Anti-DNA:RNA Hybrid [S9.6] Antibody

Overview

Reactivity

Species reactivity: General

Tested applications

DNA-RNA hybrid Immunoprecipitation (DRIP) : 1/100 - 1/200. 
Dot blot: 1/1000.

Not yet tested in other applications.

Optimal dilutions/concentrations should be determined by the end user.

Specificity

The S9.6 monoclonal recognizes DNA-RNA hybrids (also known as R-loops) and does not bind to single or double stranded DNA (PubMed IDs: 2422282, 16614443). The antibody has high affinity for DNA-RNA hybrids but also binds RNA-RNA hybrids that are AU-rich (PubMed ID: 23784994).

The specificity of the antibody appears to be determined by a combination of sequence and structural dependency since R-loop sequence affects binding affinity (PubMed ID: 28594954). 

Please see our DNA-RNA-IP (DRIP) protocol for further information.

Product Picture 1

Product Picture 2

Product Picture 3

a. S9.6 Fab region and double stranded nucleic acid sequence used in the experiment. 
 

b&c. S9.6 specifically binds to DNA-RNA hybrid duplex, while weakly binding to double stranded RNA, but not binding to double stranded DNA at all. 
 

d. The crystal structure of the complex of S9.6 and DNA-RNA hybrid duplex.

Properties

Immunogen

S9.6 ΦX174 bacteriophage-derived synthetic DNA–RNA antigen

Clonality

Monoclonal, Clone ID is S9.6

Isotype

IgG2a

Form

PBS with 0.02% Proclin 300 and 50% glycerol, pH 7.4

Storage instruction

Store at -20°C, Avoid freeze / thaw cycle.

Host

Species Immunized: Mouse(BALB/c)

Positive Controls

DRIP: R-Loop. Dot blot: R-Loop, R-Loop (RNase A treated), and R-Loop (DNase I treated).

 

The pCALM3_2 vector used to generate R-loops was kindly provided by Prof. Frederic Chedin at UC Davis. The pCALM3_2 plasmid carries a 789bp fragment of the human CALM3 region that forms R-loops (PubMed ID: 31053798) cloned between T3 and T7 RNA polymerase promoter sequences. Transcription by T3 RNA polymerase leads to R-loop formation. Transcription by T7 RNA polymerase does not allow R-loop formation. 

Applications

Highlights

1. Useful in the detection of R-loops

 

2. High specificity and affinity for DNA-RNA hybrids

 

3. Does NOT cross-react with single-stranded DNA or double-stranded DNA

 

4. Minor cross-reaction (~5-fold less) has been observed for AU-rich double-stranded RNA.

 

5. High affinity binding shown for hybrids of 8, 10, 15, and 23 base pairs in length

Application Figure 1

DNA-RNA hybrid Immunoprecipitation (DRIP) data using AT15001.

 

M1 : 100bp DNA ladder                       

 

M2 : High Range DNA Ladder 

 

Lane 1: R-Loop ApaLI (RNase A treated)

 

Lane 2: R-Loop (RNase A treated)

 

Lane 3:R-Loop (RNase A+H treated)

 

Lane 4: R-Loop ApaLI (RNase A+H treated)

 

Lane 5: AT15001 with R-Loop ApaLI (RNase A treated)

 

Lane 6: AT15001 with R-Loop(RNase A treated)

 

Lane 7: AT15001 with R-Loop(RNase A+H treated)

 

Lane 8: AT15001 with R-Loop ApaLI (RNase A+H treated)

 

Lane 9-12: Flow through from IP

 

Capture antibody: 5 μg

 

Blocking buffer and concentration: 7.5 µg ssDNA.

 

Diluting buffer and concentration: PBS containing 0.1% Triton X-100.

 

A synthetic vector (pCALM3_2) was used to generate R-loops. AT15001 immunoprecipitates R-loops in the presence or absence of prior digestion by ApaLI, which does not affect R-loop structure. Prior treatment with RNase A (digests single stranded RNA) does not affect the IP signal whereas prior treatment with RNase H (digests RNA in DNA-RNA hybrids) eliminates the signal.

 

The pCALM3_2 vector used to generate R-loops was kindly provided by Prof. Frederic Chedin at UC Davis.