RS-1 (formerly known as RAD51-Stimulatory Compound-1) is a small molecule RAD51 activator that enhances hRAD51 binding in a wide range of biochemical conditions. Moreover, it has the ability to boost the knock-in efficiencies of CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) and may have anticancer properties. It was demonstrated by salt titration experiments that RS-1 can improve filament stability. The length of protein-DNA complexes and the pitch of helical filament turns can both be increased by RS-1, according to an ultrastructural study of filaments formed on ssDNA. hRAD51-mediated homologous strand assimilation (D-loop) activity was at least 5- to 11-fold enhanced by RS-1, depending on the circumstance. It appears that the mechanism of stimulation was different from that conferred by Ca(2+) and/or inhibition of ATPase because this D-loop stimulation happened even in the presence of Ca(2+) or adenylyl-imidodiphosphate. The compound does not replace the need for a nucleotide triphosphate cofactor, as no D-loop activity was seen in the absence of one. These findings suggest that RS-1 stimulates the development of active presynaptic filaments, which in turn increases the homologous recombination activity of hRAD51. Assays for cell survival in normal newborn human dermal fibroblasts have shown that RS-1 induces a dose-dependent resistance to cisplatin, a cross-linking chemotherapy drug. RS-1 appears to function in vivo to promote homologous recombination repair proficiency, which is not surprising considering that RAD51-dependent recombination is a major determinant of cisplatin resistance. Numerous potential uses for RS-1 exist in both medical and research contexts.

Physicochemical Properties

Molecular Formula	C20H16BR2N2O3S
Molecular Weight	524.23
Exact Mass	521.925
Elemental Analysis	C, 45.82; H, 3.08; Br, 30.48; N, 5.34; O, 9.16; S, 6.12
CAS #	312756-74-4
Related CAS #	312756-74-4
PubChem CID	1039737
Appearance	White to off-white solid powder
LogP	6.487
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	6
Heavy Atom Count	28
Complexity	611
Defined Atom Stereocenter Count	0
SMILES	BrC1C([H])=C([H])C(C(N([H])C2C([H])=C([H])C(=C([H])C=2[H])Br)=O)=C([H])C=1S(N([H])C([H])([H])C1C([H])=C([H])C([H])=C([H])C=1[H])(=O)=O
InChi Key	SWKAVEUTKGKHSR-UHFFFAOYSA-N
InChi Code	InChI=1S/C20H16Br2N2O3S/c21-16-7-9-17(10-8-16)24-20(25)15-6-11-18(22)19(12-15)28(26,27)23-13-14-4-2-1-3-5-14/h1-12,23H,13H2,(H,24,25)
Chemical Name	3-(benzylsulfamoyl)-4-bromo-N-(4-bromophenyl)benzamide
Synonyms	RAD51-Stimulatory Compound-1; RS-1; RS 1; RS1
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	RAD51; CRISPR/Cas9
ln Vitro	RS-1 is a RAD51 activator that promotes hRAD51 binding to DNA with a Kd ranging from 48 nM to 107 nM when ATP or ADP is present and no nucleotide cofactor is present. This effect is not achieved by blocking ATPase activity. The helical pitch and length of hRAD51 protein-DNA complexes are influenced by RS-1 (20 μM). The strand assimilation activity of hRAD51 is stimulated by RS-1 (0, 1, 5, 10, 15, 20, and 25 μM). Human cells are more resistant to cross-linking chemotherapy when exposed to RS-1 (7.5 μM)[1]. In rabbit embryos, RS-1 (0, 7.5, and 15 μM) improves Cas9-mediated knock-in efficiencies[2].
ln Vivo	Transcription activator-like effector nuclease, zinc-finger nuclease, and CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats) are emerging as important genome editing tools. Crucially, these adaptable nucleases have allowed for the final achievement of knock-in in a number of non-rodent species, though more progress needs to be made in terms of rate improvement. We predict that the nuclease-mediated knock-in efficiency will be increased by either preventing non-homologous end joining (NHEJ) or boosting homology-directed repair (HDR). It is demonstrated here that the in vitro administration of the HDR enhancer RS-1 doubles to quintuples the knock-in efficiency at various loci, while the NHEJ inhibitor SCR7 has negligible effects. The knock-in rates have also improved multiple times since we started using RS-1 for animal production. Our research provides insights into enhancing the efficiency of gene targeting on pluripotent stem cells and offers instruments for nuclease-mediated knock-in animal production.
Enzyme Assay	In summary, preincubated for 5 minutes at 37°C, a DNA strand exchange protein (0.8 μM) is combined with 1 μM (nucleotide concentration) 32P-labeled oligonucleotide 306.7 in a reaction buffer that also contains different concentrations of RS-1 and 20 mM Hepes (pH 7.5), 1 mM DTT, 2 mM nucleotide cofactor, and 1 mM MgCl2. In experimental buffer conditions containing calcium, 1 mM CaCl2 is added to (1 mM MgCl2) or substituted (for RecA and scRAD51, in the case of hRAD51). There is also 110 nM scRAD54 in conditions with scRAD51. Following this initial binding reaction, 10.μL of target plasmid DNA (pRS306) that contains a supercoiled homologue at a base pair concentration of 19.75 μM is added, along with enough magnesium acetate to provide a final concentration of 10 mM[1].
Cell Assay	Human dermal fibroblasts from early passage neonates were kept in Medium 106 with low serum growth supplement added. The sulforhodamine B assay was used in cell-survival experiments. After being incubated for 24 hours in either cisplatin or RS-1, the cells are then left to incubate for an extra 6 days in regular media that does not contain either agent. Using a Synergy plate reader, cells stained with sulforhodamine B are quantified.
Animal Protocol
References	[1]. A chemical compound that stimulates the human homologous recombination protein RAD51. Proc Natl Acad Sci U S A. 2008 Oct 14;105(41):15848-53. [2]. RS-1 enhances CRISPR/Cas9- and TALEN-mediated knock-in efficiency. Nat Commun. 2016 Jan 28;7:10548.

Solubility Data

Solubility (In Vitro)

DMSO: ~100 mg/mL (~190.8 mM) Water: <1 mg/mL Ethanol: <1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 3 mg/mL (5.72 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 30.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 3 mg/mL (5.72 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 30.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.9076 mL	9.5378 mL	19.0756 mL
	5 mM	0.3815 mL	1.9076 mL	3.8151 mL
	10 mM	0.1908 mL	0.9538 mL	1.9076 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.