Jon Kemppainen & Gary Latham, Ambion, Inc.
TURBO DNase (patent pending) is an engineered version of DNase I that represents a major advance in DNA removal. TURBO DNase binds DNA more tightly than the wild type enzyme, enabling more efficient DNA removal over a broad range of conditions. Recently, Ambion scientists identified a small molecule enhancer of TURBO DNase activity that further magnifies the potency of this enzyme.
As interest in TURBO DNase has grown, Ambion has received a number of inquiries from researchers who require enzymatic DNA removal to enable emerging technologies. For example, several scientists have expressed interest in digesting exogenous DNA from solutions containing viable cells as a requisite bioprocessing step in the manufacturing of next generation clinical products. Current FDA regulations recommend that residual DNA be limited to <10 pg/medicinal dose for therapeutic products. Consequently, efficient DNA removal is an important regulatory and safety requirement. Residual DNA often must be cleared in solutions containing physiological levels of salt (~150 mM) which is required to maintain healthy, living cells. Wild type DNase I maintains only minimal activity in this milieu. TURBO DNase, however, is a salt-tolerant enzyme that functions optimally in physiological salt solutions.
The performance of TURBO DNase containing the new enhancer was compared to a competitors wild type DNase for removal of DNA from living cell cultures. The enzymatic removal of viral DNA in cultured cells was measured using real-time RT-PCR, and cell viability was assessed after DNase treatment. In these experiments, HeLa cells were grown to confluence for 72 hours (about 15,000 cells/well in a 96 well plate). Cell media were temporarily removed from the wells, pooled, and adjusted to the appropriate 1X buffer concentration with TURBO DNase Buffer (containing the new TURBO DNase enhancer) or the DNase Buffer supplied with the wild type DNase I. Then, either 5 units of TURBO DNase (11.6 ng), or 5 or 50 units of wild type DNase I was added (110 ng and 475 ng, respectively). The DNase-containing medium was then replaced onto the cells. After a 15 minute equilibration period at 37C, lambda genomic DNA was introduced at 200 ng/well. The DNase digestion reactions were incubated for 75 min at 37C, then the media were collected. DNase digestion products were concentrated 5-fold from the media by magnetic bead purification, and 5 l was used in real-time PCR with primer-probe sets for lambda genomic DNA (Figure 1). For viability studies, cells were treated with trypsin, resuspended in 175 l DMEM-H plus serum, and assayed by microcytometry on a Guava Technologies PCA-96 instrument.
Figure 1. TURBO DNase Eliminates More DNA Contamination than Wild Type DNase I. Even at a 10-fold lower unit concentration, TURBO DNase removed more DNA from living HeLa cell cultures. The table summarizes th e real-time PCR cycle thresholds (Ct) of residual lambda DNA detection using the conditions described in the text. Samples were amplified using an ABI 7900 real-time instrument. The Fold DNA Removal was calculated from the lambda DNA standard curve (slope=-3.3). Values are the average of replicate measurements. Duplicate real-time amplification curves for the samples presented in the table are also shown.
Real-time PCR showed that treatment of live cells with 5 units of TURBO DNase reduced the amount of DNA contamination by 17.6 Ct, or more than 200,000 fold to 990 fg (Figure 1). Treatment with 5 units of wild type DNase I only reduced the DNA contamination by 11.5 Ct, or 2893-fold to 69 pg. Addition of 10-fold more of the wild type DNase enzyme reduced DNA content by 121,072-fold to 1,650 fgand was still not as effective as TURBO DNase used at 10 times lower unit concentration. TURBO DNase was able to reduce the copy number of DNA molecules from 1 billion to less than 5000 copies. In the best case, the competitors wild type DNase I, used at 10 times the unit concentration, reduced the DNA contamination to 8000 copies. This is an important finding, since clinical bioprocessing procedures often demand that the introduction of reagents, such as enzymes, into the process stream be restricted to the lowest effective level.
The superiority of TURBO DNase is striking when the comparison is based on the mass amount of enzyme added to the reacti on. Quantitation of the total protein concentration for each DNase reveals that 1 unit of the competitors wild type DNase I contains 9.5 times more protein than 1 unit of TURBO DNase (data not shown). Thus, TURBO DNase demonstrated 663-fold better specific activity for DNA removal in this assay than the wild type DNase I tested (i.e. 9.5 x (201833/ 2893)=663 fold).
To quantify the effect of TURBO DNase treatment on the viability of the cultured cells, representative populations were stained with Viacount dyes that distinguish between living and dead cells. The results were analyzed by microcytometry using a Guava Technologies PCA-96 instrument and Cytosoft software. Treatment of the cells with 5 units or even as much as 50 units of TURBO DNase plus enhancer did not affect cell viability as compared to non-treated cells (Figure 2). In addition, scrutiny of the cell populations with the Cytosoft analysis software showed no evidence of mid-phase apoptotic cells in either treated or untreated cells.
Figure 2. Turbo DNase Treatment Does Not Affect Cell Viability. Cells (100,000/well) were incubated with 5 U of TURBO DNase, detached from the plate, and Viacount cell dye was added to differentiate live and dead cells. Cells (1000) were counted by microcytometry in a Guava Technologies PCA 96 instrument.
Ambions exclusive TURBO DNase is far superior to wild type DNase I in removing unwanted DNA contamination from viable cultured cells. In fact, even at 10 times lower unit co ncentration, TURBO DNase removes more DNA from cell culture media than the conventional DNase I. Moreover, TURBO DNase treatment does not compromise cell viability, as assayed by direct cell counting methods. Not only is TURBO DNase clearly the best choice for removing DNase in routine basic research applications, it has great potential for use in sensitive bioprocessing applications.