Aleksey Rukavishnikov

Fluorogenic cephalosporin substrates for β-lactamase TEM-1.

Cephalosporin was used to synthesize soluble and precipitating fluorogenic β-lactam substrates that demonstrated differential catalytic hydrolysis by three different subtypes of β-lactamase: TEM-1 (class A), p99 (class C), and a Bacillus cereus enzyme sold by Genzyme (class B). The most successful soluble substrate contained difluorofluorescein (Oregon Green 488) ligated to two cephalosporin moieties that, therefore, required two turnovers to produce the fluorescent Oregon Green 488 leaving group. The bis-cephalosporin modification was required so that the final reaction product was the Oregon Green 488 carboxylic acid rather than a less bright phenolic adduct of the dye. Hydrolysis in pH 5.5 Mes and pH 7.2 phosphate-buffered saline (PBS) buffers was similar, but in pH 8.0 Tris the hydrolysis rate nearly doubled. Activity of the β-lactamases on the various substrates was shown to depend highly on the linker between the cephalosporin and the fluorophore, with an allyl linker promoting faster turnover than a phenol ether linker. Measured K(m) values for dichlorofluorescein and difluorofluorescein cephalosporin substrates were approximately the same as K(m) values for penicillin G and ampicillin found in the literature (~30-40μM).

Abstract 5098: GFP compatibility with EdU cell proliferation assay

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Examining cell proliferation in GFP expressing cells is of general interest in many aspects of biology including regenerative medicine, stem cells, developmental biology and some fields of cancer research. However, visualization of GFP expression is not readily compatible with commonly used proliferation assays which incorporate a thymidine analog to directly measure S-phase proliferation of DNA. With the BrdU (bromo-deoxyuridine) assay, an antibody based detection method, alcohol for fixation and hydrochloric acid for DNA denaturation is commonly used. Neither chemical is compatible with GFP fluorescence. For imaging applications there are some methods to avoid the use of HCl as a denaturant with the BrdU assay, however; they are typically “home-brew” methods used to partially digest the DNA, adding extra steps, and not easily performed. The much faster and reliable EdU (ethynyl-deoxyuridine) cell proliferation assay which uses click chemistry for detection of S-phase proliferation of DNA, uses formaldehyde based fixation and avoids the use of HCl for DNA denaturation. However, the use of copper to catalyse the click reaction also negatively affects GFP fluorescence. Anti-GFP antibodies can be used in the click reaction work flow, to “retrieve” the lost GFP fluorescence but are not convenient and add extra steps. We present recent improvements to the click chemistry based EdU cell proliferation assay which minimizes the loss of GFP and other fluorescent proteins signals and avoids the need for “work around” methods. The resulting click reaction is both more rapid and brighter than the “classic” click EdU assay. The modifications preserve most of the GFP fluorescence and permit multiplex detection with EdU with no change in the work flow of the classic click EdU assay. We optimized components in the improved click reaction conditions and tested compatibility with various fluorescent proteins. Examples of click chemistry and GFP/RFP/mCherry compatibility are presented using the EdU cell proliferation assay in both cell culture and in GFP expressing tissue. Additionally, improvements are demonstrated in with other applications of click chemistry assays used for imaging as well as with on flow cytometry platforms where GFP, R-phycoerythrin (R-PE), or other fluorescent proteins are commonly combined with cell proliferation assays. The use of the modified click reaction is an enabling improvement over originally described copper based click reactions and will further enhance the utility of EdU based cell proliferation. Citation Format: Scott T. Clarke, Zhichao Song, Kelvin Y. Kwan, Carolyn DeMarco, Aleksey Rukavishnikov, Upinder Singh, Kyle Gee. GFP compatibility with EdU cell proliferation assay. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5098. doi:10.1158/1538-7445.AM2014-5098

Abstract 4033: Gene expression of EdU proliferating cells made possible with improved click chemistry.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Gene expression profiling of cancer cells has become increasingly common over the past five years. Especially important is identifying changes in gene expression caused by drug treatment leading to side populations with altered pathways. These populations can escape drug treatment and lead to recurrence. Characterizing gene expression of proliferating cells requires a method of identifying proliferation while simultaneously maintaining high quality RNA. Typically, cells are screened in a drug panel in the presence of tritiated thymidine or BrdU to identify the drug resistance in the proliferating population. Proliferation detection with these methods is neither rapid nor does it yield high quality RNA for profiling. A new proliferation method based on click chemistry using an alkyne modified thymidine analog (EdU), which can be used to rapidly identify replicating cells appears ideally suited for this application; however, copper used for the catalysis of the click reaction causes undesired degradation of RNA. Recent improvements to the copper based click reaction are presented here whereby the copper is tightly controlled, resulting in higher quality RNA useful for sequencing. In this study we demonstrate gene expression changes in the proliferating population of a drug resistant cancer cell line compared to the parental cell lines. Proliferating cells were identified using an improved click reaction comprising of a copper(I) ligand combined with a picolyl modified dye azide detection reagent. Total RNA from these cells was isolated and gene expression was assessed by targeted RNA sequencing using an RNA Apoptosis panel and an RNA Cancer panel using proton based sequencing technology. Use of an improved copper “safe” click reaction for detection of nascent DNA, allows for detection of incorporated click analog for measuring proliferation and subsequent isolation of high quality RNA for use in sequence analysis. Citation Format: Scott T. Clarke, Angie Cheng, Aleksey Rukavishnikov, Michelle Yan, Upinder Singh, Kyle Gee, Susan Magdaleno. Gene expression of EdU proliferating cells made possible with improved click chemistry. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4033. doi:10.1158/1538-7445.AM2013-4033