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The recent development of mRNA vaccines against the SARS-CoV-2 infection has turned the spotlight on the potential of nucleic acids as innovative prophylactic agents and as diagnostic and therapeutic tools. Until now, their use has been severely limited by their reduced half-life in the biological environment and the difficulties related to their transport to target cells. These limiting aspects can now be overcome by resorting to chemical modifications in the drug and using appropriate nanocarriers, respectively. Oligonucleotides can interact with complementary sequences of nucleic acid targets, forming stable complexes and determining their loss of function. An alternative strategy uses nucleic acid aptamers that, like the antibodies, bind to specific proteins to modulate their activity. In this review, the authors will examine the recent literature on nucleic acids-based strategies in the COVID-19 era, focusing the attention on their applications for the prophylaxis of COVID-19, but also on antisense- and aptamer-based strategies directed to the diagnosis and therapy of the coronavirus pandemic.
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Citation: Borbone, N.; Piccialli, I.;
Falanga, A.P.; Piccialli, V.; Roviello,
G.N.; Oliviero, G. Nucleic Acids as
Biotools at the Interface between
Chemistry and Nanomedicine in the
COVID-19 Era. Int. J. Mol. Sci. 2022,
23, 4359. https://doi.org/10.3390/
ijms23084359
Academic Editor: Pedro Viana
Baptista
Received: 7 March 2022
Accepted: 12 April 2022
Published: 14 April 2022
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International Journal of
Molecular Sciences
Review
Nucleic Acids as Biotools at the Interface between Chemistry
and Nanomedicine in the COVID-19 Era
Nicola Borbone 1, Ilaria Piccialli 2, Andrea Patrizia Falanga 1, Vincenzo Piccialli 3, Giovanni N. Roviello 4,*
and Giorgia Oliviero 5
1
Department of Pharmacy, University of Naples Federico II, Via Domenico Montesano 49, 80131 Naples, Italy;
nicola.borbone@unina.it (N.B.); andreapatrizia.falanga@unina.it (A.P.F.)
2Division of Pharmacology, Department of Neuroscience, Reproductive and Odontostomatological Sciences,
University of Naples Federico II, Via Sergio Pansini 5, 80131 Naples, Italy; ilaria.piccialli@unina.it
3Department of Chemical Sciences, University of Naples Federico II, Via Cintia 26, 80126 Naples, Italy;
vinpicci@unina.it
4Institute of Biostructures and Bioimaging, Italian National Council for Research (IBB-CNR), Area di Ricerca
Site and Headquarters, Via Pietro Castellino 111, 80131 Naples, Italy
5Department of Molecular Medicine and Medical Biotechnologies, University of Naples Federico II,
Via Sergio Pansini 5, 80131 Naples, Italy; giorgia.oliviero@unina.it
*Correspondence: giroviel@unina.it
Abstract:
The recent development of mRNA vaccines against the SARS-CoV-2 infection has turned
the spotlight on the potential of nucleic acids as innovative prophylactic agents and as diagnostic
and therapeutic tools. Until now, their use has been severely limited by their reduced half-life
in the biological environment and the difficulties related to their transport to target cells. These
limiting aspects can now be overcome by resorting to chemical modifications in the drug and using
appropriate nanocarriers, respectively. Oligonucleotides can interact with complementary sequences
of nucleic acid targets, forming stable complexes and determining their loss of function. An alternative
strategy uses nucleic acid aptamers that, like the antibodies, bind to specific proteins to modulate
their activity. In this review, the authors will examine the recent literature on nucleic acids-based
strategies in the COVID-19 era, focusing the attention on their applications for the prophylaxis of
COVID-19, but also on antisense- and aptamer-based strategies directed to the diagnosis and therapy
of the coronavirus pandemic.
Keywords:
DNA; RNA; oligonucleotides; nucleic acid analogs; COVID-19; antisense;
mRNA vaccines
;
antigene; nanomedicine
1. Introduction
The COVID-19 (coronavirus disease 19) pandemic, caused by severe acute respiratory
syndrome coronavirus 2 (SARS-CoV-2), is causing enormous difficulties around the globe
from both a sanitary and socioeconomic perspective [
1
10
]. Currently, the world is waiting
for effective benefits following the mass vaccination campaign conducted in some parts of
the globe using the developed anti-COVID-19 vaccines. However, despite the mass immu-
nization campaign [
11
,
12
] and the efforts of pharmaceutical companies and the scientific
community to devise effective therapies via new drug development [
13
,
14
], drug repur-
posing [
15
,
16
], herbal medicine [
17
21
] and other recently proposed approaches
[2226]
,
SARS-CoV-2 and other human coronaviruses [
27
,
28
] remain a major global issue due
to their mutations, leaving our future unclear. The recent development of DNA- and
mRNA-carrying vaccines [
29
32
] able to elicit antibody production against the SARS-CoV-2
infection has recently recalled enormous attention to the potential uses of nucleic acids
and their analogs as innovative biomedical tools. The oligonucleotide biotechnological use
has been severely limited by their reduced half-life in the biological environment and the
difficulties related to their delivery to target cells. Thus, some potent nucleic acid analogs
Int. J. Mol. Sci. 2022,23, 4359. https://doi.org/10.3390/ijms23084359 https://www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2022,23, 4359 2 of 17
(NAA) [
33
43
] are currently being utilized in anti-COVID-19 strategies because they allow
one to overcome some of these limiting aspects by resorting to chemical modifications in the
oligonucleotide and by using appropriate nanocarriers [
44
,
45
]. Like natural nucleic acids,
NAAs can interact with complementary sequences of nucleic acid targets, forming stable
complexes [
46
,
47
] and determining their loss of function [
48
]. For example, the binding of a
peptide nucleic acid (PNA), locked nucleic acid (LNA), morpholino (PMO), or another syn-
thetic analog [
49
] to the complementary nucleic acid target may determine (i) the inhibition
of mRNA translation to the corresponding protein (antisense strategy), (ii) the blocking
of gene transcription via specific binding with gene promoters (antigene strategy), and
(iii) numerous biomolecular events exploited in several diagnostic applications [
49
]. An
alternative strategy uses nucleic acids aptamers that, like the antibodies, bind to specific
proteins to modulate their activity [
50
58
]. In this regard, i-motif [
59
,
60
] and G-quadruplex
forming oligonucleotides [
61
63
] are particularly relevant as their specific role as aptamers
was explored
in vitro
and, in some cases, find applications in biomedical strategies [
64
69
].
Drug discovery campaigns against COVID-19 are targeting not only viral proteins (such as
M
pro
main protease [
70
,
71
]) but also the viral RNA genome [
72
], with particular attention
paid to highly conserved and expression-relevant tracts [73] (Figure 1).
Figure 1.
(
a
) The 28-kDa highly conserved FSE (frameshift stimulation element, PDB ID: 6XRZ https:
//www.rcsb.org/3d-view/6XRZ/1 accessed on 4 February 2022) of the SARS-CoV-2 genome is an
example of a potential candidate for targeting by small molecules and oligonucleotides as it is required
for the balanced expression of SARS-CoV-2 proteins [
73
]. (
b
) An example of quadruple helical
DNA (a monomeric parallel-stranded quadruplex in human VEGF promoter; PDB ID: 2M27 https:
//www.rcsb.org/3d-view/2M27/0 accessed on 1 April 2022;
left
) with a schematic representation of
a G4 quartet (
middle
); and a complex between a nucleic acid aptamer (RNA-aptamer K1; purple) with
its protein target (Tetracycline repressor protein; PDB ID: 6SY4 https://www.rcsb.org/3d-sequence/
6SY4?assemblyId=1 accessed on 1 April 2022; right).
Int. J. Mol. Sci. 2022,23, 4359 3 of 17
This review will examine the recent literature on nucleic acid-based technologies in
the COVID-19 era. We will focus on the main prophylactic, therapeutic and diagnostic
DNA- and RNA-based tools currently under examination or in use in the fight against
SARS-CoV-2.
2. DNA and RNA-Containing Vaccines in the Fight against COVID-19
Antiviral vaccines can generally be classified into three categories: (live-attenuated
or inactivated) virus-based, protein-based, and nucleic acid-based [
74
]. The first two
approaches have been the conventional methods that rely on unharmful forms of the
virus or proteins directly delivered as immunogens to induce the immune response in
the host. On the other hand, nucleic acid-based vaccines are delivered via nucleic acid
vectors to host cells, where DNA or RNA genes will be expressed in the host to produce
corresponding antigens that activate the adaptive and humoral immune response [
74
].
All three strategies have been explored for COVID-19, but currently (as of 4 February
2022), only gene vaccines are available in Western countries, even though the protein-based
NVX-CoV2373 vaccine developed by the American Novavax should become available
soon [
75
,
76
], and the inactivated whole virus vaccine VLA2001 from the French Valneva
could be approved in the upcoming months [
76
]. One of the advantages of nucleic-acid-
based vaccines is the easiness and relatively high rapidity of their manufacturing [
74
].
They can immediately be synthesized when the immunogen sequence is made available,
and the process can be easily scalable. With respect to DNA-based vaccines, an mRNA
vaccine expresses the antigen protein directly via translation from the mRNA after its
transfection [
74
]. These vaccines are believed to possess higher biosafety than DNA-based
vaccines because the mRNA is less likely integrated into the genome than a DNA-based
vaccine, as the translation of the antigens in the case of mRNA vaccines takes place in the
cytoplasm and not in the nucleus, where the DNA vaccines start to work [
77
]. However,
several studies suggest that the risk of genomic integration, even if diminished compared
to DNA vaccines, also remains for those based on mRNA, considering that eukaryotic cells
may exert, to some extent, a reverse transcription activity [
78
80
] that could produce DNA
theoretically starting from the vaccine-delivered mRNAs [
81
,
82
]. An advantage of nucleic
acid-based vaccines over protein-based vaccines is that they may lead to antigens better
mimicking the viral protein structure, including the post-translational modifications. In
fact, while protein-based vaccines are often produced from bacteria, mRNA vaccines are
translated by the host translation machinery. Nonetheless, the novel NVX-CoV2373 vaccine
was developed using insect cells that can perform most of the desired post-translational
modifications. As for the storage and transportation, DNA and mRNA vaccines must be
stored and transported at low or ultra-low temperatures [
83
], whereas inactivated virus-
and protein-based vaccines [
84
] require less stringent conditions. However, innovative
lipid nanoparticle technologies are significantly improving the stability characteristics of
mRNA vaccines that may require less stringent conditions [83].
From a molecular perspective, one of the most reliable strategies to fight SARS-CoV-2
consists of the administration of the nucleic information (DNA or RNA) that the host
cellular machinery uses for the production of SARS-CoV-2 spike (S) protein, which has
been generally used as the antigen of DNA vaccines used against COVID-19 (Table 1).
Once produced in human cells, S protein may provoke the immune system to respond
with cellular and humoral defenses, retaining the information in memory immune cells. In
this way, the organism is prepared to counteract virus infection in the case of subsequent
exposure to SARS-CoV-2. Generally, these prophylactic agents are administered in a two-
dose immunization scheme, with both injections being administered intramuscularly, often
within a three-week interval. Interestingly, the nucleic acid-based vaccination is currently
the most exploited anti-COVID-19 prophylaxis in the Western world, and here below, we
will give some details on the currently most in-vogue vaccines belonging to this category
of vaccines.
Int. J. Mol. Sci. 2022,23, 4359 4 of 17
Table 1. Some of the most used nucleic acid-based vaccines in Western countries.
Vaccine Name Carried Nucleic Acid Developer Confirmed Efficacy
ChAdOx1-S/AZD1222 DNA AstraZeneca + University
of Oxford
63.1%, based on a median
follow-up of 80 days
Ad26.CoV2.S DNA Janssen Pharmaceuticals
Johnson & Johnson 66.0%, 28 days post-vaccination
BNT162/Comirnaty RNA
Pfizer/BioNTech + Fosun Pharma
95.0%, measured starting from
seven days after the second dose
mRNA-1273 RNA
Moderna + National Institute of
Allergy and Infectious
Diseases (NIAID)
94.1%, measured starting from
two weeks after the second dose
2.1. DNA-Carrying COVID-19 Vaccines
They are often classified as “virally vectored vaccines” or “adenovirus vector vaccines”
being based on vaccine vectors, but they should be more correctly denominated as aden-
ovirus vector-based DNA vaccines to underline the nature of their cargo [
76
]. The most
widely utilized in Western countries, i.e., the ChAdOx1-S/AZD1222-Spike [
85
] vaccine
developed by the University of Oxford in collaboration with AstraZeneca (Cambridge, UK)
pharmaceutical company (Table 1), used a Chimpanzee non-replicating viral vector that
contains synthetic DNA encoding the S protein of SARS-CoV-2. Thus, the
ChAdOx1-S
/
AZD1222 expresses the S protein gene, which instructs the human cells to produce the
protein, allowing the body to generate an effective immune response. Clinical trials showed
efficacy in participants who received two doses of the vaccine irrespective of the interval
between the doses of about 63.1%, based on a median follow-up of 80 days or higher when
this interval was longer [
86
]. The vaccine was manufactured by SK Bioscience
Co., Ltd.
(Pangyo-ro, Korea), under the name ChAdOx1-S, and the Serum Institute of India (Pune,
India) named COVISHIELD [
87
]. Even though it is less common than the ChAdOx1-S/
AZD1222, the Ad26.CoV2.S vaccine developed by Janssen Pharmaceuticals Johnson &
Johnson (Beerse, Belgium) is worth mentioning. Ad26.CoV2.S is a non-replicating viral
vector vaccine consisting of a human adenovirus vector, with a DNA genome, into which
has been inserted the gene that encodes the S protein of SARS-CoV-2. The efficacy was
66.0% in phase-3 clinical trials (Table 1) [88].
2.2. RNA-Carrying COVID-19 Vaccines (mRNA Vaccines)
The most currently used vaccine type in Europe and North America, these vaccines
exploit mRNAs to instruct human cells to produce the S protein [
74
]. Since mRNA vac-
cines do not need to reach the cell nucleus like the DNA-based ones, they are of higher
practical significance. Although RNA is known to be a relatively unstable nucleic acid,
novel vaccine nanotechnologies were developed to improve both mRNA stability and S
protein translation efficiency, with consequent enhanced immune responses by the host
cell. More in detail, lipid-nanoparticle encapsulation [
89
92
] of the vaccine is a strategy
often used to optimize the delivery of the mRNAs for intradermal or intramuscular admin-
istration [
93
]. The leading exponents of this family of anti-COVID-19 prophylactic agents
are BNT162/Comirnaty and mRNA-1273 (Table 1) [
94
]. The former is a lipid-nanoparticle
encapsulated mRNA-based vaccine developed by BioNTech (Mainz, Germany) and Pfizer
(New York, NY, USA), partnered with Fosun Pharma (Shangai, China), which encodes the
RBD (receptor-binding domain) domain of the SARS-CoV-2 S protein. BNT162, loaded into
a patented lipid-nanoparticle composed of ionizable amino lipid, phospholipid, choles-
terol, and a PEGylated lipid (at a ratio of 50:10:38.5:1.5 mol/mol, Figure 2) [
95
], uses a
modified 4284 nucleotides long mRNA [
96
] and includes the T4 fibrin-derived trimer-
ization domain, which serves to enhance the immune response [
88
]. On the other hand,
mRNA-1273
,
developed
by Moderna (Cambridge, MA, USA) in collaboration with the
American National Institute of Allergy and Infectious Diseases (NIAID, Bethesda, MD,
USA), is a vaccine based on a 4004 nucleotides long mRNA [
97
] expressing the full-length
Int. J. Mol. Sci. 2022,23, 4359 5 of 17
prefusion stabilized S protein of SARS-CoV-2. In mRNA-1273, the mRNA strand is encapsu-
lated by two proprietary cationic lipidic nanoparticles (WO2017070626 and WO2018115527)
whose composition was described as SM-102, polyethylene glycol-2000-dimyristoyl glyc-
erol (PEG2000-DMG), cholesterol, and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC,
Figure 2) [95].
As for dosages/schedule, routes of administration, and efficacies, two intramus-
cular doses are needed for both mRNA vaccines, 21 days apart (30
µ
g per dose) for
BNT162/Comirnaty and 28 days apart (100
µ
g per dose) in the case of mRNA-1273. The
confirmed efficacy of the BNT162/Comirnaty vaccine is 95.0% (measured starting from
seven days after the second dose), while that of mRNA-1273 is 94.1% (measured starting
from two weeks after the second dose) (Table 1) [98].
2.3. Chemical and Nanotechnological Optimization of mRNA Vaccine Design
Though exempt from any risks of genomic integration [
77
], the typical vaccine de-
velopment using viruses or protein-based systems involves time-consuming steps that
appear impractical for responding rapidly to any pandemic caused by newly emerging
pathogens. As explained above, nucleic acid-based vaccines possess distinctive advantages
of rapid development and versatility that led to the development of multiple COVID-19
DNA and mRNA vaccines. Among these, mRNA vaccines seem to have higher protective
efficacy than DNA vaccines, but special strategies are needed to guarantee their safety,
stability, and consequent efficacy due to some intrinsic RNA molecular features. In fact,
RNA is particularly prone to enzymatic degradation by the RNases present in the plasma
and serum. Moreover, RNA molecules in mRNA vaccines, being exogenous molecules,
are seen by the human cellular machinery as an immunological mimic of viral infection,
which provokes an immediate immune response by host cells. Hence, the importance
of nanotechnologies aimed at maximizing the stability of RNA in mRNA vaccines and
limiting the innate immune response in the host [99,100].
Endogenously, mRNAs undergo post-transcriptional modifications, such as 5
0
-cap-
ping [
101
] and polyadenylation [
102
,
103
], needed for mRNA stabilization (protecting
mRNA from exonuclease activity) and efficient translation (facilitating pre-mRNA splicing
and serving as the binding site for the translation initiation complex). 5
0
-capping includes
the addition of 7-methylguanosine (m7G) at the 5
0
end of the first ribonucleotide of mRNA
molecules via a 5
0
to 5
0
linkage and the methylation of the 2
0
-OH of the ribose moiety
of the same ribonucleotide to form m7GpppNm (Figure 3a). Interestingly, the host can
discriminate between the self versus the exogenous mRNA thanks to the presence of
the
50-cap.
The last observation explains why adding an m7GpppNm cap to the 5
0
-end
of mRNA vaccines’ RNA strand is a highly desirable mRNA modification [
101
]. The
polyadenylation (i.e., the addition of a poly rA tail at the 3
0
-OH of pre-mRNA) is another
factor that stabilizes mRNA and promotes protein translation [
102
,
103
], as the length
of the poly(A) tail is closely associated with the translation efficiency. However, the
information on the nature of the poly(A) signal sequence was reported only in the case
of the BNT162/Comirnaty vaccine [poly rA tail: A30(GCATATGACT)A70], whereas it
remains proprietary and undisclosed for the Moderna’s counterpart [96].
Nucleoside modifications and, especially, the incorporation of pseudouridine
(Figure 3b)
into mRNA molecules may suppress the immune response by evading the activation of
TLR-3, -7, and -8. Consequently, in the BNT162/Comirnaty vaccine, uridine residues were
replaced by 1-methyl pseudouridine modification to reduce the innate immune response
and enhance the stability of the exogenous mRNA [104].
Many liposome-based transfection reagents based on cationic lipids have been formu-
lated to improve the transfection efficiency of mRNAs, which is generally low in the case of
naked oligoribonucleotides [
105
]. Generally, the lipid components of mRNA vaccines are
proprietary and include different cationic polypeptides, positively charged lipids, polymers,
dendrimers, or micelles [
106
]. Lipid nanoparticles encapsulate their mRNA cargo into
the stable lipid bilayer, which is internalized by recipient cells via endocytosis. More in
Int. J. Mol. Sci. 2022,23, 4359 6 of 17
detail, after the injection, the mRNA-lipid nanoparticle complexes enter muscle cells via
endocytosis pathways, and then the translation of the mRNA leads to translates forming a
metastable trimeric prefusion spike protein. Blood vessels adjacent to the muscles are then
believed to recruit infiltrating antigen-presenting cells [107].
Figure 2.
Chemical representations of some typical components of lipid nanoparticles as
well as specific components of the Moderna vaccine. DPPC: dipalmitoylphosphatidylcholine;
DSPC: distearoylphosphatidylcholine.
Int. J. Mol. Sci. 2022,23, 4359 7 of 17
Figure 3.
Schematic representation of some typical modifications of synthetic mRNAs contained in
the COVID-19 vaccines: (
a
) 5
0
capping via cap1 structure (m7GpppNm); (
b
) uridines are replaced
with pseudouridine or 1-methyl pseudouridine units.
2.4. Nanotechnologies in the Development of Potential COVID-19 Vaccines
Coronaviruses are nanoscale biostructures against which nanotechnology can be ex-
ploited to realize vaccines and immune engineering applications [
108
]. Live attenuated
and inactivated vaccines, viral vectors, and mRNA-lipid nanoparticles [
109
,
110
] (Figure 4a)
constitute examples of nano-biotechnological products so efficient against SARS-CoV-2.
The use of nanomaterials as carriers of antigens or prophylactic mRNAs and DNAs is
a recent biotechnological approach that proved successful in COVID-19 vaccine design
technology. In a nanotechnological vaccine, antigens and nanoparticles mutually inter-
act by adsorption, entrapment, and conjugation. As for the materials constituting the
nanoparticles, liposomes, nanopolymers, quantum dots, and inorganic nanoparticles are
conventional vehicles for subunit vaccines and nucleic acids [
111
]. Several types of lipids
are also used, including cholesterol and ionizable lipids, which, being cationic, interact
with the negatively charged RNAs.
Moreover, the conjugation of lipids to polyethylene glycol chains (Figure 4a, green)
proved effective in shielding the mRNA cargo from the host immune system, thus pro-
longing a vaccine’s lifetime following intramuscular injection. Protein nanoparticles were
also proposed in the realization of vaccines against SARS-CoV-2 (Figure 4b) [
112
]. The
vaccine strategy, in this case, was based on the display of the S protein receptor-binding
domain (RBD) on a synthetic virus-like particle platform, SpyCatcher003-mi3, using Spy-
Tag/SpyCatcher technology [112].
Int. J. Mol. Sci. 2022,23, 4359 8 of 17
Figure 4.
Structures of nanoparticles used for COVID-19 vaccine candidates: (
a
) mRNA vaccines
realized for the COVID-19 pandemic are composed of long strands of RNAs (magenta) that encode
the SARS-CoV-2 S protein enclosed in lipids (cyan), connected with lipids conjugated to polyethylene
glycol (PEG) chains (green), that deliver the RNA cargo into recipient cells. In the idealized illustration
by David S. Goodsell, RCSB Protein Data Bank [
113
], the lipids are arranged in a simplified model
of a circular bilayer surrounding the mRNAs, and the PEG chains are endowed with both folded
and extended conformations. Note how the real structure may be less regular, as suggested in the
literature [
110
]. (
b
) An example of protein nanoparticle-based vaccine proposed against
SARS-CoV-2.
It displays the S glycoprotein receptor-binding domain (RBD) on a synthetic virus-like particle
platform, SpyCatcher003-mi3, and uses SpyTag/SpyCatcher technology [
112
] (https://www.rcsb.
org/structure/7B3Y, accessed on 4 February 2022).
3. DNA and RNA Targeting in the COVID-19 Era
3.1. RNA Targeting in the Diagnostics of COVID-19
Rapid screening of infected individuals from a large population is important in epi-
demiology, especially in controlling the spread of COVID-19 infections [
114
]. The reverse
transcription polymerase chain reaction (RT-PCR) assay is the diagnostic standard for
COVID-19. In contrast, rapid antigen tests based on lateral immunochromatography and
using different matrices, including direct culture supernatants and dry swabs [
115
], could
be used as point-of-care detection of SARS-CoV-2 antigens with several advantages over the
RT-PCR assays including shorter turn-around times and lower costs [
114
]. However, their
sensitivity in detecting the SARS-CoV-2 virus remains lower than RT-PCR (0.68 compared
to RT-PCR), especially in low viral loads. Even though RT-PCR tests remain the gold
standard for population-wide screening of COVID-19 and other epidemics, significant limi-
tations prevent the large scale application of this technology, which include the significantly
higher costs and longer turnaround times due to time-consuming nucleic acid extraction
and amplification steps, and the required equipment for testing [
114
]. An RNA target-
ing strategy was thus ideated based on a direct nucleic acid assay that used a graphene
field-effect transistor (g-FET) and Y-shaped DNA dual probes [
116
]. The method relied
on Y-dual probes modified on g-FET simultaneously targeting nucleocapsid (N) and viral
replicate polyprotein open reading frame (ORF1ab) genes of the SARS-CoV-2 RNA genome.
Interestingly, the assay was associated with a high recognition ratio and a limit of detec-
tion one-two order of magnitude lower than other nucleic acid assays
(0.03 copy µL1)
.
Another
advantage of this RNA-targeting DNA-based assay was the rapidity of the nucleic
acid testing (~1 min) compared to the longer times (up to 4 h) required by the other nucleic
acid tests, along with the ultrasensitivity, easiness in operating features as well as capability
in pooled testing [116].
Int. J. Mol. Sci. 2022,23, 4359 9 of 17
3.2. COVID-19 Antisense Strategies
The outbreak of SARS in 2003 (caused by SARS-CoV-1) and the COVID-19 pandemic
sixteen years later showed the world our vulnerability to coronavirus infections [
117
].
Given that periodic outbreaks of similar pandemics could occur in the future, the scientific
community and pharmaceutical companies should be prepared to fast-track the production
of vaccines and antiviral oligonucleotides acting as RNA-targeting therapeutics in antisense
strategies directed against coronaviruses. More specifically, oligonucleotides and NAAs
can target the SARS-CoV-2 RNA genome and regulatory RNA sequences, disrupt RNA
secondary structures or host protein/virus RNA complexes, and provoke steric blocks
(Table 2). The first antisense oligonucleotides reported against SARS-CoV-1, whose genome
is closely related to SARS-CoV-2, targeted the ORF1a gene and the transcription regula-
tory RNA sequence located in the 5
0
-UTR region of the positive-sense RNA genome of
SARS-CoV-2 [
118
]. These antisense oligonucleotides (antisense morpholino oligomers
and peptide-conjugated antisense morpholino oligomers [P-PMOs], Figure 5) were en-
dowed with high antiviral activity
in vitro
, revealing the potential capacity of the antisense
nucleic acid analogs (NAA) for antiviral treatment against SARS-CoV-1 and, potentially,
SARS-CoV-2.
This work acted as a proof of concept for the efficiency of RNA-targeting
NAAs as an antiviral treatment in the context of human coronavirus infections [118].
Table 2.
Some of the most used nucleic acid analogs (NAA) and their main anti-SARS-CoV-2 applications.
NAA Full Name Properties Reference
PMO/P-PMO Morpholino/
peptide-morpholino
Targeting RNA genome/regulatory sequences/very high
nuclease stability [118]
LNA Locked nucleic acid Disrupting RNA secondary structure/provoking steric
blocks/very high nuclease stability [119,120]
20-MOE 20-Methyl O-esters
Disrupting interactions between host proteins and
SARS-CoV-2 RNA/provoking steric blocks/high
nuclease stability
[121]
PS Phosphorothioates
Disrupting interactions between host proteins and
SARS-CoV-2 RNA/provoking steric blocks/high
nuclease stability
[121]
Notably, the SARS-CoV-2 genome can be targeted at any step of its life cycle by
antisense oligonucleotides that allow for targeting any of the conserved sequences of both
positive and negative RNA. Regarding the structure of the SARS-CoV-2 genome, whose
knowledge is necessary for developing specific antisense oligonucleotides, it consists
of a single-stranded RNA of ca. 30,000 nucleotides capped at the 5
0
end and endowed
with a 3
0
poly rA-tail, as well as two short UTR-sequences [
118
]. SARS-CoV-2 RNA
genome encodes 14 ORFs, of which ORF1a and ORF1b, at the 5
0
end, encode the replicase
polyprotein comprising ~2/3 of the entire genome. In addition to these nonstructural
proteins, the remaining genome contains nine small ORFs encoding structural proteins
such as nucleocapsid (N), envelope (E), spike (S), membrane (M), and others with accessory
roles. SARS-CoV-2 RNAs include both subgenomic and genomic entities, with the first
RNA tracts being translated into structural proteins and others with accessory roles.
On the other hand, genomic RNA is involved in replicating viral RNA before its
incorporation into virions. In principle, any sequence of SARS-CoV-2 RNA genome is a
potential target of antisense oligonucleotides, but genomic RNAs and replication steps
are especially recommended for antisense strategies as targeting subgenomic entities is
associated with lower antiviral efficacies [
118
]. After infection, SARS-CoV-2 replicates its
genome inside the human cell using enzymes for replication encoded by the coronavirus
itself. More in detail, the 5
0
cap, and 3
0
poly rA tail modifications allow direct translation
of the nonstructural proteins encoded by genes ORF1a and ORF1b. Afterward, the assem-
bly of the replicase-transcriptase complex (RTC) occurs, leading to the replication of the
RNA genome alongside the discontinuous subgenomic mRNA transcription, mediated
Int. J. Mol. Sci. 2022,23, 4359 10 of 17
by short AU-rich transcription regulatory sequences, whose resulting subgenomic RNAs
are further translated into the structural proteins and the others with various accessory
roles. Huston et al. [
119
] applied a novel long amplicon strategy to resolve the secondary
RNA structure of the coronavirus genome in infected cells, which revealed an elaborate
SARS-CoV-2 genome architecture that included well-folded RNA regions, some of which
were unique while others were conserved across beta-coronaviruses. They developed
antisense oligonucleotides containing LNA (Figure 5) moieties as steric blockers and tested
them in targeting two putative RNA regions within the SARS-CoV-2 genome [
119
]. The
two oligonucleotides contained three consecutive LNAs at their 5
0
and 3
0
termini, whereas
unmodified consecutive nucleotides within the strands were limited to three to prevent
RNase-dependent activation. These NAA-based antisense strategies significantly inhibited
the growth of SARS-CoV-2, demonstrating that the two targeted secondary RNA structures
are critical for the life cycle of the coronavirus and that these LNA-containing antisense
oligonucleotides disclose potential as anti-COVID-19 therapeutics [119].
Figure 5.
Structures of nucleic acid analog [PMO (morpholino), P-PMO (peptide-morpholino), LNA
(locked nucleic acid)] moieties and modifications of the nucleic acid backbone [PS (phosphorothioate),
20-MOE (20-methyl O-ester)] employed in the antisense strategies against SARS-CoV-2.
Further investigations used antisense oligonucleotides carrying 2
0
-O-methoxyethyl
(2
0
-MOE, Figure 5) and phosphorothioate (PS, Figure 5) backbone modifications as steric
blockers to disrupt interactions between the host RNA binding proteins and SARS-CoV-2
RNA thanks to their enhanced steric blocking activity [
121
]. Moreover, RNase H-dependent
antisense oligonucleotides, carrying LNA tracts at both ends, were used to target the
SARS-CoV-2 stem-loop 2 motif (s2m) in the 3
0
-UTR of the cytosolic positive-sense RNA
strand [
120
]. In addition to the terminal LNA tracts, the antisense oligonucleotides carried
long stretches of unlocked ribonucleotides that ensured RNase H recruitment and, therefore,
viral RNA cleavage. Interestingly, as RNase H is responsible for the cleavage of the targeted
RNA in the duplexes formed by RNA and the antisense oligonucleotide, this latter remains
Int. J. Mol. Sci. 2022,23, 4359 11 of 17
intact and free to bind to other RNAs, while the SARS-CoV-2 RNA is cleaved in a sequence-
specific manner, which led to inhibition of the replication of SARS-CoV-2 in infected
cells [
120
]. Overall, these anti-COVID-19 RNA-targeting strategies seem to be promising,
especially in view of targeting conserved RNA tracts in different variants of SARS-CoV-2
and other human coronaviruses. However, these approaches still need further research
supporting their translation into clinics.
3.3. Aptamers and G-Quadruplex Structures for the Detection of SARS-CoV-2
While most anti-COVID-19 strategies were designed to target essential proteins within
the SARS-CoV-2 genome, targeting RNA structural elements is also of crucial importance
especially using the class of oligonucleotide aptamers [
122
]. This family is composed
of oligonucleotides of different nature that, similarly to antibodies, recognize specific
three-dimensional structures acting as “chemical antibodies” [
123
]. Thanks to oligonu-
cleotide aptamers’ high affinity and specificity for their targets, they offer unique chemical
and biological characteristics rendering them particularly suitable for novel biomedical
applications, including
in vitro
diagnosis, biomarkers discovery,
in vivo
imaging, and
therapy [
123
]. The highly conserved RNA structure within the s2m motif of SARS-CoV-2
was targeted by high-affinity L-DNA aptamers [
124
] to evaluate their therapeutic and
diagnostic potential. Optimized L-DNA aptamers were found to bind selectively to s2m
with affinities in the nanomolar range and proved capable of discriminating between the
monomeric s2m stem-loop and the homodimer duplex. The L-DNA mode of recognition
is highly structure-specific, allowing to differentiate s2m RNAs from different but closely
related human coronaviruses, such as SARS-CoV-1 and SARS-CoV-2, differing by only two
ribonucleotides. In addition, L-DNA aptamers induce significant conformational changes
in s2m RNA structure upon their molecular recognition, with a potential role in disrupting
or preventing protein–s2m binding [124].
G-quadruplex (G4) DNA or RNA is a non-canonical secondary structure resulting
from assembling one, two, or four guanine-rich nucleic acids strands into a quadruple
helix structure stabilized by coordination with suitable monovalent cations [
125
]. G4
structure and function are determined by factors such as the number and polarity of
nucleotide strands, the type of metal ions, as well as the structural properties of their
binding targets [
125
]. Targeting of the G4-folded SARS-CoV-2 RNA genome by specific
aptamers appears to be a promising alternative method for SARS-CoV-2 detection [
126
].
In addition to their importance as aptamer targets, G4-forming oligonucleotides can also
be used to realize G4 aptamer-based biosensors to detect SARS-CoV-2 surface proteins.
Indeed, G4-based biosensors represent a valuable alternative to antibody-based detection
of SARS-CoV-2 and other pathogens [126].
The use of aptamers for targeting SARS-CoV-2 RNA genomic tracts in COVID-19
therapy and diagnostic approaches based on the recognition of virus proteins are promis-
ing strategies thanks to the intrinsic specificity guaranteed by the aptamer technology.
Nevertheless
, the utility of these approaches is still to be evaluated in prospective clinical
and diagnostic studies.
4. Conclusions
The outbreak of the COVID-19 pandemic showed our vulnerability to coronavirus
infections, and given that other pandemics could attack humanity after the current crisis,
the scientific community, together with pharmaceutical companies, should be prepared
to fast-track the production not only of vaccines, but also of nucleic acid-based antisense
tools and aptamers acting as RNA-targeting therapeutics against coronaviruses. Overall,
the herein summarized applications of nucleic acids, and especially RNAs and NAAs, in
the context of the fight against SARS-CoV-2 demonstrate the feasibility of using nucleic
acids for mass immunization when the urgency of counteracting the virus spread does
not allow waiting for the development of long-established live attenuated and inactivated
virus- and protein-based vaccines. Additionally, the above literature reports show the
Int. J. Mol. Sci. 2022,23, 4359 12 of 17
importance of targeting SARS-CoV-2 RNA using antisense oligonucleotides and aptamers,
which has important implications in diagnosing and treating the infectious disease caused
by SARS-CoV-2. Remarkably, the high affinity and selectivity of oligonucleotide antisense
devices and aptamers, coupled with the intrinsic nuclease resistance of NAA that can be
easily introduced in their structures, enable novel opportunities for generating new tools
and probes for interrogating RNA function in SARS-CoV-2 and related coronaviruses.
Author Contributions:
All authors contributed to the conceptualization, literature collection, writing,
editing, and reviewing of the article. All authors have read and agreed to the published version of
the manuscript.
Funding:
The APC was funded by Ministero dell’Universitàe della Ricerca FOE 2017 - ISBE-IT Joint
Research Unit.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments:
We thank Antonietta Gargiulo (IBB CNR) for her help in the literature search.
We thank D.S. Goodsell, the Illustration in Figure 4a was realized by D.S. Goodsell, RCSB Protein
Data Bank (https://pdb101.rcsb.org/sci-art/goodsell-gallery/sars-cov-2-mrna-vaccine accessed on
4 February 2022).
Conflicts of Interest: The authors declare no conflict of interest.
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