A new approach to RNA synthesis: immobilization of stably and functionally co-tethered promoter DNA and T7 RNA polymerase.

Current approaches to RNA synthesis/manufacturing require substantial (and incomplete) purification post-synthesis. We have previously demonstrated the synthesis of RNA from a complex in which T7 RNA polymerase is tethered to promoter DNA. In the current work, we extend this approach to demonstrate an extremely stable system of functional co-tethered complex to a solid support. Using the system attached to magnetic beads, we carry out more than 20 rounds of synthesis using the initial polymerase-DNA construct. We further demonstrate the wide utility of this system in the synthesis of short RNA, a CRISPR guide RNA, and a protein-coding mRNA. In all cases, the generation of self-templated double stranded RNA (dsRNA) impurities are greatly reduced, by both the tethering itself and by the salt-tolerance that local co-tethering provides. Transfection of the mRNA into HEK293T cells shows a correlation between added salt in the transcription reaction (which inhibits RNA rebinding that generates RNA-templated extensions) and significantly increased expression and reduced innate immune stimulation by the mRNA reaction product. These results point in the direction of streamlined processes for synthesis/manufacturing of high-quality RNA of any length, and at greatly reduced costs.

High-salt transcription from enzymatically gapped promoters nets higher yields and purity of transcribed RNAs.

T7 RNA polymerase is commonly used to synthesize large quantities of RNA for a wide variety of applications, from basic science to mRNA therapeutics. This in vitro system, while showing high fidelity in many ways, is also well known for producing longer than encoded RNA products, particularly under high-yield reaction conditions. Specifically, the resulting product pool is contaminated by an often disperse collection of longer cis-primed extension products. In addition to reducing yield via the conversion of correctly encoded RNA to longer products, self-primed extension generates partially double-stranded RNAs that can trigger the innate immune response. Extensive and low-yield purifications are then required to produce therapeutic RNA. Under high-yield conditions, accumulating concentrations of RNA effectively compete with promoter DNA for polymerase binding, driving self-primed extension at the expense of correct initiation. In the current work, we introduce a simple and novel modification in the DNA to strengthen promoter binding, shifting the balance back toward promoter-driven synthesis and so dramatically reducing self-primed extension. The result is higher yield of the encoded RNA at the outset and reduced need for extensive purifications. The approach can readily be applied to the synthesis of mRNA-length products under high-yield conditions.

High-salt transcription of DNA cotethered with T7 RNA polymerase to beads generates increased yields of highly pure RNA.

High yields of RNA are routinely prepared following the two-step approach of high-yield in vitro transcription using T7 RNA polymerase followed by extensive purification using gel separation or chromatographic methods. We recently demonstrated that in high-yield transcription reactions, as RNA accumulates in solution, T7 RNA polymerase rebinds and extends the encoded RNA (using the RNA as a template), resulting in a product pool contaminated with longer-than-desired, (partially) double-stranded impurities. Current purification methods often fail to fully eliminate these impurities, which, if present in therapeutics, can stimulate the innate immune response with potentially fatal consequences. In this work, we introduce a novel in vitro transcription method that generates high yields of encoded RNA without double-stranded impurities, reducing the need for further purification. Transcription is carried out at high-salt conditions to eliminate RNA product rebinding, while promoter DNA and T7 RNA polymerase are cotethered in close proximity on magnetic beads to drive promoter binding and transcription initiation, resulting in an increase in overall yield and purity of only the encoded RNA. A more complete elimination of double-stranded RNA during synthesis will not only reduce overall production costs, but also should ultimately enable therapies and technologies that are currently being hampered by those impurities.

Efficient inhibition of RNA self-primed extension by addition of competing 3'-capture DNA-improved RNA synthesis by T7 RNA polymerase.

In vitro synthesized RNA is used widely in studies of RNA biology, biotechnology and RNA therapeutics. However, in vitro synthesized RNA often contains impurities, such as RNAs with lengths shorter and longer than the expected runoff RNA. We have recently confirmed that longer RNA products are formed predominantly via cis self-primed extension, in which released runoff RNA folds back on itself to prime its own RNA-templated extension. In the current work, we demonstrate that addition of a DNA oligonucleotide (capture DNA) that is complementary to the 3' end of the expected runoff RNA effectively prevents self-primed extension, even under conditions commonly used for high RNA yields. Moreover, the presence of this competing capture DNA during 'high yield' transcription, leads to an increase in the yield of expected runoff RNA by suppressing the formation of undesired longer RNA byproducts.

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

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.

Insights into the mechanism of initial transcription in Escherichia coli RNA polymerase.

It has long been known that during initial transcription of the first 8-10 bases of RNA, complexes are relatively unstable, leading to the release of short abortive RNA transcripts. An early "stressed intermediate" model led to a more specific mechanistic model proposing "scrunching" stress as the basis for the instability. Recent studies in the single subunit T7 RNA polymerase have argued against scrunching as the energetic driving force and instead argue for a model in which pushing of the RNA-DNA hybrid against a protein element associated with promoter binding, while likely driving promoter release, reciprocally leads to instability of the hybrid. In this study, we test these models in the structurally unrelated multisubunit bacterial RNA polymerase. Via the targeted introduction of mismatches and nicks in the DNA, we demonstrate that neither downstream bubble collapse nor compaction/scrunching of either the single-stranded template or nontemplate strands is a major force driving abortive instability (although collapse from the downstream end of the bubble does contribute significantly to the instability of artificially halted complexes). In contrast, pushing of the hybrid against a mobile protein element (σ3.2 in the bacterial enzyme) results in substantially increased abortive instability and is likely the primary energetic contributor to abortive cycling. The results suggest that abortive instability is a by-product of the mechanistic need to couple the energy of nucleotide addition (RNA chain growth) to driving the timed release of promoter contacts during initial transcription.

New insights into the mechanism of initial transcription: the T7 RNA polymerase mutant P266L transitions to elongation at longer RNA lengths than wild type.

RNA polymerases undergo substantial structural and functional changes in transitioning from sequence-specific initial transcription to stable and relatively sequence-independent elongation. Initially, transcribing complexes are characteristically unstable, yielding short abortive products on the path to elongation. However, protein mutations have been isolated in RNA polymerases that dramatically reduce abortive instability. Understanding these mutations is essential to understanding the energetics of initial transcription and promoter clearance. We demonstrate here that the P266L point mutation in T7 RNA polymerase, which shows dramatically reduced abortive cycling, also transitions to elongation later, i.e. at longer lengths of RNA. These two properties of the mutant are not necessarily coupled, but rather we propose that they both derive from a weakening of the barrier to RNA-DNA hybrid-driven rotation of the promoter binding N-terminal platform, a motion necessary to achieve programmatically timed release of promoter contacts in the transition to elongation. Parallels in the multisubunit RNA polymerases are discussed.

A simple approach to improving RNA synthesis: Salt inhibition of RNA rebinding coupled with strengthening promoter binding by a targeted gap in the DNA.

T7 RNA polymerase is widely used to synthesize RNA of any length, and long-standing protocols exist to efficiently generate large amounts of RNA. Such synthesis, however, is often plagued by so-called "nontemplated additions" at the 3' end, which are in fact templated by the RNA itself and give rise to double-stranded RNA impurities in RNA therapeutics. These additions are generated by RNA polymerase rebinding to the product RNA (independent of DNA) and this rebinding is in competition with promoter binding. This chapter reports on a general approach that simultaneously weakens RNA rebinding by increasing salt, while at the same time increases promoter binding through manipulating the promoter DNA structure, shifting the balance away from self-primed extension. We present two approaches for use in different regimes. For (short) RNAs using synthetic oligonucleotides as DNA, promoter binding is strengthened by using a partially single stranded promoter construct already in wide use in the field. For the synthesis of RNA (of any length), one can replicate the behavior of the first approach by introducing a targeted gap in the promoter, using a PCR primer containing an engineered deoxyuracil that is then excised by a commercially available enzyme system, to leave a promoter-strengthening gap. Both approaches are simple to implement, with only slight variations on standard synthesis approaches, making them valuable tools for a wide range of applications, from basic science to mRNA, CRISPR, lncRNA, and other therapeutics.

Direct tests of the energetic basis of abortive cycling in transcription.

Although the synthesis of RNA from a DNA template is (and must be) a generally very stable process to enable transcription of kilobase transcripts, it has long been known that during initial transcription of the first 8-10 bases of RNA complexes are relatively unstable, leading to the release of short abortive RNA transcripts. A wealth of structural data in the past decade has led to specific mechanistic models elaborating an earlier "stressed intermediate" model for initial transcription. In this study, we test fundamental predictions of each of these models in the simple model enzyme T7 RNA polymerase. Nicking or gapping the nontranscribed template DNA immediately upstream of the growing hybrid yields no systematic reduction in abortive falloff, demonstrating clearly that compaction or "scrunching" of this DNA is not a source of functional instability. Similarly, transcription on DNA in which the nontemplate strand in the initially transcribed region is either mismatched or removed altogether leads to at most modest reductions in abortive falloff, indicating that expansion or "scrunching" of the bubble is not the primary driving force for abortive cycling. Finally, energetic stress derived from the observed steric clash of the growing hybrid against the N-terminal domain contributes at most mildly to abortive cycling, as the addition of steric bulk (additional RNA bases) at the upstream end of the hybrid does not lead to predicted positional shifts in observed abortive patterns. We conclude that while structural changes (scrunching) clearly occur in initial transcription, stress from these changes is not the primary force driving abortive cycling.

Transcription elongation complex stability: the topological lock.

Transcription machinery from a variety of organisms shows striking mechanistic similarity. Both multi- and single subunit RNA polymerases have evolved an 8-10-base pair RNA-DNA hybrid as a part of a stably transcribing elongation complex. Through characterization of halted complexes that can readily carry out homopolymeric slippage synthesis, this study reveals that T7 RNA polymerase elongation complexes containing only a 4-base pair hybrid can nevertheless be more stable than those with the normal 8-base pair hybrid. We propose that a key feature of this stability is the topological threading of RNA through the complex and/or around the DNA template strand. The data are consistent with forward translocation as a mechanism to allow unthreading of the topological lock, as can occur during programmed termination of transcription.

Twisted or shifted? Fluorescence measurements of late intermediates in transcription initiation by T7 RNA polymerase.

T7 RNA polymerase undergoes dramatic structural rearrangements in the transition from initiation to elongation. Two models have been proposed for promoter-bound intermediates late in the transition. (i) A subset of promoter interactions are maintained through completion of the protein conformational (twist) change, and (ii) concerted movement (shift) of all promoter-binding elements away from the growing DNA-RNA hybrid leads to an open intermediate, with large-scale domain rotations deferred until after promoter release. Fluorescence resonance energy transfer measurements provide very strong support for the latter.

Dissociation of halted T7 RNA polymerase elongation complexes proceeds via a forward-translocation mechanism.

A recent model for the mechanism of intrinsic transcription termination involves dissociation of the RNA from forward-translocated (hypertranslocated) states of the complex [Yarnell WS, Roberts JW (1999) Science, 284:611-615]. The current study demonstrates that halted elongation complexes of T7 RNA polymerase in the absence of termination signals can also dissociate via a forward-translocation mechanism. Shortening of the downstream DNA or the introduction of a stretch of mismatched DNA immediately downstream of the halt site reduces a barrier to forward translocation and correspondingly reduces the lifetime of halted complexes. Conversely, introduction of a cross-link downstream of the halt site increases the same barrier and leads to an increase in complex lifetime. Introduction of a mismatch within the bubble reduces a driving force for forward translocation and correspondingly increases the lifetime of the complex, but only for mismatches at the upstream edge of the bubble, as predicted by the model. Mismatching only the two most upstream of the eight bases in the bubble provides a maximal increase in complex stability, suggesting that dissociation occurs primarily from early forward-translocated states. Finally, addition in trans of an oligonucleotide complementary to the nascent RNA just beyond the hybrid complements the loss of driving force derived from placement of a mismatch within the bubble, confirming the expected additivity of effects. Thus, forward translocation is likely a general mechanism for dissociation of elongation complexes, both in the presence and absence of intrinsic termination signals.

Structural confirmation of a bent and open model for the initiation complex of T7 RNA polymerase.

T7 RNA polymerase is known to induce bending of its promoter DNA upon binding, as evidenced by gel-shift assays and by recent end-to-end fluorescence energy transfer distance measurements. Crystal structures of promoter-bound and initially transcribing complexes, however, lack downstream DNA, providing no information on the overall path of the DNA through the protein. Crystal structures of the elongation complex do include downstream DNA and provide valuable guidance in the design of models for the complete melted bubble structure at initiation. In the current study, we test a specific structural model for the initiation complex, obtained by alignment of the C-terminal regions of the protein structures from both initiation and elongation and then simple transferal of the downstream DNA from the elongation complex onto the initiation complex. Fluorescence resonance energy transfer measurement of distances from a point upstream on the promoter DNA to various points along the downstream helix reproduce the expected helical periodicity in the distances and support the model's orientation and phasing of the downstream DNA. The model also makes predictions about the extent of melting downstream of the active site. By monitoring fluorescent base analogues incorporated at various positions in the DNA, we have mapped the downstream edge of the bubble, confirming the model. The initially melted bubble, in the absence of substrate, encompasses 7-8 bases and is sufficient to allow synthesis of a three base transcript before further melting is required. The results demonstrate that despite massive changes in the N-terminal portion of the protein and in the DNA upstream of the active site, the DNA downstream of the active site is virtually identical in both initiation and elongation complexes.

Observed instability of T7 RNA polymerase elongation complexes can be dominated by collision-induced "bumping".

T7 RNA polymerase elongates RNA at a relatively high rate and can displace many tightly bound protein-DNA complexes. Despite these properties, measurements of the stability of stalled elongation complexes have shown lifetimes that are much shorter than those of the multisubunit RNA polymerases. In this work, we demonstrate that the apparent instability of stalled complexes actually arises from the action of trailing RNA polymerases (traveling in the same direction) displacing the stalled complex. Moreover, the instability caused by collision between two polymerases is position dependent. A second polymerase is blocked from promoter binding when a leading complex is stalled 12 bp or less from the promoter. The trailing complex can bind and make abortive transcripts when the leading complex is between 12 and 20 bp from the promoter, but it cannot displace the first complex since it is in a unstable initiation conformation. Only when the leading complex is stalled more than 20 bp away from the promoter can a second polymerase bind, initiate, and displace the leading complex.

Mechanism of instability in abortive cycling by T7 RNA polymerase.

Abortive transcription, the premature release of short transcripts 2-8 bases in length, is a unique feature of transcription, accompanying the transition from initiation to elongation in all RNA polymerases. The current study focuses on major factors that relate to the stability of initially transcribing abortive complexes in T7 RNA polymerase. Building on previous studies, results reveal that collapse of the DNA from the downstream end of the bubble is a major contributor to the characteristic instability of abortive complexes. Furthermore, transcription from a novel DNA construct containing a nick between positions -14 and -13 of the nontemplate strand suggests that the more flexible promoter reduces somewhat the strain inherent in initially transcribing complexes, with a resulting decrease in abortive product release. Finally, as assessed by exonuclease III footprinting and transcription profiles, a DNA construct defective in bubble collapse specifically from the downstream end exhibits less abortive cycling and little perturbation of the final transition to elongation, including the process of promoter release.

Stability of gold nanoparticle-bound DNA toward biological, physical, and chemical agents.

Positively charged trimethylammonium-modified mixed monolayer protected clusters (MMPCs) interact with DNA by complementary electrostatic binding, serving as efficient DNA delivery systems. The stability of gold nanoparticle-bound DNA toward biological, physical, and chemical agents is investigated. The MMPC-bound DNA is efficiently protected from DNAse I digestion and experiences nicking/cleavage-induced morphology changes with higher concentrations of DNAse I. Significant protection of MMPC-bound DNA was also observed in a physical sonication assay. However, the MMPC-bound DNA was found to show enhanced cleavage upon exposure to chemically induced radicals. The latter may indicate that bound DNA is bent and wrapped on the surface of the cationic MMPC.