Communication

pubs.acs.org/JACS

Innate Reverse Transcriptase Activity of DNA Polymerase for

Isothermal RNA Direct Detection
Chao Shi,† Xiaotong Shen,‡ Shuyan Niu,† and Cuiping Ma*,‡
†
Key Laboratory of Sensor Analysis of Tumor Marker, Ministry of Education, College of Chemistry and Molecular Engineering,
Qingdao University of Science and Technology, Qingdao 266042, PR China
‡
Shandong Provincial Key Laboratory of Biochemical Analysis, College of Chemistry and Molecular Engineering, Qingdao University
of Science and Technology, Qingdao 266042, PR China
*
S Supporting Information

There was a surprising discovery in our lab that DNA

ABSTRACT: RNA detection has become one of the most polymerase of Klenow fragment (3′ → 5′ exo−) (Klenow exo−)
robust parts in molecular biology, medical diagnostics and exhibits innate reverse transcriptase activity, which have been
drug discovery. Conventional RNA detection methods exploited to directly detect microRNA.22 Motivated by our
involve an extra reverse transcription step, which limits ongoing interest in applying innate reverse transcriptase activity
their further application for RNA rapid detection. We of Klenow exo−, we sought to explore the possibility of other
herein report a novel finding that Bst and Klenow DNA DNA polymerases as reverse transcriptase to be used in RNA
polymerases possess innate reverse transcriptase activities, detection.
so that the reverse transcription step and next To verify if Bst DNA polymerase, large fragment (Bst LF)
amplification reaction can be combined to one step in and Vent (exo−) DNA polymerase (Vent exo−) had innate
isothermal RNA detection. We have demonstrated that Bst reverse transcriptase activities, 50 nt hepatitis C virus (HCV)
and Klenow DNA polymerases could be successfully used RNA was synthesized as a template (sequence listed in
to reverse transcribe RNA within 125-nt length by real Supplementary Table S1). The classical AMV reverse tran-
time RT-PCR and polyacrylamide gel electrophoresis scriptase (AMV) was used as a positive control here. HCV
(PAGE). Our findings will spur the development of a RNA was reverse transcribed by Vent exo−, AMV and Bst LF,
myriad of simple and easy to use RNA detection respectively, then the reaction products were further digested
technologies for isothermal RNA direct detection. This by RNase H, followed by denaturing polyacrylamide gel
will just meet the future needs of bioanalysis and clinical electrophoresis (PAGE). If RNA was successfully reverse
diagnosis to RNA rapid detection in POC settings and transcribed, hybrid of RNA and cDNA would generate. With
inspection and quarantine. addition of RNase H, RNA was digested, and only cDNA bands
appeared on gel. As shown in Figure 1, there was only primer

R NA not only can be the carrier of genetic information, but

also the executant of life functions in living cells.1−3 Thus,
RNA as an important research target has attracted more and
more attention owing to its great value in molecular biology,4
medical diagnostics5 and disease therapy.6 Several common
methods for RNA detection have been developed, such as
reverse transcription-PCR (RT-PCR),7−9 rolling-cycle amplifi- Figure 1. 17.5% denaturing PAGE of cDNAs from HCV RNA by
cation (RCA),10,11 nucleic acid sequence-based amplification different DNA polymerases. Lane 1: Primer; Lane 2: HCV RNA;
Lanes 3, 4, and 5: Reverse transcription products from Vent exo−,
(NASBA),12−14 modified invader amplification15 and loop-
AMV and Bst LF, respectively.
mediated isothermal amplification (LAMP).16,17 These meth-
ods involve an extra step of reverse transcription, in which RNA band appeared in Lane 3. This result showed no cDNA
is reverse transcribed into the complementary DNA (cDNA) generated, so Vent exo− had no reverse transcriptase activity.
strand using an additional reverse transcriptase.18 The cDNA Noticeably, there were the same cDNA bands appeared in Lane
strand serves as an initiator for next amplification reactions. 4 and 5. Thus, Bst LF possessed innate reverse transcriptase
This will no doubt bring inconveniences to the user in finding activity like AMV.
optimum assay conditions being permissive for all of the We further assess reverse transcriptase activities of a few
enzymes being used, increasing experimental costs and reaction more commonly used DNA polymerases by real-time RT-PCR
times, adding additional steps and the probability of using the 50 nt HCV RNA as target. The reverse transcription
contamination.19,20 These aforementioned limitations are reaction was first performed by different enzymes at their
completely incompatible with point-of-care (POC) or field
use,21 and not appropriate for the detection of inner RNA Received: August 3, 2015
sequence in live cells. Published: October 16, 2015

© 2015 American Chemical Society 13804 DOI: 10.1021/jacs.5b08144

J. Am. Chem. Soc. 2015, 137, 13804−13806
Journal of the American Chemical Society Communication

optimum conditions. Then, the diluted reverse transcription

products were used as template for real-time PCR. As shown in
Figure 2a, when AMV, DNA polymerase I, large (Klenow)

Figure 2. Verification of reverse transcriptase activities of multiple

DNA polymerases. (a) Verification of reverse transcriptase activities by
real-time RT-PCR. (b) Native PAGE of the RT-PCR products. Lane Figure 3. Real-time RT-PCR for RNA of different lengths from E. coli
1: AMV; Lane 2: Klenow exo−; Lane 3: Klenow LF; Lane 4: Bst LF; K-12 by different DNA polymerases. (a) 65 nt RNA (b) 125 nt RNA
Lane 5: Bst 2.0; Lane 6: WS Bst 2.0; Lane 7: DNA poly I; Lane 8: Vent (c) 262 nt RNA (d) Native PAGE of the RT-PCR products for AMV
exo−; Lane 9: Taq; M: 20-bp DNA ladder. and Bst LF. Lanes 1, 3, and 5: The products of RT-PCR by AMV from
65-, 125-, and 262-nt RNA, respectively; Lanes 2, 4, and 6: The
fragment (Klenow LF) and Klenow exo−, Bst LF, Bst 2.0 DNA products of RT-PCR by Bst LF from 65-, 125-, and 262-nt RNA,
polymerase (Bst 2.0) and Bst 2.0 WarmStart DNA polymerase respectively; Lane 7: Without addition of enzyme for conversion of
(WS Bst 2.0) were used to reverse transcribe, respectively, all of RNA template to cDNA that served as a target for RT-PCR; M: 20-bp
their corresponding fluorescence intensities of PCR signifi- DNA ladder. The primers were listed in Supplementary Table S1.
cantly increased. This result indicated that cDNA templates for
real-time PCR were produced, showing the five types of DNA about 10 cycles for tested DNA polymerases compared with
polymerases like AMV possessing reverse transcriptase AMV when fluorescence signal appeared (Figure 3c). Also, the
activities. However, no change of fluorescence intensity was reverse transcriptase activities of tested DNA polymerases were
observed, when DNA polymerase I (E. coli) (DNA poly I), decreased with the increase of the length of RNA target, and
Vent exo− or Taq DNA polymerase (Taq) was added, which there were only low reverse transcriptase activity until 262-nt
implied that these three types of DNA polymerase had no RNA template. The electrophoresis for the products of RT-
reverse transcriptase activities of their own. Surprisingly, the PCR was consistent with the fluorescence curves (Figure
fluorescence signals of RT-PCR by Bst LF, Bst 2.0 and WS Bst 3d).The products of RT-PCR by AMV and Bst LF from 65-,
2.0 appeared earlier than that of AMV. Thus, the reverse 125-, and 262-nt RNA templates, respectively, were analyzed by
transcriptase activities of a series of Bst DNA polymerase were native PAGE. As seen in Figure 3d, the same RT-PCR products
superior to that of AMV at the tested length of 50 nt. appeared in Lane 1 and 2, and Lane 3 and 4. This result showed
Native PAGE of the real time RT-PCR products was that the reverse transcription ability of Bst LF was comparable
performed (Figure 2b). RT-PCR products of 50 bp, among 40- with that of AMV for 65- and 125-nt RNA templates. However,
and 60-bp band of the ladder, appeared in Lanes 1−6, when with the increase of RNA template to 262-nt, RT-PCR product
AMV, Klenow exo−, Klenow LF, Bst LF, Bst 2.0, and WS Bst was only formed in Lane 5, and weak product appeared in Lane
2.0, respectively, were used to reverse transcribe. This result 6. This also verified that Bst LF was only low reverse
demonstrated that successful transcription of RNA generated transcriptase activity for 262-nt RNA template. Without
cDNA as a template for RT-PCR, showing Klenow exo−, addition of enzyme for conversion of RNA template to
Klenow LF, Bst LF, Bst 2.0, and WS Bst 2.0 possessed innate cDNA that served as a target for real time PCR, there was no
reverse transcriptase activities like AMV. On the contrary, there products in Lane 7, showing high quality of extracted RNA and
were no RT-PCR products generated in Lane 7−9, which the templates of real time PCR indeed were from reverse
implied DNA Poly I, Vent exo−, and Taq could not reverse transcription of extracted RNA in Lane 1−6.
transcribe RNA to cDNA, and had no innate reverse In order to verify the effectiveness of cDNA from the
transcriptase activities. This electrophoresis result was con- extracted RNA as template in the subsequent amplification
sistent with that of real time RT-PCR above-described. experiment, 10-fold dilution of 65-nt cDNA from the
To assess the reverse transcription ability to RNA of different conversion of the extracted RNA of E. coli K-12 by WS Bst
lengths, we designed different primers for reverse transcribing 2.0 was detected by RT-PCR (Figure 4). The fluorescence
different lengths of 16S rRNA from the extracted total RNA of curves showed good regularity with the increase of amount of
Escherichia coli (E. coli) K-12. As shown in Figure 3, real time cDNA (Figure 4a), demonstrating that WS Bst 2.0 could be
RT-PCR was performed for 65-, 125- and 262-nt RNA, used to reverse transcribe RNA instead of common reverse
respectively. The reverse transcription abilities of Klenow LF, transcriptase for RT-PCR. The time corresponding to the
Klenow exo−, Bst LF, Bst 2.0, and WS Bst 2.0 were comparable maximum slope in the fluorescence curve as well as the point of
with that of AMV for 65-nt RNA in Figure 3a. When the target inflection (POI) was linearly related to the negative logarithmic
was increased to 125-nt RNA, the levels of their reverse (lg) moles of cDNA in the tested concentration range from 10
transcription abilities were decreased to a certain extent (Figure fmol to 1 amol (Figure 4b). The correlation equation was
3b). When being increased to 262-nt RNA, their reverse found to be POI = −51.553−4.00547 lgA (mol) (A was the
transcription abilities were decreased to approximately a moles of cDNA, R2 = 0.9912). Thus, the cDNA reverse
thousand to one of that of AMV according to increasing transcribed from the extracted RNA by WS Bst 2.0 can be used
13805 DOI: 10.1021/jacs.5b08144
J. Am. Chem. Soc. 2015, 137, 13804−13806
Journal of the American Chemical Society Communication

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*
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AUTHOR INFORMATION
Corresponding Author
*[email protected]
Notes
The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS
The work was supported by the National Natural Science
Foundation of China (31170758, 21375071, and 21307064).

■ REFERENCES
(1) Moreira, S.; Breton, S.; Burger, G. Wiley Interdiscip Rev. RNA
2012, 3, 213.

13806 DOI: 10.1021/jacs.5b08144

J. Am. Chem. Soc. 2015, 137, 13804−13806