1page Paper RESEARCH ARTICLE High-salt transcription of DNA cotethered with T7 RNA polymerase to beads generates increased yields of highly pure RNA Recei

1page Paper RESEARCH ARTICLE
High-salt transcription of DNA cotethered with T7 RNA
polymerase to beads generates increased yields of highly
pure RNA
Received for publication, April 29, 2021, and in revised form, July 13, 2021 Published, Papers in Press, July 22, 2021,
https://doi.org/10.1016/j.jbc.2021.100999

Elvan Cavac1 , Luis E. Ramírez-Tapia2, and Craig T. Martin1,2,*
From the 1Department of Chemistry, 2Graduate Program in Molecular & Cellular Biology, University of Massachusetts Amherst,
Amherst, Massachusetts, USA

Edited by Craig Cameron
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 demon-
strated that in high-yield transcription reactions, as RNA ac-
cumulates 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 purifi-
cation methods often fail to fully eliminate these impurities,
which, if present in therapeutics, can stimulate the innate im-
mune 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. Tran-
scription 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.

Our understanding of RNA’s central role in biology continues
to expand and be exploited. Researchers across a wide swath of
basic science and applied technologies require high yields of
pure RNA. Solid-phase chemical synthesis can, in principle,
generate RNAs up to 50 to 100 nt in length, but both yield and
purity decrease with increasing lengths (1–3). Enzymatic syn-
thesis in vitro by T7 RNA polymerase is widely used to syn-
thesize high yields of RNA of all lengths for structural studies,
basic RNA biology (splicing, riboswitches, CRISPR, lncRNA),
therapeutics applications (mRNA vaccines and therapies,
siRNA, gRNA for CRISPR), and nanotechnology (4–7).
* For correspondence: Craig T. Martin, cmartin@chem.umass.edu.
Present address for Luis E. Ramírez-Tapia: Green Light Biosciences, Boston,

Massachusetts 02118, USA.

© 2021 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
T7 RNA polymerase binds its consensus promoter sequence
with near nanomolar affinity in vitro (8, 9), initiates transcription
at a unique site in the DNA, transitions to stable elongation, and
runs off the end of a linear DNA template to synthesize the
encoded RNA (4, 6, 10). It has long been known that in addition
to the full-length encoded RNA, T7 RNA polymerase produces
short abortive RNAs 2 to 7 nucleotides in length (6, 11) and
RNAs longer than the encoded length. These longer products
have been proposed to be generated through templated and
nontemplated additions (6, 12–14), cis or trans primed extension
of RNA (15–22), or strand jumping (23). Our lab recently
demonstrated that in high-yield transcription reactions, the most
significant contribution to the longer, undesired RNA products is
through the cis self-primed extension mechanism (16, 24). As
high yields of encoded RNA accumulate in solution, mass action
drives the polymerase to rebind the accumulated RNA at its 30

end and self-extend via a nonpromoter-dependent mechanism.
The process is heterogeneous and distributive, leading to a
diverse pool of products often abundant in (partially) double-
stranded RNAs longer than the encoded length.

In RNA therapeutics applications, dsRNA contamination
from in vitro synthesized RNA can invoke the innate immune
response, as dsRNA is classified as a potent pathogen-associated
molecular pattern in the body. This can happen by way of
activating natural sensors such as retinoic acid inducible gene
(RIG-I) (25, 26), Toll-like receptor 3 (TLR-3) (27–29), and
protein melanoma-differentiation-associated antigen 5
(MDA5) (25). It can cause the production of type I interferon,
which can inhibit translation through the activation and upre-
gulation of protein kinase R (PKR) (30). It can also cause cellular
mRNA degradation by activating the 20-50 oligoadenylate syn-
thetase (OAS) enzyme family (31). Therefore, therapeutics re-
searchers must follow up in vitro T7 RNA polymerase
transcription with often very extensive purification methods
(32–36). Gel or chromatic (HPLC) purification methods are
time-consuming, result in a loss of yield, and are imprecise, as
the encoded product may not always be readily identified. At
long RNA lengths, the resolution in these separations becomes
progressively worse, making the purification of the precisely
encoded RNA unattainable. A recently developed method
removes dsRNA impurities from the in vitro transcription pool
J. Biol. Chem. (2021) 297(3) 100999 1
Biochemistry and Molecular Biology. This is an open access article under the CC

https://doi.org/10.1016/j.jbc.2021.100999

https://orcid.org/0000-0001-9284-745X

Delta:1_given name

Delta:1_surname

https://orcid.org/0000-0003-1029-5239

mailto:cmartin@chem.umass.edu

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Cotethered transcription at high salt
by their selective binding to cellulose in an ethanol-based buffer
(37). However, the effectiveness of this method is expected to
depend on the relative lengths of double-stranded regions and
may remove desired RNAs with natively structured regions.

Although researchers have long focused on improving the
overall yield of in vitro transcription reactions, we have
confirmed with RNA-Seq that the very conditions of high-yield
synthesis often drive the correct product into primer extended,
double-stranded impurities (24). This not only impacts the
overall purity, but also the yield of the encoded RNA.

In this research, we present a novel method of in vitro
transcription that allows promoter-directed transcription
while preventing primer extension activity, thereby dramati-
cally reducing double-stranded impurities. In brief, by
increasing salt concentrations in solution, we reduce all
protein–nucleic acid interactions. To selectively restore pro-
moter binding, we tether both T7 RNA polymerase and pro-
moter DNA to a solid support (beads). This drives their
association even at high salt concentrations. Near elimination
of RNA rebinding and extension not only results in a dramatic
reduction in longer, primer-extended products, but also nets a
dramatic increase in the yield of encoded RNA.

Results

The goal of this study is to eliminate RNA product
rebinding and subsequent extension activities of T7 RNA
polymerase, while retaining promoter-directed transcription.
Like almost all protein–nucleic acid interactions, both initial
binding of T7 RNA polymerase to its promoter and rebinding
of product RNA are stabilized in part by electrostatic in-
teractions between positively charged residues on the RNA
polymerase surface and the negatively charged phosphate
backbone of the DNA or RNA (38–41). As a result, increasing
salt concentrations should destabilize both promoter DNA
binding and product RNA rebinding. We have previously
shown that covalently cross-linking an engineered cysteine
(A94C) in the N-terminal domain of T7 RNA polymerase to a
30 thiol-modified template DNA creates a locally high con-
centration of the promoter near its binding site, allowing
promoter binding, even at high salt (42). Initiation proceeds
well and at least some of these complexes transition to the
stable elongation phase (42). Elongation by T7 RNA poly-
merase is stabilized by the topological locking of the RNA
around the template DNA in the enzyme active site (10) and
elongation has been shown to be resistant to added salt con-
centrations up to at least 0.2 M NaCl (40, 43).

By tethering T7 RNA polymerase to its DNA promoter and
carrying out transcription at elevated salt concentrations, we can
achieve promoter-initiated transcription while preventing
product RNA rebinding that otherwise would lead to cis primed
extension activity. We expect this approach to reduce substan-
tially the production of longer, double-stranded RNA impurities.

Design of a tethered in vitro transcription system

In order to tether the polymerase to the promoter DNA and
still allow functional initiation and substantial transition to
2 J. Biol. Chem. (2021) 297(3) 100999
elongation, we bound each, independently to Strep-TactinXT
magnetic beads. The N-terminal domain of T7 RNA poly-
merase (together with a hairpin loop from the C-terminal
domain) forms the promoter binding platform (44, 45), and
many N-terminal fusions of T7 RNA polymerase function well
in promoter-directed transcription (46–48). Thus, we fused
the Strep-tag II peptide (WSHPQFEK), followed by a short and
flexible peptide linker (GGS), to the N-terminus of recombi-
nant T7 RNA polymerase (49). Herein, we will call this Strep-
tagged variant Strep-T7 RNA polymerase. The Strep-tag II
peptide has nanomolar binding affinity to specifically engi-
neered Strep-TactinXT-coated magnetic beads (50). We also
used 50-biotinylated nontemplate DNA to independently bind
the promoter DNA to the Strep-TactinXT beads. Biotin is
reported to have picomolar binding affinity to Strep-
TactinXT-coated magnetic beads (50).

To confirm that the Strep-tag II peptide addition at the
N-terminus of T7 RNA polymerase does not affect transcrip-
tion activity, we performed in vitro transcription reactions
using Strep-T7 RNA polymerase and promoter DNA (without
a biotin tag) encoding a 24 base RNA (RNA-24) under high-
yield transcription conditions. The gel analysis in Fig. S2
demonstrates identical transcription profiles using T7 RNA
polymerase and Strep-T7 RNA polymerase. This confirms that
the addition of the Strep-tag II peptide has no adverse effect on
the activity of T7 RNA polymerase. Similarly, biotinylating the
upstream end of the promoter has no effect on promoter
function, as also shown in Fig. S2. Finally, we tested these
constructs for transcription activity at 0.4 M added NaCl and,
as expected for the uncoupled species, observed essentially
complete inhibition of transcription in all constructs.
Tethered system favors promoter-directed transcription at
high salt

Having demonstrated that the DNA and protein modifica-
tions do not perturb promoter binding and transcription, we
proceeded to test the cotethered system for function. Given
that at low salt the dissociation constant for duplex promoter
binding by T7 RNA polymerase is ≈4 nM (9) we first pre-
incubated the 50-biotinylated nontemplate strand, template
strand encoding a 24 base RNA and Strep-T7 RNA polymerase
at final equimolar concentrations, as described in the Methods
section. We then incubated the assembled promoter complex
with tetrameric Strep-TactinXT-coated magnetic beads to
form the tethered in vitro transcription system illustrated in
Figure 1.

While elevated salt concentrations weaken both promoter
binding and RNA rebinding activities of T7 RNA polymerase,
indirectly tethering the polymerase to the promoter (as demon-
strated in Fig. 1) should restore promoter binding by increasing
the local concentration of promoter DNA compared with free
RNA in solution, as observed previously using a direct tethering
approach (42). To test the hypothesis with our tethered in vitro
transcription system, we performed a comparative analysis of
transcription between tethered and untethered systems as a
function of increasing concentrations of added NaCl. In order to

Strep-T7RP

Strep Tag II®

iSp18

Biotin

M
ag

n
et

ic
B

ea
d Nontemplate

Template

Strep Tactin-XT®

Figure 1. Cross-linked transcription complex. T7 RNA polymerase con-
taining an N-terminal Strep-tag II peptide and duplex DNA labeled with
biotin at the 50 end of the nontemplate strand are bound to (tetravalent)
Strep-TactinXT coated magnetic beads.

Cotethered transcription at high salt
see the direct effect on cis primed extension activity, we selected a
template strand that encodes a 24 base RNA (RNA-24) known to
serve effectively in 30 self-extension (24). The gel analysis pre-
sented in Figure 2A shows that as added NaCl concentration is
increased from 0 M to 0.4 M in 0.1 M intervals, all transcription
activity decreases for the untethered system and is negligible at
0.4 M added NaCl. In the tethered system, promoter binding (and
transcription) is relatively resistant to increasing concentrations
of added salt, as expected. The data also confirm that product
RNA rebinding to polymerase is inhibited, as there is a dramatic
reduction in the formation of primer extension products. At
0.3 M added NaCl, most of the primer extension activity is
inhibited, leading directly to an increase in the encoded RNA
yield. At 0.4 M added NaCl, the overall yield is decreased
somewhat relative to that at 0.3 M, but the purity of the encoded
RNA is at its highest (and the concentration of the encoded RNA
is substantially higher than in the untethered control). Overall,
the tethered transcription system under high salt produces
significantly improved purity and increased yield of the correct
length product.
Synthesis of encoded RNA is not impaired by tethering

Not all encoded RNAs participate in 30 self-extension (24).
To confirm that the described tethering does not have an effect
24

Untethered Tethered

0 0.1 0.2 0.3 0.4

[NaCl] (M)

0 0.1 0.2 0.3 0.4

20

30

40

D
N

A
st

an
da

rd
s

50

E
xt

e
n
d
e
d

[NaCl] (M)

A B

Figure 2. Tethered, high-salt transcription dramatically reduces primer e
tethered complexes analyzed by 20%, 7 M urea denaturing gel electrophores
added to the standard reaction mixture are shown. B, quantification of individ
on the fundamental efficiency of promoter-directed tran-
scription, we repeated the above comparative analysis with a
template strand encoding another 24 base RNA (RNA-24Alt)
that is known not to participate substantially in 30 self-
extension (24). For the untethered system, gel analysis of the
products presented in Figure 3A (and quantified in 3B) shows
an overall loss of yield in RNA transcription with increasing
added NaCl concentration, as expected (more subtly, there is
an initial increase in 24 base RNA at 0.1 M added NaCl, which
then decreases to barely detectable levels by 0.4 M added
NaCl).

In contrast, Figure 3, A and B show that the tethered system,
while also initially showing an increase in the yield of 24 base
RNA with increasing NaCl, continues to transcribe well up to
at least 0.3 M added NaCl and produces more 24 base RNA at
0.4 M than at 0 M added NaCl. Close inspection of the gel
lanes in Figure 3A suggests that the RNA-24Alt sequence is in
fact producing primer extended products at 0 M added NaCl,
as evidenced by a broad smear above the 24 base RNA band
that decreases with increasing NaCl. Quantification of the
extended products is shown in gray at 10× magnification in
Figure 3B to better depict this observation. Thus, even for this
construct, the intensity of the 24 base RNA band un-
derestimates the total RNA produced. According to the model,
inhibition of the primer extension that produces the broad
smear would result in higher net amounts of the initially
synthesized 24 base RNA, as observed.
Generality of the system

The results presented in Figures 2 and 3 use DNAs encoding
24 base RNAs with different sequences. To further test the
generality of this system, we took the RNA-24 template
sequence introduced in Figure 2 and inserted ten bases at
position +8 to yield DNA that encodes a 34 base RNA (RNA-
34), as shown in Fig. S1. Paralleling the experiment of Figure 2,
we compare in Figure 4 untethered and tethered transcription
of RNA-34 at low (0 M) and high salt (0.4 M) added NaCl
concentrations. As predicted by the general model, the
0 M

0.1 M

0.2 M

0.3 M

0.4 M

[NaCl]

24

Extended

24

Extended

Untethered Tethered

xtension. A, salt dependence of transcription profiles for untethered and
is, labeled via incorporation of [α-32P]ATP. The final concentrations of NaCl
ual gel lanes in A.

J. Biol. Chem. (2021) 297(3) 100999 3

0 0.1 0.2 0.3 0.4

[NaCl] (M)

0 0.1 0.2 0.3 0.4

20

30

40
D

N
A

st
an

da
rd

s

50

[NaCl] (M)

A B
[NaCl]

24

Untethered Tethered Untethered Tethered

10X 0 M

0.1 M

0.2 M

0.3 M

0.4 M

4242

E
xt

e
n

d
e

d

Extended Extended

Figure 3. Transcription by tethered complexes is salt-resistant. A, salt dependence of transcription profiles for untethered and tethered complexes
encoding RNA-24Alt, analyzed as in Figure 2. B, quantification of individual lanes in A. Extended products shown in gray at 10× scale.

Cotethered transcription at high salt
tethered in vitro transcription system produces primarily the
encoded 34 base RNA at high salt. This result confirms that
the system can be used for RNA of longer lengths to generate
high yields of encoded RNA while preventing the formation of
self-extended longer RNA impurities.
Biotinylated DNA and Strep-T7 RNA polymerase bind at
nearby tetrameric sites

To encourage both enzyme and DNA to attach to the same
Strep-TactinXT tetramer in the above experiments, we pre-
incubated (at low salt) Strep-T7 RNA polymerase and bio-
tinylated promoter DNA before adding the Strep-TactinXT
magnetic beads. Following assembly, a high salt wash was used
to remove components (free polymerase and DNA) not
strongly bound to the beads. As controls, we prepared tethered
transcription complexes using DNA and T7 RNA polymerase
with only one or neither of the two modifications. The
20

30

40

50

D
N

A
st

an
da

rd
s

60

34

E
xt

e
n

d
e

d

[NaCl] (M) 0.40 0.40
TetheredUn

tet
he

red

0 M

0.4 M

0 M

0.4 M

[NaCl]

34

Extended

U
n

te
th

e
re

d
T
e

th
e

re
d

A B

Figure 4. Improvements are independent of RNA length. A, low- and
high-salt transcription profiles for tethered and untethered complexes
analyzed as in Figure 2. B, gel quantification of A.

4 J. Biol. Chem. (2021) 297(3) 100999
resulting in vitro transcription reactions with DNA encoding
RNA-24 under high-yield conditions, shown in Fig. S3,
confirm that the absence of one or both of the modifications
destroys RNA synthesis, both at low and high salt concentra-
tions, as expected.

Despite the high affinity of T7 RNA polymerase for its
promoter, and the strategic preforming of the promoter
complex before tethering to the beads, we expected that some
enzyme might couple to beads lacking nearby DNA or vice
versa. Without a partner, on well-washed beads, these species
should be inactive in transcription. To test for enzyme
immobilized without a promoter DNA partner, we challenged
an assembled and washed system encoding RNA-24Alt by
introducing in solution unmodified promoter DNA-34Alt that
encodes RNA-34Alt. At low salt, RNA polymerase without a
locally (and functionally) tethered 24-Alt DNA partner should
bind the free DNA-34Alt and synthesize a 34 base RNA. The
results in Fig. S4 demonstrate this hypothesis to be correct.
While at low salt, both 24 base and 34 base RNAs are pro-
duced at levels similar to that of untethered transcription, at
high salt, only the 24 base RNA from the tethered DNA
template is observed.

To further confirm that (RNA-34-encoding) DNA-34 is
binding to and reacting with enzyme lacking a DNA partner,
we increased the ratio of labeled 24 base encoding DNA-24 to
protein from 1:1 to 2:1, followed by a high-salt wash to remove
all unbound DNA. The results shown in Fig. S5 reveal an
approximately twofold increase in the overall RNA-24Alt
production for the 2:1 prep compared with the 1:1 prep,
while RNA-34Alt production decreases only slightly. This
suggests that under these conditions, a significant portion of
enzyme bound to beads may not have a functionally tethered
DNA nearby. Alternatively, biotinylated DNA-24 could be
binding to an empty binding site in the tetramer, further
increasing the DNA concentration near tethered polymerase.
Future development in this system will focus on optimizing
binding capacity.

Cotethered transcription at high salt
Generally speaking, since promoter DNA and product RNA
are in competition for binding to the polymerase active site,
excess RNA polymerase (relative to the DNA) should always
be avoided, as there will necessarily be free enzyme available to
bind and extend RNA in solution. While one might argue that
excess DNA is to be preferred, DNA can be resource limiting.
In any case, a tethered 1:1 complex should provide the highest
efficiency (relative to either DNA or polymerase), while at the
same time proximity tethering of the DNA helps it compete
with RNA for rebinding to the enzyme.
The system is stable and reusable

In this system, Strep-T7 RNA polymerase and promoter
nontemplate DNA are immobilized on Strep-TactinXT mag-
netic beads. This allows that this bead-immobilized tran-
scription complex could be reutilized for multiple rounds of
transcription. To test this, we carried out three rounds of
transcription using the above bead tethered transcription
complex, as described in further detail in the Methods section
and illustrated as a scheme in Figure 5A. Results in Figure 5B
show that there is a significant loss in overall transcription
after each round. We hypothesized that this could be due to
washing off of the template DNA with each round, as template
DNA is only bound to the tethered system via the strong
promoter contacts. Although promoter binding is tight, off
rates of the enzyme are fast (51–53). This suggests that with
each round of transcription, it is not unreasonable to expect
some template DNA to wash off of the tethered system if
promoter contacts are lost during the off states.

With the observation that there is significant loss in overall
transcription at each round, we hypothesized that strength-
ening promoter contacts might allow greater persistence of the
complex on washing. In the two domain model of the 17-base
pair consensus T7 promoter, the duplex recognition element
(responsible for binding) stretches from position −17 upstream
to position −5, while the AT rich melting region (required for
optimal catalysis) is situated between positions −4 and −1 (54).
0.4 0.40 0

Use 1 Use 2

+ Tx Buffer
+ NTPs

React
2 h

37 °C

Gel
analysis

supernatant

heat to 95 °C

beads BA pss[+2]

20

30

40

50

D
N

A
st

an
da

rd
s

Figure 5. The tethered system can be reused. A, reusability experimental or
new substrate NTP solution was added to initiate a 2 h “Use two” reaction (a
acrylamide denaturing urea gel stained with SYBR Gold Nucleic Acid Gel Stain.
transcription of RNA-24 using pss[+2], analyzed as in Figure 2. C, fifteen percen
Gel stain. Transcription profiles at low (0 M) and high (0.4 M) added NaCl for
Part of the binding energy from the strong duplex binding
interactions upstream of (and including) position −5 is used to
institute conformational change in the downstream DNA,
essentially predicted to melt bases from position −4 to
about +3 (9, 55). As a result, the enzyme binds a partially
single-stranded promoter DNA that has no bases downstream
of position −5 in the nontemplate strand (referred to here as
pss[−5]) at least four times tighter than it binds double-
stranded (or pss[+2], as used here) promoter (9, 55). With
significantly slower off rates (51–53) and strengthened pro-
moter binding, use of the pss[−5] promoter in the tethered
system should reduce loss by washing and result in greater
overall retention of transcription activity after each round of
transcription.

Comparison of the results in Figure 5C with those in
Figure 5B confirms that use of the pss[−5] construct signifi-
cantly increases the reusability of the tethered system, at both
low and high salt concentrations. The gel analysis compares
the products from each reaction cycle, using the template
encoding RNA-24, under both low (0 M) and high (0.4 M)
added salt conditions, and nontemplate DNA pss[+2] and pss
[−5] respectively. The overall transcription yield decreases with
each cycle using pss[+2] and is nearly lost by round 3. In
contrast, using pss[−5] DNA, the overall transcription yield at
each added salt concentration remains reasonably constant
through three rounds of washing and reuse. The losses asso-
ciated with the pss[+2] reuse point out that yields will likely
depend on the extent of washing of the system. The pss[−5]
system appears more resistant to such losses and so can be
used at least three times, with essentially no loss in yield, by
simply removing the product and adding transcription buffer
with fresh NTPs.
Discussion

Transcription in vitro by T7 RNA polymerase is a long-
established method to synthesize RNAs of diverse lengths
and sequences, due to the promoter specificity and robust
0.4 0.40 0 [NaCl] (M) 0.40

Use 1 Use 2 Use 3
[NaCl] (M) 0.40

Use 3

24

C

E
xt

en
de

d

24

E
xt

en
de

d

pss[-5]

20

30

40

50

D
N

A
st

an
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rd
s

der scheme. After 2 h of “Use 1,” the reaction solution was removed, and a
nd repeated to initiate a 2 h “Use three” reaction). B, fifteen percent poly-
Transcription profiles at low (0 M) and high (0.4 M) added NaCl for tethered
t polyacrylamide denaturing urea gel, stained with SYBR Gold Nucleic Acid
tethered transcription of RNA-24 using pss[−5].

J. Biol. Chem. (2021) 297(3) 100999 5

Cotethered transcription at high salt
nature of this system (6). Over the past 30 years, researchers
have noted nonpromoter-driven activities of this enzyme that
contaminate the product pool with other than encoded RNA
products, often termed incorrectly as nontemplated additions
(6, 12–14). The quest for high-yield RNA synthesis only ex-
acerbates the production of undesired, longer products (24).
As high concentrations of encoded RNA accumulate, T7 RNA
polymerase binds RNA at its 30 end and self-extends via cis
primed extension, now independent of the promoter.

High-yield RNA synthesis efforts to date have focused on a
two-step approach: 1) high-yield transcription using T7 RNA
polymerase, followed by 2) (sometimes extensive) purification
of the encoded RNA using gel or chromatic purification
methods. High-yield conditions drive primer extension,
resulting in the correct length RNA product being converted
into heterogeneous, longer than desired double-stranded RNA
contaminants. The very nature of high-yield reactions leads
directly to longer, double-stranded impurities and reduces the
encoded RNA yield. This process is sequence-dependent, and
in cases where the encoded RNA product does not signifi-
cantly participate in cis primed self-extension, gel electro-
phoretic or chromatographic purification methods may be
adequate to address the purity problem. However, each puri-
fication step generally reduces overall product yield. Moreover,
electrophoretic or chromatographic purification approaches
have highest success for relatively short RNAs. Preparative
purification of longer RNA (e.g., separating an encoded 300
base RNA from products extended by 20–30 bases) is often
difficult, if not impossible. With increased emphasis on
mRNAs many kilobases in length, the problem becomes
increasingly difficult.

The goal then is to prevent the rebinding of the synthesized
RNA from the outset, to then prevent the RNA-primed self-
extension that leads to longer impurities. Noting that high
ionic strength inhibits all polymerase–nucleic acid in-
teractions, we introduce here a novel in vitro transcription
method, transcribing at high salt to limit RNA product
rebinding. To restore promoter binding and initiation of
transcription, we cotether promoter DNA and T7 RNA poly-
merase to beads. Specifically, we cotether Strep-T7 RNA po-
lymerase and biotinylated promoter DNA to Strep-TactinXT
beads, bringing the enzyme in close proximity to its promoter
to restore promoter binding, even at high ionic strength. This
results in overall higher yields of the desired RNA at greater
initial purity, reducing the need for subsequent (and low yield)
purification steps.
Tethering plus salt leads to improved transcription purity and
yield

Increasing salt concentrations leads to complete inhibition
of all enzymatic activity in the untethered system, as shown in
Figures 2–4. In the tethered system, high salt inhibits only the
undesired cis-primed extension activity of T7 RNA polymer-
ase, while allowing promoter-directed transcription. This re-
flects as a dramatic decrease of the longer, double-stranded
impurities. Since production of the longer double-stranded
6 J. Biol. Chem. (2021) 297(3) 100999
products derives from and therefore consumes encoded-
length RNAs, tethered system recovers higher yields of the
desired (directly encoded) product. As expected, at sufficiently
high concentrations of added salt, promoter-driven tran-
scription begins to be inhibited, even for the tethered system.
For these constructs and these conditions, a practical optimum
of about …

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