Molecular Biology and Nucleic Acid 2022
Development of RNA G-quadruplex
(rG4)-targeting L-RNA aptamers by rG4-SELEX
Introduction:
Guanine (G)-rich sequences
of single-stranded DNA and RNA can fold into stable, intra- or intermolecular
secondary structures called G-quadruplexes (dG4s and rG4s). These nucleic acid
structure scaffolds are composed of stacks of G-quartets and can be further
stabilized in the presence of monovalent ions, preferentially K+ or Na+ but
not Li. Earlier findings have shown that G4s play important roles in various
cellular events, including but not limited to DNA replication, DNA damage
repair, transcription, translation, RNA metabolism and epigenetic remodelling.
The ability to regulate fundamental biological processes, as well as their
chemically interesting structures, makes G4s promising targets for potential
cancer, antimicrobial and antiviral treatments. With the mounting interests in
the biological role of G4s, more structure-specific, sensitive and
low-cytotoxicity tools are needed to not only differentiate between G4s and
duplexes, but also among different subtypes of G4s, to allow gene/transcript
control and manipulation by selective targeting of specific G4 structure in any
gene/transcript of interest.
Naturally
occurring nucleic acids are homochiral and are built from the monomers of D-DNA
and D-RNA nucleotides. Earlier studies have shown that D-DNA/RNA
oligonucleotides are incapable of forming contiguous Watson–Crick base pairing
with their enantiomeric counterparts, L-DNA/RNA oligonucleotides. As they are
unnatural, L-nucleic acids are unrecognizable by natural nucleases,
which enable them to have extended half-lives for cellular and in vivo studies.
These special properties motivate researchers to develop various biological
tools based on L-nucleic acids. One of the common
examples is an L-RNA aptamer, also known as a spiegelmer.
Speigelmers, first reported by Furste and colleagues, evolved from a modified
version of systematic evolution of ligands through exponential enrichment
(SELEX). To date, spiegelmers have been selected to recognize a range of
targets, including small molecules, peptides and proteins. In addition, a few
spiegelmers are currently under clinical trial phases I or II. Inspired by the
earlier works of Sczepanski and Joyce to use spiegelmers to target canonical
RNA structure motifs such as hairpin and stem-loop RNAs, and also by the
protocol of Lorenz et al. to use natural RNAs to target proteins of interest,
we were motivated to investigate whether such a strategy can be adopted,
refined and generally applicable in developing a new class of targeting tool
for noncanonical RNA structures such as rG4 motifs. The rationale behind
structured RNA targeting spiegelmers is based on the non-Watson–Crick
base-pairing principle of nucleic acids with opposite chirality. The
experimental details shown below have been demonstrated and reported in our
recent publications that employed (UUAGGG)4,
which is part of the telomeric repeat-containing RNA (TERRA) sequence,
and the human telomerase RNA (hTERC rG4) as our targets, and in
this Protocol Extension we have summarized this information as a protocol and
resource for the scientific community.
Applications of the
method
As we have successfully showcased the validity of rG4-SELEX using
different rG4 targets in our proof-of-concept studies, we believe this method
can be a novel strategy to create highly specific, noncytotoxic and
nuclease-resistant rG4-targeting probes. The unique tertiary interaction
between each L-aptamer (L-Apt.) and D-RNA G4 can
potentially achieve an unprecedented specificity in G4 targeting, i.e.,
distinguishing an individual rG4 from other RNA structure motifs such as
hairpins and stem loops, or even between dG4s and rG4s. In terms of potential
biological applications, we have demonstrated that these spiegelmers can
interfere with the binding of the target rG4s with biologically relevant
peptides or proteins, with a half-maximal inhibitory concentration (IC50) comparable to
state-of-the-art small-molecule G4 ligands, which may be used as a strategy for
G4 targeting therapeutics. The nuclease-resistant nature of these
rG4-targeting L-RNA aptamers also allows them to be promising
probes for developing trackers of G4 folding and unfolding dynamics, or
high-specificity vehicles for delivery in cells by coupling them with
fluorophores or other moieties of interest, respectively. Besides that, these
rG4-targeting L-RNA aptamers can also be employed as rG4 ligands
to regulate rG4-mediated gene expression and RNA metabolism, as well as control
of gene activity for diverse applications. Recently, Tolnai developed a
sandwich detection assay based on two different speigelmers that bind to the C-
and N- termini of cardiac troponin I, respectively, which suggests spiegelmers
can be developed into highly sensitive and specific sensors for biopolymers. A
similar strategy may be designed for biosensing of rG4 using rG4-targeting
spiegelmers.
Even though our method was developed and optimized for rG4s, it is
likely that our strategy can be applied to other noncanonical nucleic acids
structures such as pseudo knots and i-motifs, potentially by refining
the selection conditions to meet the folding of these specific types of
structure motifs. In particular, pseudo knot RNAs were reported to be a highly
conservative motif in the 3′ untranslated region (3′UTR) of coronaviruses and
other regions that regulate gene expressions in viruses through mechanisms like
frame shifting. They were also found to be an essential element for
standby-mediated translation in bacteria and regulation of human
telomerase RNA activity. DNA i-motifs, whose in vivo existence has long been
controversial due to their structural instability at physiologically relevant
pH range, were recently shown to be detectable in the nuclei of living cells.
This noncanonical structure generally appears in the complementary strand of
dG4s with high proximity, although its biological role is still elusive and
requires further investigation. While discovery of small-molecule ligands for
these noncanonical nucleic acid structural motifs is feasible and on-going, it
may be a challenging process, and often requires laborious chemical synthesis,
purification and characterization; thus, we think that our spiegelmer approach
may serve as an alternative and practical option, especially for biochemists
and molecular biologists.
Comparison with
other methods
A large collection of G4 detection methods is available, with many that
show specificity toward G4 over non-G4 structural motifs such as duplexes and
hairpins. One of the most commonly employed G4 detection tools is to use
small-molecule ligands—low molecular weight (LMW) organic or inorganic
compounds that typically show notable fluorescence enhancement or increase in thermo
stability in the form of melting temperature (Tm), or stronger
binding affinity (lower dissociation constant Kd), while
interacting with G4s. The use of G4 ligands has been widely adopted both in
vitro and in cells for diverse chemical and biological applications, and some
of these G4 ligands can stabilize the formation of G4s and dissociate G4–G4
binding protein complexes, which may be further developed as therapeutics. For
instance, commercially available ligands like N-methyl
mesoporphyrin IX (NMM) and Thioflavin T (ThT) have
shown promising G4 sensitivity in vitro; thus, their fluorescence signal has
widely been applied as one of the G4 detection methods. Yet, unlike
the L-RNA aptamers that we are presenting here, most ligands lack the ability
to achieve selective G4 structure binding, i.e., selectively bind to an
individual G4 target over other closely related G4s; recently, however, a few
small-molecule ligands were reported to have a higher specificity toward an
individual G4 target, such as the telomeric multimeric dG4 and the c-MYC dG4.
These ligands showed preferred binding preference toward one specific G4 over
other G4s, highlighting that individual G4 targeting should be feasible with G4
ligands, potentially by rationally designing ligand side chains to recognize
the groove and loops of G4 to provide additional specificity. However, the
rationale behind such a property is not fully explained and whether such a
strategy can be applied easily to any other G4 targets is unclear given the
limited data. Apart from ligands, other G4 detection approaches using
G4-specific antibodies and G4-specific peptides are also available. Similar to
G4 ligands, they showed excellent binding toward G4s over non-G4s; however, so
far there are only limited data on their ability to distinguish individual G4s
over other G4s. Notably, Chen et al. utilized a supramolecular host–guest
sensing array that allows efficient distinction of G4s of different topologies,
while a few recent studies have reported guanine- or guanine analog–conjugated
antisense oligonucleotides or peptides as new strategies to achieve greater G4
target specificity. Interestingly, some innovative methods like targeting G4
with small-molecule ligand-labelled oligonucleotides or G-rich displacing
oligonucleotides with either modified nucleic acids or its analogs have shown
some success in distinguishing G4 targets with high sequence specificity.
However, these conjugate probes require additional recognition sites with the
G4 targeting module to achieve such specificity. Compared with these
above-mentioned approaches, the unique tertiary interaction between an L-RNA aptamer
and D-RNA G4 provides uncharted territory to explore the possibility of
achieving selective rG4 targeting through specific interaction between D- and L-nucleic acids,
potentially with higher G4 specificity than other currently available
approaches without the additional recognition sites.
Limitations
The synthesis of L-RNA for long transcripts (e.g., 50 nt or above)
cannot be easily achieved, making it difficult to target long RNAs, and the
associated costs are generally more expensive compared with D-RNA; therefore, it
is recommended to design the target and optimize the aptamer to be shorter in
length. Another potential solution is to evolve and optimize DNA/RNA polymerase
and reverse transcriptase that can incorporate L-DNA/RNA
nucleotides, such that the rG4-SELEX can be carried out using L-DNA/RNA instead
of D-DNA/RNA. During the negative selection step, the original SELEX
protocol used nitrocellulose membrane to remove membrane-specific RNAs,
whereas here we use magnetic beads to remove bead-specific RNAs because the use
of nitrocellulose membrane is not feasible when targeting RNAs rather than
proteins. However, the magnetic beads approach is not sufficient to prevent the
selection of nonspecific RNAs, i.e., sequences that do not bind to our intended
target. From our lab’s experience, we found that these nonspecific RNAs arise
due to imperfect selection and over-amplification in the rG4-SELEX process;
thus, these nonspecific sequences also showed up in the final sequencing
results. Hence, in addition to beads, the use of scramble RNAs or mutant rG4 in
the negative selection can potentially improve the selection of highly specific
aptamers. In addition, minimal number of PCR cycles should be used in the
amplification steps to minimize bias introduced by PCR amplification. Like the
original protocol, we have successfully employed Sanger sequencing platform to
sequence and identify potential aptamer candidates; however, this low-throughput
sequencing platform can only provide limited number of sequencing data, which
restricts the number of potential candidates for consideration. A possible
solution is to employ a next-generation sequencing platform, which can provide
a much larger candidate pool for analysis and further verification.
Source: https://4dg2.short.gy/MehKl7
Conference Name: 6th International Conference on Molecular Biology and Nucleic
Acids
Date: August 15-16, 2022
Palace: Chicago, USA
Whtsapp
URL: https://4dg2.short.gy/gl1Hac
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