Efficient construction of a stable linear gene based on a TNA loop modified primer pair for gene delivery

Article abstract of DOI:10.1039/d0cc04356g

A terminal-closed linear gene with strong exonuclease resistance and serum stability was successfully constructed by polymerase chain reaction (PCR) with an α-l-threose nucleic acid (TNA) loop modified primer pair, which can be used as an efficient gene e

Full text of DOI:10.1039/d0cc04356g

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Efficient construction of a stable linear gene
based on a TNA loop modified primer pair for
Cite this:DOI: 10.1039/d0cc04356g
gene delivery†
Received 23rd June 2020,
Accepted 18th July 2020
Xuehe Lu,ab Xiaohui Wu,bc Tiantian Wu,bc Lin Han,ab Jianbing Liu*bc and
abc
Baoquan Ding
*
DOI: 10.1039/d0cc04356g
rsc.li/chemcomm
A terminal-closed linear gene with strong exonuclease resistance adverse immune response.7 To address this issue, polymerase
and serum stability was successfully constructed by polymerase chain reaction (PCR) amplified linear gene expression cassettes
chain reaction (PCR) with an a-L-threose nucleic acid (TNA) loop including only the necessary transcription unit (promoter and
modified primer pair, which can be used as an efficient gene target gene) are attracting much interest.8 However, the poor
expression system in eukaryotic cells for gene delivery.
stability of the linear gene expression cassettes is the main
challenge for their further application in gene delivery. Much
Gene therapy is a promising strategy for the treatment of effort has been devoted to improving the stability of this linear
various human diseases at the genetic level through the regula- nucleic acid, such as the construction of a dumbbell-shaped
tion of target gene expression.1 Tailored genetic manipulation nucleic acid.9 This kind of terminal-closed linear gene is
technologies have been developed for gene therapy, such as conventionally fabricated step by step through PCR amplifica-
gene delivery (target gene and mRNA), gene silencing (anti- tion, restriction endonuclease digestion, and T4 ligation pro-
sense and small interference RNA), and gene editing (sgRNA/ cesses, which are complex and time-consuming. We, therefore,
Cas9).2 Construction of a safe and efficient delivery system for hypothesize that PCR amplification with the unnatural nucleic
these nucleic acid drugs is the first key step for the develop- acid loop modified DNA primer pair may result in the facile and
ment of gene therapy in clinical application. Till now, the efficient construction of a stable terminal-closed linear gene in
developed gene vectors are broadly classified as viral and large scale for gene delivery.
non-viral types.3 The viral gene vectors with efficient cellular
Xeno-nucleic acids (XNAs) are synthetic nucleic acid analo-
uptake have been approved for clinical treatments, such as gues with unnatural sugar backbones.10 Because of their high
glybera for lipoprotein lipase deficiency (LPLD) and strimvelis stability towards the nuclease, XNAs have been widely employed
for adenosine deaminase-deficient severe combined immune in various biomedical applications, such as bio-imaging, diagno-
deficiency (ADA-SCID).4 Considering potential immunogenicity sis, and therapeutics.11 In particular, a-L-threose nucleic acid
and insertional mutagenesis, the wide application of viral gene (TNA), first synthesized by Eschenmoser’s group twenty years
vectors is limited.5 Much attention has been drawn to the ago, demonstrates several unique properties for biological appli-
fabrication of various non-viral vectors.6 The traditional circu- cation, such as excellent biocompatibility, strong enzymatic resis-
lar plasmid is the most representative non-viral vector in the tance, and efficient cellular uptake.12 TNA also possesses the
research of gene delivery. The obstinate bacterial remnants ability to form a stable antiparallel Watson–Crick base pairing
such as lipopolysaccharides (LPS) and numerous unmethylated with itself and natural nucleic acids (DNA and RNA), which can be
CpG motifs in the backbone of the plasmid can induce an tailored to fabricate unnatural nucleic acid loop modified linear
genes. To the best of our knowledge, the construction of stable
TNA loop modified linear gene expression cassettes for gene
delivery has not been previously reported.
a School of Materials Science and Engineering, Henan Institute of Advanced
Technology, Zhengzhou University, Zhengzhou, 450001, China
b CAS Key Laboratory of Nanosystem and Hierarchical Fabrication, CAS Center for
Excellence in Nanoscience, National Center for Nanoscience and Technology,
Beijing, 100190, China. E-mail: liujb@nanoctr.cn, dingbq@nanoctr.cn
c University of Chinese Academy of Sciences, Beijing, 100049, China
† Electronic supplementary information (ESI) available: Experimental details of
the DNA sequence information, synthetic method of primer, PCR process and
additional figures for analysis. See DOI: 10.1039/d0cc04356g
Herein, we describe a facile and universal strategy to con-
struct a terminal-closed linear gene with two TNA loops for
efficient gene expression (Scheme 1). A TNA loop modified primer
pair is rationally designed and employed to amplify the linear
gene through PCR. After T4 ligation, the terminal-closed linear
gene with strong exonuclease resistance and serum stability is
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Scheme 1 Schematic representation of the construction of the terminal-
closed linear gene through PCR amplification with a TNA loop modified
primer pair for gene expression.
successfully fabricated with a high yield. The stable gene expres-
sion cassette elicits an efficient and long-term gene expression in
eukaryotic cells. Our report is the first example of the use of a TNA
loop modified primer pair to amplify the target gene as the
efficient gene expression system.
We initially synthesized the four 20-phosphoramidites based
on Chaput’s reports (TNA-A, TNA-T, TNA-G, and TNA-C mono-
mers, Scheme S1, ESI†) for the solid-phase synthesis of TNA.13
These TNA monomers were characterized using 1H, 13C,
Fig. 1 Synthesis of the terminal-closed linear gene. (a) MALDI-TOF-MS
analysis of F3 and R3. (b) 8% native polyacrylamide gel electrophoresis
analysis of primer pairs, including F1 and R1 (DNA20), F2 and R2 (TNA20), and
F3 and R3 (30-DNA20-TNA26-DNA4-P-50). The gel was stained by Stains-All
(M: DNA marker). (c) 1% Agarose gel electrophoresis analysis of PCR
products (F1 and R1, F2 and R2, and F3 and R3) of the linear CMV-EGFP
template based on the primer pairs (F1 and R1, F2 and R2, and F3 and R3),
respectively. The gel was stained using EB (M: DNA marker).
31P NMR and ESI-MS analyses (shown in ESI†). Then, we designed groups of primer pairs using 8% native PAGE, as shown in
three groups of primer pairs including F1 and R1 (DNA20, the Fig. 1b. In order to clearly visualize the TNA involved primers,
subscript represents the base number of the nucleic acid we employed an efficient nucleic acid dye (Stains-All) for gel
sequences), F2 and R2 (TNA20), and F3 and R3 (30-DNA20-TNA26- staining. With the same nucleic acid sequences, the TNA
DNA4-P-50) for PCR analysis (detailed nucleic acid sequences are primer pair (F2 and R2) showed slightly faster mobility than
shown in the ESI†). The common DNA primer pair (F1 and R1) was the DNA primer pair (F1 and R1) as a whole, which may be
commercially available to amplify the naked linear gene (F1 + R1). attributed to the one less methylene group on the TNA mole-
The TNA primer pair (F2 and R2) was employed as a control to cular backbone compared with DNA. We also observed the clear
evaluate the tolerability of DNA polymerase (La-Taq) towards the bands of the TNA loop modified primer pair (F3 and R3) in the
TNA primer. The TNA loop modified primer pair (F3 and R3) gel image (Fig. 1b). These results of MALDI-TOF MS and gel
includes four tailored functional components to facilitate the characterization indicated that the TNA loop modified primer
construction of the terminal-closed linear gene (Scheme 1). In pair (F3 and R3) was successfully synthesized.
the 30 to 50 direction of F3 and R3, a common DNA primer with the
To evaluate the PCR amplification ability of these obtained
same sequences as that of F1 and R1 for PCR (green and blue), a primer pairs, we constructed a linear CMV-EGFP gene template
TNA loop for terminal closing and PCR stopping with the same (1679 bp) as a proof of concept (detailed gene sequences are
sequences as that of eukaryotic tRNA D-loop (red), four DNA bases presented in the ESI†). As shown in the 1% agarose gel electro-
[GCGC] for efficient base pairing with the stem region of TNA loop phoresis analysis (Fig. 1c), the naked linear DNA template (CMV-
(black), and a phosphate group for T4 ligation (yellow) were all EGFP) can be efficiently amplified using the common DNA primer
covalently linked step by step in one pot for solid-phase synthesis pair (F1 and R1). However, no PCR product was observed in the
(detailed synthetic method is shown in the ESI†).
PCR system with the TNA primer pair (F2 and R2). This negative
After the desalination process using a SEP-PAK classic C18 result indicated that the TNA sequences cannot be recognized and
column, we further employed 15% denatured polyacrylamide subsequently elongated using La-Taq DNA polymerase. Because of
gel electrophoresis (PAGE) to purify the primer pairs obtained this PCR blocking phenomenon induced by TNA, we rationally
by solid-phase synthesis. The purified primer pairs (F2, R2, F3 organized the TNA loop in the 50 terminal of the common DNA
and R3) were subsequently characterized using MALDI-TOF primer for terminal closing and PCR stopping. As shown in
mass spectrometry (Fig. 1a and Fig. S1, ESI†). We found that Fig. 1c, we observed an obvious target PCR product with the
the observed values of molecular weights are consistent with TNA loop modified primer pair (F3 and R3) in the agarose gel
the calculated values. We subsequently analyzed these three image. This result demonstrated that the terminal TNA loop
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modifications of DNA primers will not affect the PCR process to After incubation for 6 h, a clear band of the terminal-closed linear
amplify the target gene.
EGFP gene (F3 + R3) could still be observed. After statistical
After obtaining the terminal TNA loop modified EGFP gene, analysis of the bands using Image J (Fig. S3, ESI†), an intuitive
the terminal-closed linear EGFP gene (F3 + R3) was subse- increase of serum stability was shown in the group of the
quently fabricated using T4 DNA ligation at 25 1C for 5.0 h. terminal-closed linear EGFP gene (F3 + R3). These results indicated
This ligation process was facilitated by the efficient comple- that the terminal-closed TNA loop modification is a universal
mentary base pairing between the 50 terminal DNA [GCGC] and strategy to enhance the stability of a target linear gene against the
the stem region of the TNA loop involved in the primer pair digestion of exonuclease and serum.
(F3 and R3). A 1% denatured agarose gel electrophoresis analy-
Encouraged by the enhanced exonuclease resistance and
sis and exonuclease III (a kind of 30 to 50 exonuclease) digestion serum stability, we subsequently evaluated the gene expression
assay were performed to evaluate the efficiency of terminal- potential of this terminal-closed linear EGFP gene (F3 + R3) in
closed ligation. After T4 ligation, the PCR product based on the eukaryotic cells (HEK-293T). The eukaryotic CMV promoter and
primer pair F3 and R3 showed obviously slower mobility than the SV40 poly A section were both involved in the linear DNA
non-ligated control group (Fig. S2a, ESI†). The enhanced resis- template (CMV-EGFP) to ensure successful gene expression in
tance to exonuclease III digestion also confirmed the successful the eukaryotic cells. 0.5 pmol naked linear EGFP gene (F1 + R1)
construction of the terminal-closed linear gene (F3 + R3) (Fig. S2b, and terminal-closed linear EGFP gene (F3 + R3) were transfected
ESI†). After purification using a gel extraction kit, the terminal- into the HEK-293T cells using Lipofectamine 2000, respectively.
closed linear gene (F3 + R3) can be directly used in the following The transfected HEK-293T cells were directly imaged using a
evaluations.
fluorescence microscope with an excitation wavelength of
We next investigated the exonuclease resistance and serum 488 nm at different time periods to visualize the expression
stability of the terminal-closed linear gene (F3 + R3). The level of the target EGFP gene. We observed an intense EGFP
constructed linear EGFP gene was incubated with different fluorescence in the group of the terminal-closed linear EGFP
units of lambda exonuclease (a kind of 50 to 30 exonuclease) gene (F3 + R3, Fig. 3a), indicating that the terminal-closed TNA
at 37 1C for 1.5 h. As shown in Fig. 2a, the naked linear EGFP gene loop modification of the linear gene will not obviously affect the
(F1 + R1) was almost completely digested after the treatment of efficiency of gene expression in the cellular environment.
lambda exonuclease in a relatively low concentration (5.0 U). In
Furthermore, compared with the naked linear EGFP gene
contrast, the terminal-closed linear EGFP gene (F3 + R3) demon- (F1 + R1), our terminal-closed EGFP expression cassette (F3 + R3)
strated a strong exonuclease resistance even at a higher concen- elicited a more efficient and long-lasting EGFP gene expression
tration of lambda exonuclease (10.0 U). The efficient terminal- (Fig. 3b). After incubation at 37 1C for 60 h, we employed flow
closing by the stable TNA loop is responsible for the greatly cytometry to quantitatively analyze the level of EGFP expression
enhanced exonuclease resistance. Meanwhile, we incubated the with the treatments of these constructed gene expression
linear EGFP gene with 10% FBS in DMEM at 37 1C for different cassettes (Fig. 3c and Fig. S4, ESI†). About a 36% enhancement
time periods to evaluate the serum stability. After incubation for of the EGFP positive cells was observed upon the treatment of
4 h, most of the naked linear EGFP gene (F1 + R1) disappeared and the terminal-closed linear EGFP gene (Fig. S5, ESI†). This
an evident smear band of digested gene was detected (Fig. 2b). efficient and long-lasting gene expression may be attributed
to the strong exonuclease resistance and serum stability of
the terminal-closed linear EGFP gene with the stable TNA loop
modification.
In summary, we have developed a universal and efficient
strategy for the construction of a terminal-closed linear gene
through PCR with a TNA loop modified primer pair. This
terminal-closed linear gene demonstrates several desirable
features for gene delivery. First, this terminal-closed linear gene
can be easily produced by PCR amplification in a short time
without the requirement of restriction endonuclease. Second, this
chemically well-defined linear gene shows obviously enhanced
exonuclease resistance and serum stability. Finally, our stable
terminal-closed linear gene can elicit an efficient and long-lasting
gene expression in eukaryotic cells. This construction strategy for a
stable gene expression cassette can be potentially generalized to
other target genes, which will open a new avenue for the develop-
ment of gene therapy.
Fig. 2 Evaluation of exonuclease resistance and serum stability. (a) 1%
Agarose gel electrophoresis analysis for the exonuclease resistance of the
naked linear EGFP gene (F1 + R1) and terminal-closed linear EGFP gene
(F3 + R3). (b) 1% Agarose gel electrophoresis analysis for the serum stability
of the naked linear EGFP gene (F1 + R1) and terminal-closed linear EGFP
gene (F3 + R3) (M: DNA marker).
This work is supported by the National Natural Science
Foundation of China (21708004 and 21721002), the National Basic
Research Program of China (2016YFA0201601 and 2018YFA0-
208900), Beijing Municipal Science & Technology Commission
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Fig. 3 Gene expression analysis in HEK-293T cells. (a) Fluorescence
microscope images of the expression of EGFP in HEK-293T cells trans-
fected with 0.5 pmol naked linear EGFP gene (F1 + R1) and terminal-closed
linear EGFP gene (F3 + R3) for different time periods. Scale bar: 50 mm.
(b) Statistical result of the relative EGFP intensity of HEK-293T cells treated
with F1 + R1 and F3 + R3 using Image J analysis (*P o 0.05). (c) Flow
cytometry analysis of the expression of EGFP in HEK-293T cells trans-
fected with PBS, F1 + R1, and F3 + R3 for 60 h.
(Z191100004819008), the Strategic Priority Research Program of
Chinese Academy of Sciences (XDB36000000), Key Research Pro-
gram of Frontier Sciences, CAS, Grant QYZDB-SSW-SLH029, CAS
Interdisciplinary Innovation Team and K. C. Wong Education
Foundation (GJTD-2018-03).
Conflicts of interest
There are no conflicts to declare.
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