Available online at www.sciencedirect.
com
ScienceDirect
Computational approaches in design of nucleic
acid-based therapeutics
Mark Sherman1 and Lydia Contreras2
Recent advances in computational and experimental methods
have led to novel avenues for therapeutic development.
Utilization of nucleic acids as therapeutic agents and/or targets
has been recently gaining attention due to their potential as
high-affinity, selective molecular building blocks for various
therapies. Notably, development of computational algorithms
for predicting accessible RNA binding sites, identifying
therapeutic target sequences, modeling delivery into tissues,
and designing binding aptamers have enhanced therapeutic
potential for this new drug category. Here, we review trends in
drug development within the pharmaceutical industry and ways
by which nucleic acid-based drugs have arisen as effective
therapeutic candidates. In particular, we focus on
computational and experimental approaches to nucleic acid-
based drug design, commenting on challenges and outlooks
for future applications.
Addresses
1
Cell and Molecular Biology Graduate Program, University of Texas at
Austin, 100 E. 24th Street, A6500, Austin, TX 78712, USA
2
McKetta Department of Chemical Engineering, University of Texas at
Austin, 200 E. Dean Keeton St., Stop C0400, Austin, TX 78712, USA
Corresponding author: Contreras, Lydia (lcontrer@utexas.edu)
Current Opinion in Biotechnology 2018, 53:232–239
This review comes from a themed issue on Pharmaceutical
biotechnology
Edited by Amanda Lewis and Nripen Singh
https://doi.org/10.1016/j.copbio.2017.12.001
0958-1669/ã 2018 Published by Elsevier Ltd.
Introduction
Over almost 80 years of FDA regulation, the field of drug
discovery and development has transformed its focus from
pain management of disease symptoms to systematic
identification of causative factors and targeted mediation
(Figure 1). Early pharmaceutical development stemmed
from the isolation and concentration of active agents from
natural products by chance discovery. Although the ther-
apeutic mechanisms of these substances were unclear at
the time, most contemporary drug development efforts
aim to target specific disease-associated proteins. The
majority of approaches involve screening small molecule
Current Opinion in Biotechnology 2018, 53:232–239
libraries for target protein inhibition that lead to thera-
peutic effects. While these efforts have resulted in hun-
dreds of new drug entities, the journey to approval for the
average drug is increasingly costly [1

]. The rising cost of
drug development can, in part, be attributed to increased
screening efforts due to low hit rates and to misdirected
investigations based on proteins that show no interactions
with current candidate drug molecule libraries; other
reasons include the high probability of failure during
the scale-up process and the escalating demand by regu-
latory bodies for more safety and efficacy data prior to
approval [1

,2]. Furthermore, many diseases simply can-
not be influenced by small molecules due to the inability
to identify a binding pocket in key proteins underlying
disease pathways to serve as a suitable drug target. In
addition, challenges to small molecules arise from the
possibility that disease phenotypes are caused by mecha-
nisms upstream of protein production, such as improper
gene splicing, without a suitable small molecule target.
In recent years, drug development efforts have become
more sophisticated, relying less on large-scale screening
of chemical compounds and more on targeted develop-
ment of drugs toward specific molecular entities. Modern
drug development pipelines attempt to target a wider
spectrum of molecules beyond traditional enzyme targets,
including metabolites [3], protein–protein interactions
[4], and nucleic acids [5]. These advances have enabled
treatment of various diseases through the development
and engineering of new vaccines (with over 30 active
vaccine-associated clinical trials currently underway (clin-
icaltrials.gov)), peptide therapeutics such as Parsabiv [6],
nucleotide prodrugs such as Vosevi [7], as well as a variety
of antibody-based technologies including antibody–drug
conjugates (used in Mylotarg) [8,9], phage display (used
in development of Humira) [10,11
,12], and radiolabelled
diagnostic antibodies as used in Netspot [13–15].
Incentives and challenges to nucleic-acid
therapeutic development
While protein-based therapeutics (antibodies, recombi-
nant proteins, and peptides) have made up over a quarter
of FDA approved drugs since 2015 (FDA.gov), they have
relatively a short shelf life, require refrigerated transport,
and rely on live organisms for production, making
manufacturing them expensive, contamination prone,
and variable across batches [16

]. In response to these
shortcomings, recent efforts have been made toward
development of nucleic acid-based drugs, offering the
key advantage of chemical manufacturing. As such, they
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 Computational approaches in nucleic acid therapeutics Sherman and Contreras 233

Figure 1

1939-1959 1960-1999 1990-2017

N= 27 N=111 N=357
Current Opinion in Biotechnology

An analysis of indications and usage descriptions from FDA approved drug labels across three time periods from 1939 to 2017. Between
1939 and 1959, approved drugs appear to be associated with pain management and symptomatic relief. Between 1960 and 1989, small molecule
inhibitors appear to become mainstream and birth control is developed. Between 1990 and 2017, inhibition continued to be a major theme for
pharmaceutical development and drugs became more specific toward particular diseases, including viruses such as HIV. During the last 30 years,
the emergence of nucleic acids as targets (nucleosides) also represents a newer phenomenon. Data generated by an analysis on a collection of
drug labels through U.S. National Library of Medicine’s DailyMed website.

maintain a relatively long half life, are easily transport-
able, are consistent from batch to batch, and have the
potential to introduce new therapeutic chemistries [17] to
the current, inhibitor-dominated field (Figure 1). For
instance, DNA has been identified as a target for drug
development [18] for its gene therapy ability via manip-
ulation of gene expression or editing of defective genes
[19
]. Likewise, RNA variants have been implicated in a
variety of disease phenotypes such as Spinal Muscular
Atrophy (SMA) [20], Duchenne Muscular Dystrophy
(DMD) [21,22

], Alzheimer’s Disease, and cancer
[23,24
]. Therefore, development of nucleic acid-based
drugs hold promise to provide high affinity and high
specificity entities for therapeutic applications; however,
progress in this field has been limited by challenges in
delivery to specific tissues, a very active area of research
(discussed later) [25–31].
In 2016, three nucleic acid-based therapies gained FDA
approval: Exondys-51 [22

] (for treatment of DMD),
Spinraza [20] (for treatment of SMA), and Defitelio [32]
(for treatment of renal or pulmonary dysfunction). Both
Spinraza and Exondys-51 are antisense oligonucleotides
that therapeutically influence splicing patterns of dis-
eased proteins. The mechanism for Defitelio has not
been elucidated [32]. Several other RNA-based or
RNA-targeting drugs are currently in clinical trials
(Table 1). The majority of these candidate drugs involve
infusion of chimeric antigen receptor (CAR) T cells
while others are siRNA (small interfering RNA), shRNA
(small hairpin RNA), and mRNA (messenger RNA)
based. CAR T cells are derived from the patients own
T cells which are genetically modified ex vivo and intro-
duced back into the body with enhanced therapeutic
functions. Currently approved RNA-based drugs are
administered as local injections or systemic infusions
(Table 1). As research in nucleic acid-based therapeutics
progresses, it holds promise to develop drugs that can not
only inhibit action of a protein, but could alter gene
expression and metabolic flux through development of
aptamers (RNA analogs of antibodies), prodrugs,
siRNAs, miRNAs, and new vaccines with more efficient
modes of delivery.
Current strategies toward aptamer-based
therapeutic development
Due to their ability to bind to diverse targets such as ions,
dyes, amino acids, RNAs, oligosaccharides, antibodies,
and cells, aptamers have become the main focus of
nucleic acid-based therapeutic development [33]. Apta-
mers are short, single-stranded DNA or RNA oligonu-
cleotides that bind to a variety of target molecules with
high affinity and specificity, dependent on their tertiary
structure [34]. The majority of DNA and RNA aptamers
are experimentally developed via Systematic Evolution
of Ligands by Exponential Enrichment (SELEX)

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234 Pharmaceutical biotechnology

Table 1

Nucleic acid-based drugs recently approved or currently in clinical trials

Current Active ingredient/indicated study Indicated condition Mode of delivery Intervention

phase
Early Non-Viral, RNA-Redirected Hodgkin Lymphoma Intravenous (IV) infusion CD19 RNA redirected
Phase 1 Autologous T Cells autologous T-cells (RNA
CART19 cells)
Early Autologous T Cells Expressing MET Malignant Melanoma Breast Intravenous (IV) infusion T cells modified with RNA
Phase 1 scFv CAR (RNA CART-cMET) Cancer anti-cMET CAR
Phase 1 RNActive1 Rabies Vaccine (CV7201) Rabies Intravenous (IV) injection mRNA
Phase 1 Hypoxia-inducible Factor 1a (HIF1A) Carcinoma, Hepatocellular Intravenous (IV) infusion mRNA Antagonist
Phase 1 Pbi-shRNATM EWS/FLI1 Type 1 LPX Ewing’s Sarcoma Intravenous (IV) infusion pbi-shRNATM EWS/FLI1
Type 1 LPX
Phase 1 Neutral Liposomal Small Interfering Advanced Cancers Intravenous (IV) infusion siRNA-EphA2-DOPC
RNA Delivery of EphA2
Phase 1 cMet CAR RNA T Cells Metastatic Breast Cancer Intratumor administration cMet RNA CAR T cells
Triple Negative Breast
Cancer
Phase 1 Basiliximab in Treating Patients With Malignant Neoplasms Brain Intravenous (IV) infusion RNA-loaded dendritic cell
Newly Diagnosed Glioblastoma vaccine; Basiliximab
Multiforme Undergoing Targeted
Immunotherapy and
Temozolomide-Caused
Lymphopenia
Phase 1 Gene Transfer Sickle Cell Disease Intravenous (IV) infusion CD34+ HSC cells
transduced with lentiviral
vector containing shRNA
targeting BCL11a
Phase 1 CD19+ CAR T Cells Leukemia Lymphoma Intravenous (IV) infusion Fludarabine
monophosphate;
Cyclophosphamide
Phase 3 Dendritic Cells loaded with Uveal Melanoma Intravenous (IV) infusion Autologous Dendritic Cells
Autologous Tumor RNA loaded with autologous
Tumor RNA
Approved Nusinersen (Spinraza) Spinal Muscular Atrophy Intrathecal injection Antisense Oligonucleotide
(SMA)
Approved Eteplirsen (Exondys 51) Duchenne muscular Intravenous (IV) infusion Antisense Oligonucleotide
dystrophy (DMD)
Approved Defibrotid (Defiteli) Severe hepatic veno- Intravenous (IV) infusion Oligonucleotide
occlusive disease (sVOD)

Data collected from FDA Novel Drug Approval summaries (FDA.gov) and ClinicalTrials.gov.

[35
,36], a process that utilizes multiple rounds of ligand
selection and amplification of RNA or DNA library var-
iants to raise aptamers with high affinity and selectivity to
a target ligand. Aptamers can bind in the pM range of
affinity and exhibit higher specificity and lower off-target
effects than their protein counterparts [37]. The utiliza-
tion of SELEX has contributed greatly in our understand-
ing of RNA biology and development of nucleic acid-
based therapeutics; however, its successful application to
aptamers with desirable properties requires 10–20 rounds
of selection, representing weeks to months of develop-
ment effort [16

]. While the coupling of SELEX with
other molecular technologies such as high throughput
sequencing, microarrays and bioinformatics have shown
a reduction in selection efforts [38–41], computational
modeling approaches for the rational design of aptamers,
particularly RNA aptamers, show promise to greatly
reduce development time and associated costs [16

].
Role of computational approaches in
aptamer-based therapeutic design
The structural makeup of aptamers are key for their use as
effective therapeutics [33,34]; therefore, development of
structure prediction algorithms represents a significant
undertaking in the rational design of aptamer therapeutics.
Structural predictions from nucleic acid sequences are
challenging due to structural differences observed in vivo
versus in vitro [42

], a high number of transitional states
[43

], and the influence of metal ions, other RNAs, small
organic molecules, and proteins on 3D RNA structure
[33,34,44]. To address these challenges, bioinformatics
and computational modeling approaches have been con-
structed to statistically analyze existing datasets to increase
the likelihood of successful aptamer design. In particular,
databases of RNA-RNA interactions [45], RNA-DNA
interactions [46], and nucleic acid-protein interactions
[47] have been compiled to assist in these efforts [16

].

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 Computational approaches in nucleic acid therapeutics Sherman and Contreras 235
In this scheme, once a target for the aptamer has been
identified, structural data of both the target and the ideal
aptamer is gathered or generated. This is generally done
through crystallography-based approaches [48], homology
modeling using modeling software like ModeRNA [49–
51], or through the use of a thermodynamics-based model
such as RNAup [52] (Table 2). Furthermore, several
models have been introduced in recent years to derive
secondary structure from primary RNA sequence such as
mFold [53] and ViennaRNA [54] (Table 2). In general,
these models take into account a series of user defined
constraints to predict RNA secondary structure through
energy minimization algorithms. Models have also been
developed to predict 3D tertiary structure from secondary
structure (MC-FOLDjMC-Sym [55]) and even directly
from primary sequence using coarse grained molecular
dynamics approaches (NAST [56], ERNWIN [57])
(Table 2). A graphical summary of these models based
on complexity is shown in Figure 2. For modeling RNA–
RNA interactions of uncharacterized RNAs, a desirable
metric is the identification of accessible regions of target
RNAs in vivo [50]. To address this challenge, models have
been developed and benchmarked to investigate the
likelihood for RNA–RNA binding. These include
IntaRNA [58], an RNA–RNA prediction algorithm based
on seed constrains, and InTherAcc [43

], an informed
biophysical model based on biological data collected in
vivo capturing RNA accessibility for binding using an in
vivo RNA Structural Sensing System [42

]. To help
inform crystal structure determination and provide experi-
mental validation for models, several biochemical techni-
ques have been useful; these include hydroxyl radical
footprinting (probing for protected nucleotides), selective
20 OH acylation by primer extension (SHAPE) (chemical
probing for backbone flexibility), and dimethysulfate
sequencing (probing for H bonding) [59].
Table 2
A collection of modeling programs referenced in this article, their URL address, and a short description of their function
Model Website
ModeRNA http://genesilico.pl/moderna/
MC-FOLDjMC-Sym http://www.major.iric.ca/MC-Fold/
NAST https://simtk.org/projects/nast
ERNWIN http://rna.tbi.univie.ac.at/ernwin
IntaRNA http://rna.informatik.uni-freiburg.de/IntaRNA/
Input.jsp
RNAup http://rna.tbi.univie.ac.at/cgi-bin/
RNAWebSuite/RNAup.cgi
mFold http://unafold.rna.albany.edu/?q=mfold
ViennaRNA https://www.tbi.univie.ac.at/RNA/
AutoDock Vina http://vina.scripps.edu/
HADDOCK http://www.bonvinlab.org/software/haddock2.
2/haddock-start/
PatchDock https://bioinfo3d.cs.tau.ac.il/PatchDock/
Data collected from model-associated publications and their web portals.
www.sciencedirect.com
Once tertiary structure is established, models can be
utilized to predict docking location and strength of bind-
ing of nucleic acids and their targets based on atomistic
simulations. These simulations calculate minimum free
energy based on parameters such as electrostatic inter-
actions, Van der Waals forces, and hydrophobic interac-
tions. Atomistic modeling tools such as CHARMM [60]
and AMBER [61] have been used in this fashion to
simulate RNA–metal interactions and several examples
of this approach have recently been reviewed in Sun et al.
[50]. For instance, molecular simulations and Markov
state modeling have been applied to predict the kinetics
of RNA conformational state changes, supporting experi-
mental observations that the binding of specific RNAs can
be relatively slow and involves large conformational rear-
rangements after initial binding [62]. Understanding
these dynamics can allow for efficient use of informatics
tools toward rational design of therapeutic aptamers, a
process analogous to approaches used in protein engi-
neering for therapeutics [63].
Rational design of nucleic acid therapeutic
delivery systems
While the molecular toolkit of nucleic acid-based drugs is
being diversified and potential therapeutic mechanisms
are being elucidated, the challenge to effectively deliver
these therapeutic agents to the site of treatment remains a
major hurdle. That is, for a therapeutic molecule to reach
its target site, several aspects need to be addressed; a few
key ones include prevention of immunogenicity, escape
from degradation enzymes, penetration through cellular
barriers that inhibit uptake, release from liposomes before
degradation, and (depending on its mechanism) transport
to the nucleus to perform intended functions [64,65].
While nucleic acid-based drugs have been experimentally
optimized for individual elements [64], there is not yet an
Description
Comparative modeling of RNA 3D structures
Predicts secondary structures from sequence
Uses coarse grained molecular dynamics and a knowledge-based
force field to generate RNA structures
Predicts RNA 3D structure using a coarse-grain helix-centered model
Prediction of interactions between two RNA molecules
Predicts RNA-RNA interactions
Predicts nucleic acid folding and hybridization
A C code library and stand-alone programs for prediction and
comparison of RNA secondary structures
Molecular docking program to calculate free energy scores
High Ambiguity Driven Biomolecular Docking based on Biochemical
and/or biophysical information
Molecular Docking Algorithm Based on Shape Complementarity
Principles
Current Opinion in Biotechnology 2018, 53:232–239
236 Pharmaceutical biotechnology
Figure 2
Graphical depiction of nucleic acid-based models and their uses across three levels of complexity: 2D RNA structure prediction from primary
sequence, prediction of RNA–RNA interactions, and prediction of RNA–protein interactions. Data collected from model-associated publications
and their web portals.
effective vehicle for targeted drug delivery. Current
therapeutic delivery relies on transport of naked particles
or suboptimal modes of delivery, such as PEGylation and
liposomal based delivery [27,66]. Multi-scale computa-
tional approaches to these issues could complement
experimental methods. Indeed, these approaches have
been implicated both as tools to gain mechanistic insights
on biological properties and as inspirations to develop
novel systems for nanoparticle delivery [27]. Specifically,
the coarse grained molecular dynamics simulation model,
MARTINI [67], has been used to model RNA interac-
tions with other biomolecules and has been implicated for
use in nanoparticle-mediated delivery modeling
[27,68,69].
Future directions of nucleic acid-based
therapeutics
Developments in DNA and RNA sequencing technolo-
gies, genome editing capabilities, and bioinformatics
approaches hold promise to replace dependence of a
comfortable lifestyle on pharmaceuticals with advanced
control of gene expression, particularly for complex dis-
eases caused by genetic anomalies [70]. As computational
technologies become inextricably incorporated into soci-
ety, their role in biotechnology and therapeutics is likely
to expand. In the near future, these new technologies
raise the possibility that a drop of blood placed on a device
similar to a glucose monitor could screen the genome,
transcriptome, proteome and metabolome for abnormali-
ties and those anomalies could be medicated appropri-
ately before the onset of disease. In a perhaps not so
Current Opinion in Biotechnology 2018, 53:232–239
Current Opinion in Biotechnology
distant future, it is possible that genetically engineered
replacement organs could be delivered overnight for
transplantation the following day [71
]. The societal
outcome of these revolutions may transition the role of
health care from one of fixing health issues to one of
surveillance followed by immediate treatment in
response to detection of early disease indicators. Nucleic
acid-based technologies, such as viral gene therapy and
genome editing, enable the potential for permanent
removal of diseases without sustained medication nor
drug development for treatment of disease phenotypes.
This type of therapy could reduce health care costs and
could curtail the impact of complex diseases worldwide.
Conclusions
Nucleic acid-based therapeutics are quickly becoming
relevant within the pharmaceutical industry. With three
oligonucleotide based drugs approved in 2016 and over
40 privately in development [72] (ClinicalTrials.gov), the
nucleic acid-based therapeutic industry appears to be
gathering momentum. Additionally, increasing attention
by the academic research community toward new tools
and delivery mechanisms for DNA and RNA oligonucleo-
tides suggests that we are not far off from advances in
delivery to specific cell types and tissues, providing a new
suite of therapeutic chemistries with fewer side effects
and more efficient modes of action, rivaling antibody-
based therapeutics. The development of computational
tools to identify target molecules (DNA, RNA, protein,
small molecules) and modeling approaches to effectively
predict 3D structure from sequence will likely assist in
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 Computational approaches in nucleic acid therapeutics Sherman and Contreras 237
this effort, potentially identifying targets leading to can-
cer-specific treatments, orphan disease therapies, and
effective precision medicine. Finally, advancements in
computational approaches that enable genome-wide
identification of disease characteristics could provide
early detection of diseases to induce lifestyle changes
for longer, healthier livelihoods.
Funding
Welch Foundation [F-1756]; Funding for open access
charge: Welch Foundation [F-1756]; National Science
Foundation CAREER Program [CBET-1254754].
Acknowledgements
We would like to thank Dr. Elebeoba May for careful reading of the
document and providing feedback and Daniel Herrera for assistance with
data analysis. We would also like to thank K. Alexandra Krippner for
assisting with illustrations.
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