Expertise
Proprietary algorithms predict optimal quencher-fluorophore pairs, minimizing steric hindrance for efficient signal output.
Rapid Turnaround
Standard probes synthesized in <8 weeks, scalable from mg to gram quantities for research and early clinical needs.
Ultra-High Purity
ISO-certified facilities use dual HPLC purification for >99.5% purity, removing impurities that affect performance.
Cross Modification Synergy
Seamlessly integrates quenchers with PS, LNA, or GalNAc for multifunctional oligonucleotides with enhanced stability and targeting.
End to End Support
Assistance from in silico modeling to in vivo validation, ensuring compliance with FDA/EMA guidelines.
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Customer Reviews
FAQs
Q1: How do you ensure complete quenching of background fluorescence and consistent signal activation?
A: We use FRET-based assays to validate ≥95% fluorescence suppression in unbound states and ≥50× signal enhancement upon target binding. Our proprietary coupling chemistry ensures precise quencher placement, with batch-to-batch variability <3% verified by HPLC and mass spectrometry.
Q2: Will the quencher modification affect oligonucleotide binding affinity or downstream applications?
A: Our designs minimize steric interference; terminal quencher placement avoids disrupting base pairing. We provide Tm data and SPR validation showing <1°C shift in melting temperature. Compatibility with enzymes (e.g., RNase H) and transfection reagents is routinely confirmed, with no reduction in activity.
Q3: Are there limitations to the number or type of modifications that can be combined with quenchers?
A: No. We routinely integrate quenchers with phosphorothioate backbones, 2'-O-methyl sugars, LNA, or GalNAc conjugates. Our synthesis platform ensures compatibility, though we recommend pre-testing novel combinations via small-scale pilot runs.
Q4: Can you validate quencher performance in my specific assay conditions?
A: Yes. We offer customized validation services, including spiking experiments in your matrix of choice (e.g., plasma, cell lysate) to confirm signal-to-noise ratios and dynamic range. This ensures compatibility with your workflow.
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