3′ end additions by T7 RNA polymerase are RNA self-templated, distributive and diverse in character—RNA-Seq analyses

Abstract

Synthetic RNA is widely used in basic science, nanotechnology and therapeutics research. The vast majority of this RNA is synthesized in vitro by T7 RNA polymerase or one of its close family members. However, the desired RNA is generally contaminated with products longer and shorter than the DNA-encoded product. To better understand these undesired byproducts and the processes that generate them, we analyze in vitro transcription reactions using RNA-Seq as a tool. The results unambiguously confirm that product RNA rebinds to the polymerase and self-primes (in cis) generation of a hairpin duplex, a process that favorably competes with promoter driven synthesis under high yield reaction conditions. While certain priming modes can be favored, the process is heterogeneous, both in initial priming and in the extent of priming, and already extended products can rebind for further extension, in a distributive process. Furthermore, addition of one or a few nucleotides, previously termed ‘nontemplated addition,’ also occurs via templated primer extension. At last, this work demonstrates the utility of RNA-Seq as a tool for in vitro mechanistic studies, providing information far beyond that provided by traditional gel electrophoresis.

Synthesis of low immunogenicity RNA with high-temperature in vitro transcription

“…Altogether, the intact mass and denaturing gel analyses of the short IVT templates suggest that during IVT spurious, 3'-extended products are synthesized in the reaction and some of these can form dsRNA structures, supported by their RNase III sensitivity. These observations are consistent with a recent study that demonstrated the synthesis of a heterogeneous population of 3'extended RNA products during IVT from a completely unrelated DNA template [20].…”

“…In contrast, reactions performed at 50°C did not show any detectable 3'-extended products ( Figure 3D). In conclusion we find that consistent with previous studies, T7 RNA polymerase can efficiently 3'-extend run-off transcripts [20], and that high-temperature IVT reduces the 3'extension of the run-off product.…”

“…Earlier work has demonstrated that the 3'-extended byproducts are synthesized by self-extension of the run-off transcript, leading to synthesis of considerably longer RNA products during IVT [11,13,20]. Therefore, we tested whether the 3'-extended byproducts we observed from our templates were synthesized by the same mechanism and whether high-temperature IVT prevented the self-extension of the run-off product by T7 RNAP.…”

“…Because wild-type T7 RNAP has the propensity to make spurious 3'-extended byproducts, we investigated whether changing the reaction conditions could alter the formation of these byproducts. One of the proposed mechanisms for formation of 3'-extended byproducts is the rebinding of the run-off transcript by T7 RNA polymerase followed by self-priming [13,20]. We hypothesized that altering the reaction temperature could prevent rebinding of the RNA polymerase to the run-off transcript and thereby reduce the formation of the 3'-extended contaminants.…”

“…Recent studies have identified two main types of byproducts in the IVT reaction that result in formation of dsRNA molecules. The first is formed by 3'-extension of the run-off products annealing to complementary sequences in the body of the run-off transcript either in cis (by folding back on the same RNA molecule) or trans (annealing to a second RNA molecule) to form extended duplexes [11,20]. The second type of dsRNA molecules is formed by hybridization of an antisense RNA molecule to the run-off transcript.…”