Poly(dA:dT)

Synthetic analog of B-DNA

Poly(dA:dT) is a poly(deoxyadenylic-deoxythymidylic) acid sodium salt. It is a repetitive synthetic double-stranded DNA sequence of poly(dA-dT):poly(dT-dA) and a synthetic analog of B-DNA.

Poly(dA:dT)  is recognized by multiple PRRs:
- Sensing by cytosolic DNA sensors (CDS), including cGAS, AIM2, DAI, DDX41, IFI16, and LRRFIP1, triggers the production of type I interferons [1-3].
- Sensing by the cytosolic DNA sensor AIM2 triggers the formation of an inflammasome and the subsequent secretion of IL-1β and IL-18 [4].
- Indirect sensing by the cytosolic RNA sensor RIG-I can occur upon the transcription of Poly(dA:dT) into dsRNA with a 5'triphosphate moiety (5'ppp-dsRNA) by the RNA polymerase III [5,6]. This indirect sensing by RIG-I leads to the production of type I interferons [7].

To achieve stimulation of cytosolic PRRs, Poly(dA:dT) must be delivered into the cytoplasm, for example by using a transfection agent, such as LyoVec™.

Key features:

• Multi-PRR agonist
• For your convenience, pre-complexed Poly(dA:dT)/ LyoVec™ is also available.
• Each lot is functionally tested

References:

1. Unterholzner L., 2013. The interferon response to intracellular DNA: why so many receptors? Immunobiology. 218(11):1312-21.
2. Zhang Z. et al., 2011. The helicase DDX41 senses intracellular DNA mediated by the adaptor STING in dendritic cells. Nat Immunol. 12(10):959-65.
3. Wu J. et al., 2013. Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA. Science. 339(6121):826-30.
4. Jones JW. et al., 2010. Absent in melanoma 2 is required for innate immune recognition of Francisella tularensis. PNAS, 107(21):9771-6.
5. Ablasser A. et al., 2009. RIG-I-dependent sensing of poly(dA:dT) through the induction of an RNA polymerase III-transcribed RNA intermediate. Nat Immunol. 10(10):1065-72.
6. Chiu YH. et al., 2009. RNA polymerase III detects cytosolic DNA and induces type I interferons through the RIG-I pathway. Cell. 138(3):576-91.
7. Takeshita F. & Ishii KJ., 2008. Intracellular DNA sensors in immunity. Curr Opin Immunol. 20(4):383-8.

Figures

Responses of B16-Blue™ IFNα/β cells to dsDNA and dsRNA:

B16-Blue™ IFNα/β cells were stimulated with increasing concentrations of poly(dA:dT), poly(dG:dC) or 5’ppp-dsRNA complexed extemporaneously with the transfection reagent LyoVec™.
After 24h incubation, induction of type I IFNs was assessed by determining the levels of SEAP using QUANTI-Blue™, a SEAP detection medium.

Pyroptosis response of THP1-HMGB1-Lucia™ cells. Cells were primed with 1 μg/ml LPS-EK for 3 hours and then incubated with inflammasome inducers : 0.5 μg/ml complexed Poly(dA:dT) (left panel) or 8 μM Nigericin (right panel). Lucia luciferase activity and LDH release in the supernatant were quantified at 2, 3, 4, 5 and 6 hours post-induction. The Lucia luciferase activity was determined by measuring relative light units (RLUs) in a luminometer using QUANTI-Luc™ detection reagent. Results are presented as the fold increase over non-induced cells. The LDH release was determined using a commercially available LDH cytotoxicity assay. Results are presented as percentage of the maximal LDH release measured in positive control.

Specifications

Specificity: RIG-I & CDS agonist, AIM2 inflammasome inducer

Formulation: Naked or pre-complexed with LyoVec™

CAS number: 86828-69-5 (for Naked Poly(dA:dT))

Working Concentrations:

• 10 ng- 1 μg/ml for Naked Poly(dA:dT) for CDS or RLR stimulation.
• 1 µg- 5 μg/ml for Naked Poly(dA:dT) for inflammasome induction.
• 10 ng- 10 μg/ml for pre-complexed Poly(dA:dT)/LyoVec™ for CDS or RLR stimulation.
• 1 µg- 5 μg/ml for pre-complexed Poly(dA:dT)/LyoVec™ for inflammasome induction.

Solubility:

• 2 mg/ml in water for Naked Poly(dA:dT)
• 50 µg/ml in water for pre-complexed Poly(dA:dT)/LyoVec™

Quality Control:

• The biological activity has been verified using cellular assays.
• The absence of bacterial contamination (lipoproteins and endotoxins) has been confirmed using HEK-Blue™ TLR2 and HEK-Blue™ TLR4 cells.

Contents

Poly(dA:dT) Naked is available in two quantities:

tlrl-patn:

• 200 µg lyophilised poly(dA:dT) naked
• 1.5 ml sterile endotoxin-free water 

tlrl-patn-1:

• 1 mg lyophilised poly(dA:dT) naked
• 1.5 ml sterile endotoxin-free water

Poly(dA:dT)/LyoVec™ is available in one quantity:

tlrl-patc:

• 4 x (25 µg Poly(dA:dT) complexed with 50 µg of LyoVec™), lyophilized
• 10 ml sterile endotoxin-free water

 Poly(dA:dT) and  Poly(dA:dT)/LyoVec™ are provided lyophilized and shipped at room temperature.

 Store lyophilized Poly(dA:dT) and  Poly(dA:dT)/LyoVec™ at -20°C.

 Lyophilized product is stable for 1 year when stored -20°C.