Bioengineered

ISSN: 2165-5979 (Print) 2165-5987 (Online) Journal homepage: http://www.tandfonline.com/loi/kbie20

Polyvinylsulfonic acid: A Low-cost RNase inhibitor

for enhanced RNA preservation and cell-free
protein translation

Conner C. Earl, Mark T. Smith, Richard A. Lease & Bradley C. Bundy

To cite this article: Conner C. Earl, Mark T. Smith, Richard A. Lease & Bradley C. Bundy (2018)
Polyvinylsulfonic acid: A Low-cost RNase inhibitor for enhanced RNA preservation and cell-free
protein translation, Bioengineered, 9:1, 90-97, DOI: 10.1080/21655979.2017.1313648

To link to this article: https://doi.org/10.1080/21655979.2017.1313648

© 2018 The Author(s). Published with

license by Taylor & Francis© Conner C. Earl,
Mark T. Smith, Richard A. Lease, and Bradley
C. Bundy

Published online: 29 Jun 2017.

Submit your article to this journal

Article views: 841

View Crossmark data

Citing articles: 1 View citing articles

Full Terms & Conditions of access and use can be found at

http://www.tandfonline.com/action/journalInformation?journalCode=kbie20
BIOENGINEERED
2018, VOL. 9, NO. 1, 90–97
https://doi.org/10.1080/21655979.2017.1313648

RESEARCH PAPER

Polyvinylsulfonic acid: A Low-cost RNase inhibitor for enhanced RNA

preservation and cell-free protein translation
a
Conner C. Earl , Mark T. Smitha, Richard A. Lease b
, and Bradley C. Bundy a

a
Department of Chemical Engineering, Brigham Young University, Provo, UT, USA; bDepartment of Chemical and Biomolecular Engineering, The
Ohio State University, Columbus OH USA

ABSTRACT ARTICLE HISTORY

The effectiveness and economics of polyvinyl sulfonic acid (PVSA) as a ribonuclease inhibitor for in Received 3 February 2017
vitro systems is reported. PVSA was shown to inhibit RNA cleavage in the presence of RNase A as Revised 15 March 2017
well as in the presence of Escherichia coli lysate, suggesting that PVSA can act as a broader Accepted 21 March 2017
ribonuclease inhibitor. In addition, PVSA was shown to improve the integrity of mRNA transcripts by KEYWORDS
up to 5-fold in vitro as measured by their translational viability. Improved preservation of mRNA in vitro transcription and
transcripts in the presence of PVSA under common RNA storage conditions is also reported. A cost translation; nuclease
comparison with commercially available RNAse inhibitors indicates the economic practicality of inhibition; polyvinyl sulfonic
PVSA which is approximately 1,700 times less expensive than commonly used ribonuclease acid; RNase inhibitor; RNA
inhibitors. PVSA can also be separated from RNA by alcohol precipitation for applications that may storage
be sensitive to the presence of PVSA.

Introduction
(DEPC), which is effective for ribonuclease inhibition.9,10
RNA plays a vital role in myriad biologic processes One issue with this solution, however, is that DEPC and
including protein translation, gene regulation, and other similar chemicals are known carcinogens and
gene expression. Beyond its natural functions, RNA require caution and training for their use. These chemi-
has been engineered for diverse applications including cals also react quite readily with amine, thiol, and alcohol
therapeutics development, medical diagnostics, and groups and cannot be used in many biologic experiments
protein engineering.1-4 In particular, messenger RNA where buffers and biologic reagents being used and pro-
(mRNA) is naturally prone to rapid degradation by duced often contain these side groups. DEPC can also
ubiquitous Ribonucleases (RNases) as its degradation alkylate RNA which renders it unusable for some applica-
is essential to the regulation of protein expression.5 tions.11 Biologically produced RNase inhibitors may also
RNA degradation is a major challenge for in vitro effectively inhibit ribonucleases, but their action is often
applications such as cell-free protein synthesis (CFPS), specific to certain types of ribonucleases and they are
reverse transcription polymerase chain reaction (RT- often very expensive.9,12,13
PCR), quantitative RT-PCR (qRT-PCR), RNA-Seq and One promising solution to some of these challenges
Northern Blot analysis, all of which rely on RNA integ- is the use of inexpensive chemical (non-biologic) RNase
rity and purity.5-8 Maintaining the integrity of RNA inhibitors. Utilizing anionic polymers as a tool for
molecules during storage is also a challenge, as com- RNase A inhibition is one chemical method that was
plete removal/inactivation of RNases is difficult with- initially tested over 50 years ago.14,15 More recently, it
out damaging or denaturing the RNA sample or using was reported that polyvinyl sulfonic acid (PVSA; aver-
toxic chemicals such as phenol and chloroform. age MW »2–5 kDa), a negatively charged polymer
Techniques to mitigate RNA degradation in vitro have with sulfate branches, is a potent inhibitor of RNase
a long history. One prominent solution is the pretreat- A16. The repeating sulfate units resemble the repeating
ment of samples and solutions with diethylpyrocarbonate phosphate units that form the backbone of RNA and

CONTACT Bradley C. Bundy [email protected] Brigham Young University, Department of Chemical Engineering, 350S Clyde Building, Provo, Utah 84602.
© 2018 Conner C. Earl, Mark T. Smith, Richard A. Lease, and Bradley C. Bundy. Published with license by Taylor & Francis
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
 BIOENGINEERED 91

are thought to form competitive coulombic interactions

with RNase A, thereby occupying its RNA-binding sites
and effectively inhibiting RNase A.16,17
Here we describe experiments performed to assess
the viability of PVSA beyond RNase A, as an inexpen-
sive, safe, and effective inhibitor against bacterial
RNases. We examine PVSA’s effects in RNA stabiliza-
tion in common in vitro applications, such as in vitro
transcription (IVT) and coupled and decoupled in
vitro transcription and translation. We further analyze
the economic viability of using this polymeric RNase
inhibitor. Our results suggest that certain applications,
particularly RNA storage and in vitro transcription,
can benefit from low-cost RNase inhibition through
the use of PVSA. Figure 1. Inhibition of RNase Activity with PVSA. The relative
RNase Activity of both RNase A and E. coli lysate was measured at
varying concentrations of PVSA using RNaseAlertÒ (Ambion). The
Results amount of PVSA required for 50% inhibition (IC50, inset) was deter-
mined from normalized data fit to a reciprocal semi-log response
PVSA-mediated inhibition of RNase activity in curve (n D 3, error bars represent 1 standard deviation).
bacterial lysate evident (IC50 value of 1.03 mg/mL) and essentially
To examine the RNase inhibitory potency of PVSA, we no protein synthesis was observed at 10 mg/mL
measured the ribonuclease activity of RNase A and E. coli PVSA.
lysate in the presence of PVSA. The assays were per-
formed using Ambion’s RNaseAlertÒ assay kit (IDT, IA, Decoupled in vitro transcription and translation
USA). Inhibition of RNase A (0.75 nM) was examined
with increasing concentrations of PVSA (0.001 mg/mL – To determine the basis of PVSA inhibition in the
50 mg/mL). Consistent with a previous report,16 PVSA CFPS system, the processes of mRNA transcription
effectively inhibited RNase A (Fig. 1; IC50 of 0.15 mg/mL and translation were decoupled (Fig. 3A). mRNA
PVSA with greater than 95% inhibition occurring at con- encoding GFP for subsequent translation was pre-
centrations greater than 13 mg/mL of PVSA). We also pared in the presence of PVSA at varying concentra-
tested the inhibition potency of PVSA against a bacterial tions by in vitro transcription (IVT) using the same
lysate from E. coli, which contains diverse bacterial ribo- plasmid (pY71-sfGFP) and RNA polymerase (T7
nucleases. Many of these intrinsic ribonucleases are RNA polymerase) used in the coupled results above.
known to escape inhibition from biologic RNase A inhib- An aliquot of these reactions was purified by precipita-
itors.18-20 The results showed significant inhibition of the tion with isopropanol, and the resuspended mRNA
lysate’s combined RNase activities with an IC50 of was assessed for storage stability and retained func-
0.43 mg/mL PVSA (Fig. 1). tion. Gel electrophoresis suggests IVT reaction prod-
ucts stored for 7 d with 5 mg/mL PVSA had
approximately 2 to 4 times the amount of mRNA as
Coupled in vitro transcription and translation
those without PVSA.
Next, PVSA’s inhibitory capacities were explored To determine whether the mRNA transcripts tran-
in an E. coli-based cell-free protein synthesis scribed in the presence of PVSA were viable for trans-
(CFPS) reaction where transcription and transla- lation, equal-volume aliquots of mRNA produced and
tion were coupled. We introduced PVSA at vary- purified from PVSA-treated and untreated IVT reac-
ing concentrations to the coupled in vitro reaction tions were introduced into an E. coli-based cell-free
and measured the total green fluorescent protein translation system. The yield of GFP translated from
(GFP) synthesis by its fluorescence (Fig. 2). As the mRNA transcripts were normalized to the yield of
increasing concentrations of PVSA were added, a GFP from mRNA produced without PVSA (Fig. 3C;
strong inhibitory effect on protein synthesis was Day 0, 0 mg/mL PVSA). This normalization facilitates
92 C. C. EARL ET AL.

comparison of relative protein yields as a response to inhibitor. The use of PVSA at 10 mg/mL for protec-
increasing PVSA concentration and storage time. tion of mRNA during IVT is over 1,700 times less
The mRNA produced with PVSA had significantly costly than using the manufacturer’s recommended
better protein translation yields in the decoupled concentration for either the Murine RNase inhibitorÒ
CFPS reaction than mRNA produced without PVSA. (NEB) or the Recombinant RNase inhibitor (Takara).
The protein yield from mRNA transcribed with For reference, using 10 mg/mL PVSA in place of the
10 mg/mL PVSA resulted in a greater than 500% manufacturer’s recommended concentration of
increase in protein translation when the mRNA was Murine RNase inhibitorÒ or Recombinant RNase
directly translated following transcription and purifi- inhibitor reduces the cost of IVT reagents by more
cation (Fig. 3C). Storage of mRNA for 7 d at ¡20 C than 95%. Further, we compared the costs per mass of
did result in a decrease in the protein synthesis levels; protein produced in a decoupled protein transcription
however, including 10 g/mL PVSA resulted in a and translation system with adding 10 mg/mL PVSA
2,000% increase in protein production relative to and without adding PVSA during transcription
mRNA transcribed without PVSA. These results sug- (Table 1). Using PVSA proved to be significantly less
gest PVSA does not inhibit T7 RNA polymerase- expensive (approximately 6-fold) on a cost basis per
mediated transcription, but likely inhibits a translation mg of protein produced. Estimated costs per mg pro-
mechanism in the coupled transcription and transla- tein with and without PVSA were $13 and $86 respec-
tion system. Pretreatment of the IVT with PVSA and tively (Table 1) as calculated using in vitro
subsequent isopropanol precipitation does not appear transcription/translation reagent costs published
to inhibit the RNA-programmed CFPS reaction and previously.21
improves both mRNA and protein yield from the
programmed lysate. Thus, PVSA appears to not co-
precipitate with the RNA at significant concentrations Discussion
during isopropanol precipitation and can be efficiently Coupled and decoupled protein synthesis
separated in this manner.
Biological protein-based RNase inhibitors typically
evolved to target relatively small groups of RNases.12
Economic viability
For example, commercially available murine RNase
We performed an economic analysis to determine the inhibitor is known to specifically inhibit RNases A, B,
relative cost-effectiveness of PVSA as a ribonuclease and C, however it does not inhibit RNases T1 and

Figure 2. Inhibitory Effects of PVSA on Coupled in vitro Transcription and Translation Reactions. Varying concentrations of PVSA were
added to an E. coli-based cell-free coupled transcription and translation system and GFP production yield is shown relative to production
without PVSA. The amount of PVSA required for 50% inhibition (IC50, inset) was determined from normalized data fit to a reciprocal
semi-log response curve (n D 3, error bars represent 1 standard deviation).
 BIOENGINEERED 93

Figure 3. PVSA Effect on Decoupled in vitro Transcription with Subsequent Translation. (A) A schematic illustrates in vitro transcription
(IVT) and subsequent purification with isopropanol precipitation and in vitro translation. (B) Image of mRNA product from IVT after aga-
rose gel electrophoresis and staining with ethidium bromide. Lane 1 is the nucleic acid marker of double stranded DNA with bands cor-
responding to 400, 500, 600, 700, and 800 base pairs from bottom to top. Lane 2 is the IVT product where no PVSA was added. Lane 3
is the IVT product with 5 mg/mL PVSA. The expected migration location for the 898 nucleotide long mRNA is shown by arrow and corre-
sponds to »600 base pairs of double stranded DNA due to the mRNA’s single stranded and only partially hybridized nature. (C) Relative
GFP protein yields as translated with mRNA produced by IVT in the presence of 0, 5, or 10 mg/mL PVSA and after 0 or 7 d of storage
(n D 6, error bars represent one standard deviation).

H20. Thus multiple and diverse RNase inhibitors may of RNase A activity, its activity against other RNases
be required for protection from multiple sources of has not been previously reported.14,16 We have dem-
RNases. Non-biologic inhibitors offer alternatives that onstrated that PVSA has inhibitory properties against
have broader potential due to the lowered specificity RNase A as well as RNases in bacterial lysates (Fig. 1).
of inhibitory interactions and reduced costs (Table 1). Although this evidence is not exhaustive, the activity
Although PVSA has been reported to be an inhibitor of PVSA against a spectrum of RNases suggests that
94 C. C. EARL ET AL.

Table 1. Economic Analysis of Decoupled Protein Synthesis with of mRNA (about 35 kDa in this work) permits it to
PVSA. solubilize effectively in alcohol. It is also thought that
Decoupled Protein Synthesis the charge to mass ratio of PVSA, about 2.5 times
Protein Yield Estimated Cost/ greater than that of RNA also plays a role in permit-
(mg/mL)a mg Protein ting solubilization. These unique qualities that facili-
With PVSA 0.409 C/¡ 0.095 $13 tate PVSA separation by alcohol precipitation could
Without PVSA 0.062 C/¡ 0.009 $86 potentially be exploited to separate PVSA by ion
a
Protein Yield C/¡ Standard Deviation (nD6) exchange chromatography which is commonly used
to purify nucleic acids. This evidence supports the
its mechanism of inhibition allows it to be a potent judicious use of PVSA in IVT systems, where follow-
inhibitor of several biologic ribonucleases with diverse on enzymatic treatment of RNA after production (e.g.,
catalytic mechanisms, substrate sequences and active translation, end-labeling or ligation) could be per-
sites. formed after removal of PVSA, but is contraindicated
The idea that PVSA can be an inhibitor of RNase A in those systems that rely on translation or other
has previously been demonstrated, however, its appli- RNA-binding proteins.
cations for in vitro biologic systems were unclear.16
An increase of mRNA yield from IVT with PVSA and RNA storage
the resulting mRNA’s viability for in vitro translation
after isopropanol precipitation was demonstrated in A comparison of relative yields of protein at different
this work. As mentioned, however, a limitation of time points (Fig. 3) suggests that addition of PVSA
PVSA use was shown in its inhibition of a coupled can enhance preservation of viable mRNA transcript
cell-free protein synthesis reaction. Such a deleterious under ¡20 C storage conditions. Currently, one com-
effect could be explained by PVSA interfering with mon method of storing RNA involves solubilizing
one or more mechanisms of translation. This is espe- RNA in water treated with diethylpyrocarbonate
cially likely considering PVSA’s hypothesized mecha- (DEPC).24 However, as discussed previously, DEPC
nism of inhibition where PVSA’s polyanionic nature and similar chemicals are known carcinogens, are
in solution causes it to resemble a ribonucleic acid and unable to treat certain buffers that may contain amine,
competitively bind to RNases.16 As more PVSA binds thiol, or alcohol groups and cannot be directly added
with the ribonuclease, less RNA would be degraded, to a reaction solution without side-reactions,10 includ-
which allows for a higher relative yield of mRNA. ing modification of RNA.11 Results from our
However, the RNA-mimicking structure of PVSA decoupled translation system demonstrated that for
could also inhibit the ribosome and other RNA-bind- samples containing no PVSA, relative yields at 2 time
ing proteins in addition to RNases. Interestingly, points (days 0 and 7) were similar, indicating similar
PVSA does not inhibit T7 RNA polymerase during RNA recovery, with significantly better RNA recovery
IVT, suggesting that the inhibitory properties of at concentrations of 5–10 mg/mL of PVSA. Such evi-
PVSA are not generalizable to all nucleic acid-protein dence suggests PVSA may act as a potent inhibitor of
interactions and PVSA does not likely mimic double- ribonucleases to protect mRNA degradation in storage
stranded DNA. It is worth noting that another chemi- conditions.
cal RNase inhibitor, aurin tricarboxylic acid (ATA)
inhibits interactions between RNA polymerase and its Materials and methods
DNA template.22 Further, ATA binds both RNA and
Inhibition of RNases
DNA, can partition into the insoluble fraction in an
alcohol precipitation, and can be only partially Ambion’s RNaseAlertÒ kit (Catalog number:
removed by size-exclusion chromatography.23 There- AM1964) comprising a double-labeled oligoribonu-
fore, PVSA occupies a different niche from ATA in cleotide with both fluorescent and quenching moie-
applications of RNase inhibitors as PVSA appears to ties was used as a substrate for treatment with
be readily separable from nucleic acids by alcohol pre- RNases in the presence or absence of Polyvinyl Sul-
cipitation. It is thought that the relatively small molec- fonic Acid (PVSA; average MW »2–5 kDa, Sigma
ular weight of PVSA (2 to 5 kDa) compared with that Aldrich, Catalog Number: 278424) as a
 BIOENGINEERED 95

ribonuclease inhibitor. Cleavage of this RNA deriv- The 550 mL IVT reactions were templated using the
ative substrate molecule separates the fluorophore 50 mg/mL pY71-sfGFP plasmid 29 and incubated for
from the quenching moiety and enables detection 90 minutes at 37 C with vigorous agitation (200 rpm).
of fluorescence, as monitored with a BiotekÒ Syn- Separate aliquots were then purified and resuspended
ergy MX plate reader spectrophotometer in in 55 mL RNase free water using an isopropanol pre-
fluorimetry mode using CorningÒ 96 well black cipitation procedure 30 or placed in storage (¡20 C,
plates. Excitation and emission peaks were mea- 7 days) until precipitation. Aliquots of the IVT
sured at 480/520 nm respectively. Relative activity reaction after 7 d of storage and after purification by
for both RNase A and RNase activity in bacterial isopropanol precipitation were imaged with electro-
lysate were measured at varying concentrations of phoreses on a 1% agarose gel stained with ethidium
PVSA. The data were normalized and fit to a recip- bromide using a Tris-Acetate-EDTA (TAE) buffer
rocal semi-log response curve to determine IC50 (166 V constant, 1 hour). 1Kb Plus DNA Ladder (Invi-
values (concentration of inhibitor required to trogen) was used as a nucleic acid reference. The gel
inhibit the reaction by 50%). was then imaged using a Fluor-S BioRad Multiimager
Bacterial lysate was prepared as described previ- with UV filter to visualize mRNA bands (898 nt).
ously25-27 using the Escherichia coli B strain BL21 Densitometry was performed to estimate the relative
StarTM (DE3) (Life Technologies C601003; BL21 amount of mRNA.
(DE3) StarTM is rne131 F¡ ompT gal dcm lon Analysis of reagent costs were determined with the
hsdSB(rB¡mB¡) λ(DE3 [lacI lacUV5-T7p07 ind1 sam7 prices listed by Sigma Aldrich, Bachem, NEB, and
nin5]) [malBC]K-12 (λS)). The bacterial cells were Takara and calculated using other in vitro transcrip-
grown, harvested, and lysed using an Avestin tion/translation reagent costs published previously.21
EmulsiFlexÒ B-15 Homogenizer with 3 passes at In comparing the cost of using Murine RNase inhib-
21,000 psi. The lysate was clarified by centrifugation itorÒ (NEB) or Recombinant RNase inhibitor
at 16,000 g, 4 C for 30 min and the supernatant was (Takara), the manufacturers’ recommendation of
collected, aliquotted, flash frozen, and stored at 1,000 unit/mL for each was applied.
¡80 C until use. The clarified lysate was then added
to the RNaseAlertÒ reaction at a 0.1% (v/v) concentra- Decoupled in vitro transcription and translation
tion. Ribonuclease A (Sigma Aldrich, Catalog Num-
The mRNA transcripts, produced by IVT and purified
ber: R6513) was added to 0.75 nM.
by precipitation as described above, were then added
in equal volume (15 mL) aliquots to an E. coli-based
In vitro transcription (IVT) cell-free translation PANOxSP system (150 mL final
volume). The cell-free reactions were then performed
In vitro Transcription (IVT) reactions were pre-
as described previously with the exception that plas-
pared with varying concentrations of PVSA. Reac-
mid DNA was not added to the reaction.25-27 Relative
tions were prepared with nucleotide triphosphate
GFP protein yields were determined using a Biotek
mixture containing 2 mM each ATP, GTP, CTP,
Synergy MX plate reader spectrophotometer in
and UTP (Sigma Aldrich catalog numbers A2383,
fluorimetry mode to measure emission at 510 nm
G8877, 30320, 94370, respectively), T7 RNA poly-
(excitation at 485 nm). Excitation data were normal-
merase buffer containing 50 mM Tris-Cl (Sigma
ized to the sample with no PVSA added (Day 0 sample
Aldrich RES3098T-B7) at a pH of 7.5, 15 mM
with 0 mg/mL PVSA).
MgCl2 (as hexahydrate; Sigma Aldrich 1374248),
5 mM dithiothreitol (Bachem USA, Q-1225), and
Coupled in vitro transcription and translation (cell-
2 mM spermidine (Sigma Aldrich S2626). A mix-
free protein synthesis)
ture of 3 mM spermidine and 2 mM putrecine
(Sigma Aldrich P5780) was also used in the reac- E. coli-based cell-free protein synthesis reactions based
tion. T7 RNA polymerase enzyme was prepared as on the PANOxSP were performed as described previ-
described previously28 and added at 2% v/v. PVSA ously25-27 and using the same reagents used in the
was added at concentrations of 0, 5, and 10 mg/ decoupled in vitro translation system described above.
mL. As an exception the pY71-sfGFP plasmid, which is
96 C. C. EARL ET AL.

transcribed by T7 RNA polymerase in concert with References

translation, was added instead of adding mRNA tran-
[1] Kreiter S, Diken M, Pascolo S, Nair SK, Thielemans KM,
scribed by T7 RNA polymerase from pY71-sfGFP in a Geall A. RNA vaccination therapy: Advances in an emerg-
prior IVT reaction. The coupled CFPS reaction with- ing field. J Immunol Res 2016; 2016:9703914;
out PVSA was compared with the coupled system PMID:27019856; https://doi.org/10.1155/2016/9703914
where PVSA was added at varying concentrations [2] Sullenger BA, Gilboa E. Emerging clinical applications of
(0.0001–100 mg/ml) and the yield was normalized to RNA. Nature 2002; 418:252-8; PMID:12110902; https://
doi.org/10.1038/418252a
GFP production without PVSA. Protein production
[3] Tao H, Yang JJ, Zhou X, Deng ZY, Shi KH, Li J.
yields of GFP were determined with fluorescence as Emerging role of long noncoding RNAs in lung can-
described above. cer: Current status and future prospects. Respir Med
2016; 110:12-9; PMID:26603340; https://doi.org/
Conclusion 10.1016/j.rmed.2015.10.006
[4] Spencer JD, Schwaderer AL, Eichler T, Wang H, Kline J,
Here we demonstrate the potential of PVSA as a Justice SS, Cohen DM, Hains DS. An endogenous ribo-
low cost chemical alternative to biologic RNase nuclease inhibitor regulates the antimicrobial activity of
inhibitors and present data suggesting its ability to ribonuclease 7 in the human urinary tract. Kidney Int
2014; 85:1179-91; PMID:24107847; https://doi.org/
inhibit RNases beyond RNase A. While PVSA is
10.1038/ki.2013.395
1,700-fold less expensive and was shown to suc- [5] Condon C. Maturation and degradation of RNA in bacteria.
cessfully preserve mRNA, PVSA appears to inhibit Curr Opin Microbiol 2007; 10:271-8; PMID:17560162;
mechanisms that involve RNA binding such as https://doi.org/10.1016/j.mib.2007.05.008
translation. To overcome this limitation, the suc- [6] Wang Z, Yang B. MicroRNA expression detection meth-
cessful removal of PVSA using a simple alcohol ods. Springer Berlin, Heidelberg, Germany, 2010.
[7] Ranji A, Wu JC, Bundy BC, Jewett MC. Transforming
precipitation mechanism is reported. It is impor-
synthetic biology with cell-free systems. Elsevier,
tant to note that this is a proof-of-concept study Waltham, MA 2013.
demonstrating the potential of the chemical RNase [8] Smith MT, Wilding KM, Hunt JM, Bennett AM, Bundy
inhibitor PVSA, and more head-to-head studies BC. The emerging age of cell-free synthetic biology. FEBS
comparing PVSA directly with biologic RNase Lett 2014; 588:2755-61; PMID:24931378; https://doi.org/
inhibitors is needed before widespread use. How- 10.1016/j.febslet.2014.05.062
[9] Yakovlev G, Mitkevich V, Makarov A. Ribonuclease
ever, here we further report the potential of chemi-
inhibitors. Mol Biol 2006; 40:867-74; https://doi.org/
cal inhibitors such as PVSA to drastically decrease 10.1134/S0026893306060045
the costs and complexity of preserving RNA in an [10] Wolf B, Lesnaw JA, Reichmann ME. A mechanism of the
RNase ubiquitous world. irreversible inactivation of bovine pancreatic ribonucle-
ase by diethylpyrocarbonate. Euro J Biochem 1970;
Disclosure of potential conflicts of interest 13:519-25; PMID:5444158; https://doi.org/10.1111/
j.1432-1033.1970.tb00955.x
No potential conflicts of interest were disclosed. [11] Nilsen TW. Chemical modification interference. Cold
Spring Harb Protoc 2015; 2015:599-603;
Funding PMID:26034302
[12] Lomax JE, Bianchetti CM, Chang A, Phillips GN, Fox
The authors would like to acknowledge our funding sources: BG, Raines RT. Functional evolution of ribonuclease
DARPA Young Faculty Award (#D13AP000037) and National inhibitor: insights from birds and reptiles. J Mol Biol
Science Foundation CBET Division CAREER Award 2014; 426:3041-56; PMID:24941155; https://doi.org/
(#1254148). Any opinions, findings, and conclusions or recom- 10.1016/j.jmb.2014.06.007
mendations expressed in this material are those of the authors [13] Johnson RJ, McCoy JG, Bingman CA, Phillips GN, Jr,
and do not necessarily reflect the views of the National Science Raines RT. Inhibition of human pancreatic ribonuclease
Foundation or DARPA. by the human ribonuclease inhibitor protein. J Mol Biol
2007; 368:434-49; PMID:17350650; https://doi.org/
ORCID 10.1016/j.jmb.2007.02.005
[14] Fellig J, Wiley CE. The inhibition of pancreatic ribonucle-
Conner C. Earl http://orcid.org/0000-0002-7916-3165 ase by anionic polymers. Arch Biochem Biophys 1959;
Richard A. Lease http://orcid.org/0000-0002-9242-0023 85:313-6; PMID:13822112; https://doi.org/10.1016/0003-
Bradley C. Bundy http://orcid.org/0000-0003-4438-183X 9861(59)90496-5
 BIOENGINEERED 97

[15] Littauer UZ, Sela M. An ultracentrifugal study of the effi- the isolation of RNA from animal tissues. Biochem J
ciency of some macromolecular inhibitors of ribonucle- 1989; 263:73-80; PMID:2481441; https://doi.org/10.1042/
ase. Biochimica et Biophysica Acta (BBA)-Specialized bj2630073
Section on Nucleic Acids Related Subjects 1962; 61:609- [24] Hemmerling J, Jansen J, M€ uller M, Haller D. Fetal gut
11; https://doi.org/10.1016/0926-6550(62)90114-7 laser microdissection in combination with RNA pream-
[16] Smith BD, Soellner MB, Raines RT. Potent inhibition of plification enables epithelial-specific transcriptional
ribonuclease A by oligo (vinylsulfonic acid). J Biol Chem profiling. J Immunol Methods 2015; 416:189-92;
2003; 278:20934-8; PMID:12649287; https://doi.org/ PMID:25462537; https://doi.org/10.1016/j.jim.2014.11.
10.1074/jbc.M301852200 008
[17] Raines RT, Smith BD, Soellner MB, Lynn DM. (2013) [25] Smith MT, Bennett AM, Hunt JM, Bundy BC. Creating a
Nuclease inhibitors and methods for their use. US Patent completely “cell-free” system for protein synthesis. Bio-
# US 20130344563 A1. technol Prog 2015; 31:1716-9; PMID:26289032; https://
[18] Regonesi ME, Del Favero M, Basilico F, Briani F, Benazzi doi.org/10.1002/btpr.2157
L, Tortora P, Mauri P, Deh
o G. Analysis of the Escheri- [26] Salehi AS, Earl CC, Muhlestein C, Bundy BC. Escherichia
chia coli RNA degradosome composition by a proteomic coli-based cell-free extract development for protein-based
approach. Biochimie 2006; 88:151-61; PMID:16139413; cancer therapeutic production. Int J Dev Biol 2016;
https://doi.org/10.1016/j.biochi.2005.07.012 60:237-43; PMID:27251070; https://doi.org/10.1387/ijdb.
[19] Deutscher MP. Maturation and degradation of ribosomal 160125bb
RNA in bacteria. Prog Mol Biol Transl Sci 2009; 85:369- [27] Salehi AS, Smith MT, Bennett AM, Williams JB, Pitt
91; PMID:19215777 WG, Bundy BC. Cell-free protein synthesis of a cyto-
[20] Kim BM, Schultz LW, Raines RT. Variants of ribonucle- toxic cancer therapeutic: Onconase production and a
ase inhibitor that resist oxidation. Protein Sci 1999; just-add-water cell-free system. Biotechnol J 2016;
8:430-4; PMID:10048337; https://doi.org/10.1110/ps.8.2. 11:274-81; PMID:26380966; https://doi.org/10.1002/
430 biot.201500237
[21] Shrestha P, Smith MT, Bundy BC. Cell-free unnatural [28] Bundy BC, Franciszkowicz MJ, Swartz JR. Escherichia
amino acid incorporation with alternative energy systems coli-based cell-free synthesis of virus-like particles. Bio-
and linear expression templates. New Biotechnol 2014; technol Bioeng 2008; 100:28-37; PMID:18023052;
31:28-34; PMID:24103470; https://doi.org/10.1016/j. https://doi.org/10.1002/bit.21716
nbt.2013.09.002 [29] Bundy BC, Swartz JR. Site-specific incorporation of p-
[22] Iapalucci-Espinoza S, Haim L, Franze-Fernandez MT. propargyloxyphenylalanine in a cell-free environment for
Aurintricarboxilic acid as a tool for investigating the tem- direct protein¡ protein click conjugation. Bioconjug
plate-bound and unbound forms of RNA polymerase I in Chem 2010; 21:255-63; PMID:20099875; https://doi.org/
permeabilized cells. Mol Cell Biochem 1983; 55:41-7; 10.1021/bc9002844
PMID:6621519; https://doi.org/10.1007/BF00229241 [30] Martins R, Queiroz J, Sousa F. Ribonucleic acid purifica-
[23] Skidmore AF, Beebee TJ. Characterization and use of the tion. J Chromatogr A 2014; 1355:1-14; PMID:24951289;
potent ribonuclease inhibitor aurintricarboxylic acid for https://doi.org/10.1016/j.chroma.2014.05.075