Every in vitro transcription (IVT) reaction starts with DNA you put there on purpose. A linearized plasmid or polymerase chain reaction (PCR) template goes in, the polymerase does its work and a few hours later you have RNA, often a lot of it. The template’s job is done.
In theory, a quick DNase step takes care of the rest.
In practice, the template doesn’t always leave quietly.
The DNA you added is the hardest DNA to remove
In an IVT reaction, the DNA was added deliberately, often in microgram amounts. It sits in a buffer designed to make the polymerase as productive as possible, which is not the same as making DNase productive.
That’s the trade-off at the center of IVT cleanup. The easiest place to digest the template is the reaction tube, right after transcription, with no extra purification step. But IVT reactions carry high ionic strength and a crowded mix of other components, conditions that suppress the activity of many DNase I enzymes (1,2). The reaction is good at making RNA and bad at getting rid of what made it.
If you run IVT, you have probably worked around this with more enzyme, longer incubations, or a cleanup step before digestion, which costs time and RNA. Even then, the same protocol can leave different amounts of template behind.
The consequences reach past the tube. Residual DNA can inflate yield estimates, so downstream amounts are based on more RNA than you actually have (3). When IVT RNA goes into cells, co-delivered DNA can trigger the cell’s own DNA-sensing pathways (4). For anyone whose work is heading toward therapeutic or vaccine applications, the stakes are higher: residual template DNA is a quality attribute regulators expect to see measured and controlled (4).
A DNase I built for IVT conditions
The usual answer has been to manage carryover with additional steps, increased time and extra labor after the fact. These steps help, but they don’t change how well the enzyme works in the conditions where it’s actually used.
That’s the problem RQ2.0 DNase I was designed to address. It’s a recombinant DNase I, produced in Pichia pastoris under current Good Manufacturing Practice (cGMP) conditions without animal-origin materials or antibiotics (1). Making the enzyme under cGMP in a controlled fermentation system produces an enzyme with lot-to-lot consistency and functionality under harsh buffer conditions.
In head-to-head testing, we spiked a model DNA template into a buffer modeled on typical IVT reaction conditions. We then compared RQ2.0 DNase I with three other recombinant DNase I products, measuring residual DNA by quantitative PCR (qPCR) (1). RQ2.0 demonstrated robust nuclease activity at moderate (40 mM) and high (330mM) ionic strength and under low-magnesium conditions (1).
RQ2.0 also works fast and over a range of temperatures. In the same IVT-modeled buffer, it removed a substantial amount of template in as little as 5 minutes and stayed active from room temperature to 42°C (1).
You can also digest the template directly in the reaction without a buffer exchange or without doubling the enzyme to make up for the conditions (1,2). Consistent unit activity from lot to lot means the digestion you set up today performs the same way with the next lot (1).
What changes at the bench
The benefit is an IVT reaction you can trust. Yield numbers reflect transcript, not leftover template. Cell-based experiments have one less confounding variable, and PCR-based assays on your RNA start with a cleaner baseline. RQ2.0 pairs with the RiboMAX™ Large-Scale RNA Production Systems for research-scale work (5). It also pairs with the RiboMAX™ AOF RNA Production System, built from animal-origin-free, cGMP-manufactured components (2,6). If your RNA heads toward the clinic, cGMP versions with added testing and documentation are available (6).
Every IVT reaction begins with a template, and for a long time some of that template has stayed behind. RQ2.0 DNase I doesn’t change why the DNA is there in the first place, but it provides a clear exit to the door.
References
- Promega Corporation. (2026). RQ2.0 DNase I: More complete DNA removal. cGMP manufactured. Built for your process (Publication No. GFN411): https://promega.widen.net/s/zddplsdx2w/rq2_0_dnase_i_flyer_gfn411
- Promega Corporation. (2026). RQ2.0 DNase I quick protocol (Publication No. FB276). https://www.promega.com/-/media/files/resources/protcards/rq2-dnase1-quick-protocol-fb276.pdf
- Wieczorek, D., Delauriere, L., & Schagat, T. (n.d.). Methods of RNA quality assessment. Promega Corporation. https://worldwide.promega.com/resources/pubhub/methods-of-rna-quality-assessment/
- Nowak, C. J. A., Liu, S., Falconer, R. J., & Gerstweiler, L. (2026). Process and analytical strategies for the safe production of mRNA vaccines and therapeutics. Molecular Biology Reports, 53, Article 306. https://doi.org/10.1007/s11033-026-11455-0
- Promega Corporation. (n.d.). RQ2.0 DNase I. Retrieved October 20, 2026, from https://www.promega.com/products/cloning-and-dna-markers/molecular-biology-enzymes-and-reagents/rq2-0-dnase-i/
- Promega Corporation. (n.d.). RiboMAX™ AOF RNA Production System. Retrieved October 20, 2026, from https://www.promega.com/products/rna-analysis/in-vitro-transcription/ribomax-aof-rna-production-system/
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