RNA As a Stable Polymer to Build Controllable and Defined

Nano Today (2015) 10, 631—655

Available

online at www.sciencedirect.com

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j ournal homepage: www.elsevier.com/locate/nanotoday

REVIEW

RNA as a stable polymer to build

controllable and deﬁned nanostructures for

material and biomedical applications

a a,b c a

Hui Li , Taek Lee , Thomas Dziubla , Fengmei Pi ,

a,d e e a

Sijin Guo , Jing Xu , Chan Li , Farzin Haque ,

e a,∗

Xing-Jie Liang , Peixuan Guo

Nanobiotechnology Center, Markey Cancer Center, and Department of Pharmaceutical Sciences,

University of Kentucky, Lexington, KY 40536, USA

Department of Chemical and Biomolecular Engineering, Sogang University, Seoul, Republic of Korea

Department of Chemical and Materials Engineering, University of Kentucky, Lexington, KY 40506, USA

Department of Chemistry, University of Kentucky, Lexington, KY 40506, USA

Laboratory of Nanomedicine and Nanosafety, CAS Key Laboratory for Biomedical Effects of Nanomaterials

and Nanosafety, National Center for Nanoscience and Technology of China, Beijing 100190, China

Received 14 July 2015; received in revised form 9 September 2015; accepted 14 September 2015

Available online 26 October 2015

KEYWORDS Summary The value of polymers is manifested in their vital use as building blocks in mate-

Biopolymer; rial and life sciences. Ribonucleic acid (RNA) is a polynucleic acid, but its polymeric nature

in materials and technological applications is often overlooked due to an impression that RNA

RNA nanotechnology;

is seemingly unstable. Recent ﬁndings that certain modiﬁcations can make RNA resistant to

RNA nanostructure;

RNase degradation while retaining its authentic folding property and biological function, and

RNA therapeutics;

the discovery of ultra-thermostable RNA motifs have adequately addressed the concerns of RNA

phi29 DNA packaging

motor; unstability. RNA can serve as a unique polymeric material to build varieties of nanostructures

pRNA including nanoparticles, polygons, arrays, bundles, membrane, and microsponges that have

potential applications in biomedical and material sciences. Since 2005, more than a thousand

publications on RNA nanostructures have been published in diverse ﬁelds, indicating a remark-

able increase of interest in the emerging ﬁeld of RNA nanotechnology. In this review, we aim

to: delineate the physical and chemical properties of polymers that can be applied to RNA;

introduce the unique properties of RNA as a polymer; review the current methods for the con-

struction of RNA nanostructures; describe its applications in material, biomedical and computer

sciences; and, discuss the challenges and future prospects in this ﬁeld.

BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

∗

Corresponding author. Tel.: +1 859 218 0128; fax: +1 859 257 1307.

E-mail address: [email protected] (P. Guo).

http://dx.doi.org/10.1016/j.nantod.2015.09.003

632 H. Li et al.

Introduction chemical diversity offer a rich repertoire of available prop-

erties from which one can custom-design a polymer for a

desired application.

Polymers have been extensively used by humans since the

Biological macromolecules, as natural building blocks,

earliest recorded history. Tree saps, tar and plant ﬁbers, all

are critical for the functioning of living organisms. RNA is

represent the earliest natural polymeric materials used by

one of the ﬁve most important biological macromolecules

humans [1].

in addition to DNA, proteins, lipids and carbohydrates.

Since the 1950s, through continued synthesis and

With some aspects similar to DNA, RNA, composed of four

manufacturing improvements, synthetic polymers have dra-

nucleotides including adenosine (A), cytosine (C), guanosine

matically improved in quality and strength to the point

(G) and uridine (U), is special in its homogeneity. RNA is

where they, in many cases, can have mechanical properties

a homopolymer of nucleotide, but is also a heteropolymer

that better match desired use, as compared to alternative

of A, U, G, and C. Each nucleotide contains a ribose sugar,

natural counterparts. Synthetic polymers have the manufac-

a nucleobase, and a phosphate group. The nucleotides are

turing advantages of rapid production rates and synthetic

covalently linked together through 3 → 5 phosphodiester

simplicity. However, owing to the inherently random nature

bonds between adjacent ribose units, giving the direction-

of most polymerization mechanisms, chain polydispersity

ality to the sugar-phosphate backbone that deﬁnes RNA as

and controlled shaping remain two of the key limiting

a polynucleic acid. The phosphate moieties in the backbone

hurdles that have led to increased variable performance

are negatively charged, making RNA a polyanionic macro-

and overall reduced control of the ﬁnal properties [2—4].

molecule at physiological pH. RNA molecules are typically

As such, there has been much effort towards develop-

single-stranded; however, Watson-Crick (canonical) base-

ing synthetic building blocks and schemes that can create

pair interactions (A:U and G:C), wobble base pairing (such

monodisperse polymer systems [5—7]. These, however, are

as G:U) [24] or other non-canonical base pairing such as

typically very expensive, time consuming and difﬁcult to

control. twelve basic geometric families of edge-to-edge interac-

tion (Watson-Crick, Hoogsteen/CH or sugar edge) with the

As a result of these developments, we are at another

orientation of glycosidic bonds relative to the hydrogen

unique turning point in the evolution and design of polymers.

bonds (cis or trans) [25—28], all together give rise to various

We once again turn towards nature as our inspiration for the

structural conformations exhibiting loops, hairpins, bulges,

development of advanced biomaterials. By applying what we

stems, pseudoknots, junctions, etc., which are essential ele-

have learned about the complexity and versatility of nucleic

ments to guide and drive RNA molecules to assemble into

acid structures, it is now possible to create materials with

desired structures [12,29—31].

revolutionary properties previously considered unattainable

On the contrary, synthetic polymers are usually formed

through synthetic strategies. Recent advances in RNA chem-

via polymerization reaction, a chemical process through

istry, RNA biology and RNA nanotechnology have shown that

which many monomers form covalent bonds between each

RNA as a biopolymer not only shares the common character-

other and ﬁnally form a long chain or network, imparting

istics of other polymers, but also possesses a range of unique

them with their desired structural properties.

properties advantageous for applications in nanotechnology

Generally, the properties of polymers are strongly

as well as biomedical and material sciences [8—14].

dependent on their monomer chemistry. Moreover, the

In this review, we will (1) delineate the physiochemical

molecular weight and 2D structure (e.g., linear chain-

properties of polymers that can be applied to RNA, (2) intro-

shaped structure or branch-shaped structure) also play

duce the unique properties of RNA as a polymer, (3) review

important roles in determining the characteristics and

the current methods for constructing RNA nanostructures for

properties of a speciﬁc polymer. Important properties of

diverse applications, and (4) discuss the future prospects

chemical polymers include [32]: (1) rheological properties,

including challenges, solutions and new directions of this

(2) solubility, (3) volumetric and viscometric properties,

nascent ﬁeld.

(4) stress-strain relationships, (5) electrical properties, (6)

thermal properties, (7) optical properties, (8) stiffness, (9)

Physical and chemical properties of polymers

ﬂex life, (10) hardness, (11) chemical resistance, etc. Vari-

in relationship to RNA nanotechnology ous physical or chemical tests can be readily performed to

characterize these properties for RNA polymers.

The word (poly)-(mer) means (many)-(parts) and refers to Traditional synthetic polymers are heterogeneous and

macromolecules consisting of a large number of repeating have typical polydispersity index greater than 1 [33,34].

elementary units covalently joined together. These ‘mers’ In contrast, RNA is synthesized by either in vitro tran-

control the inter-chain interactions, and dictate the ﬁnal scription or solid phase chemistry. Both approaches are

polymer characteristics. Given the large variations in poly- based on stepwise reactions and will generate RNA poly-

mer morphologies, multiple ways are used to describe mers with deﬁned sequences, structures, and molecular

polymer structure. Polymers can be formed from one type weights. This leads to a polydispersity index of 1 of a speciﬁc

of monomer (homopolymers) or of various monomers (het- RNA. Structurally, the average molecular weight deter-

eropolymers). Heteropolymers can be further described mines the mechanical property of the polymer. However,

by the arrangement of the monomers along the back- as polydispersity increases, the increased lower molecu-

bone, including alternating, random, and block formations lar weight chains can act as a lubricating/solvating system,

and also adopt more elaborate structures, including star, reducing its mechanical strength and integrity. As increases

ring, branched, dendrimeric, crosslinked, ladder, and oth- in temperature result in increased molecular motion,

ers [15—23]. These large arrays of potential structures and these weak associations begin to destabilize and reduce

RNA as a stable polymer 633

mechanical integrity. Such polymers are classically referred Thermodynamic stability of RNA

to as thermoplastics [35], as their mechanical strength

weakens with increased temperature. However, this prop- From a thermodynamic point of view, the stability of

erty also means they can be intentionally deformed at higher the double helix of RNA can be evaluated by measur-

temperatures and then regain their structural properties ing the Gibbs free energy (G ) required for double

once cooled. Thermodynamically, RNA molecule is relatively helix formation, or conversely, double helix unwinding

0 0 0

more stable at lower temperature and intends to dissoci- −

( G = G helix formation = G helix unwinding). Remarkably, the

ate or misfold at higher temperatures. Thus, RNA displays RNA double helix is more thermodynamically stable than the

the typical thermoplasticity of polymers. Boiling-resistant DNA double helix considering G for RNA double helix for-

RNA has been discovered and constructed [36], suggesting a mation is, on the average, −3.6 to −8.5 kJ/mol per base

potential application of RNA in molding technology. pair stacked and G for DNA double helix formation is

−

Chemical polymers have a wide range of applications 1.4 kJ/mol per base pair stacked [52]. Moreover, the pres-

in biomedical and material sciences. Speciﬁc applications ence of special motifs such as bends, stacks, junctions and

include, but not limited to [1,37—50]: (1) sutures for surgery, loops in the tertiary structure of RNA may also further

(2) dental devices, (3) orthopedic ﬁxation devices, (4) scaf- improve its stability [8]. In addition, various proteins, such

folds for tissue engineering, (5) drug formulation (e.g., as RNA chaperone proteins, and metal ions, such as Mg

controlled release of drugs, excipients for drug stabiliza- may also interact or coordinate with RNA and signiﬁcantly

tion or solubilization), (6) biodegradable vascular stents, (7) contribute to the stability of RNA [53,54].

biodegradable soft tissue anchors, (8) implantable biomed-

ical devices, (9) ﬂexible transparent displays, (10) ﬁeld

effect transistors, (11) solar cell panels, (12) printing elec- Enzymatic stability of RNA

tronic circuits, (13) organic light-emitting diodes, (14)

supercapacitors, etc. Usually, an ideal chemical polymer In biological systems, the instability of RNA is mainly the

for biomedical and industrial applications should exhibit result of enzymatic degradation by ribonucleases (RNases).

the majority of the following properties: (1) non-toxic, (2) The presence of RNases in living organisms is universal,

no or low immunogenicity (if applicable, for in vivo use), suggesting that RNase-mediated RNA degradation is a vital

(3) biodegradable and metabolizable, (4) mechanical fea- process for normal biological function. Several important

tures that ﬁt the needs of the speciﬁc application, (5) easily roles of RNases have been revealed, including degrading sur-

sterilizable, (6) processability, (7) scalability, (8) electrical plus cellular RNA [55,56], editing messenger RNA, processing

conductivity, and (9) thermostabilty. RNA as a polymer favor- microRNA and other non-coding RNA during their maturation

ably displays many of these properties, as described below. [57,58], defending against the invasion of viral RNA [59],

and providing crucial machinery for RNA interference (RNAi)

[60,61]. RNases can be divided into two major categories: (1)

endo-ribonucleases that cleave phosphodiester bonds within

Unique properties of RNA as a polymer to

the RNA backbone [62]. Examples include RNase A, RNase H,

build nanostructures

RNase I, RNase III, RNase L, RNase P, RNase PhyM, RNase T1,

RNase T2, RNase U2, RNase V, etc.; (2) exo-ribonucleases

The 2 -hydroxyl group makes RNA dramatically

that cleave phosphodiester bonds at either the 5 or the 3

different from DNA

end of an RNA chain [63]. Examples include polynucleotide

phosphorylase (PNPase), RNase PH, RNase II, RNase R, RNase

The characteristic of RNA that deﬁnes and differentiates it

D, RNase T, oligoribonuclease, exoribonuclease I, exoribonu-

from DNA is the 2 -hydroxyl on each ribose sugar of the back-

clease II, etc. RNA is indeed very sensitive to degradation by

bone. The 2 -OH group offers RNA a special property, which

RNases, which confers a very short half-life and thus a poor

can be either an advantage or a disadvantage. From a struc-

pharmacokinetic proﬁle to most RNA molecules. This degra-

tural point of view, the advantage of this additional hydroxyl

dation limits the in vivo application of RNA molecules as

group is that it locks the ribose sugar into a 3 -endo chair

therapeutics. However, chemical modiﬁcations of RNA can

conformation. As a result, it is structurally favorable for the

overcome this shortcoming. For example, the substitution

RNA double helix to adopt the A-form which is ∼20% shorter

of the 2 hydroxyl group with a Fluorine (2 -F), O-methyl

and wider rather than the B-form that is typically present in

(2 -O-Me) or Amine (2 -NH2) dramatically increases the sta-

the DNA double helix. Moreover, the 2 -OH group in RNA is

bility of RNA in vivo by preventing degradation by RNases

chemically active and is able to initiate a nucleophilic attack

[64—66]. Recent studies also showed that the stability of

on the adjacent 3 phosphodiester bond in an S 2 reaction.

N siRNA in serum is also highly depended on the speciﬁc RNA

This cleaves the RNA sugar-phosphate backbone and this

sequences and the degradation of both short and long RNA

chemical mechanism underlies the basis of catalytic self-

duplexes mostly occurred at UA/UA or CA/UG sites [67].

cleavage observed in ribozymes [51]. The disadvantage is

Synthetic polymers always show good stability against

that the 2 -OH group makes the RNA susceptible to nuclease

various enzymes. However, biodegradable polymers that can

digestion since many RNases recognize the structure of RNAs

be degraded by enzymes such as proteases, oxidoreduc-

including the 2 -OH group as speciﬁc binding sites. However,

tases and phospholipases are more beneﬁcial for therapeutic

such enzymatic instability has been overcome by applying

applications [68]. The nanocarriers composed of enzyme

chemical modiﬁcation of the 2 -OH group (see the sections

sensitive polymers can control the site of cellular uptake

Enzymatic stability of RNA and Methodology II: Chemical

of carriers and drug release to improve the drug efﬁcacy

modiﬁcation of RNA).

[69—71]. Synthetic polymers can also be designed to be

634 H. Li et al.

◦ ◦ ◦

non-degradable or degradable under certain physiological phi29 DNA packaging motor from 60 to 90 to 108 to transit

conditions. For example, Poly(lactic-co-glycolic acid) (PLGA) from triangle, square, and pentagon structures, respectively

(which is generally regarded as safe) is approved by FDA, as [92].

it can be degraded in the body by hydrolysis and has now

been widely used to fabricate nanoparticles for drug deliv-

RNA can form complex 3D structures in nanoscale

ery. Poly (beta-amino ester) is another class of degradable

biomaterial developed by Langer group [72], which can bind

While most biologically active DNAs are double-stranded, in

negatively-charged RNA or DNA and facilitate gene trans-

contrast, most RNAs in living organisms are single-stranded.

fections. Although the biodegradable polymers are highly

However, RNA molecules contain self-complementary

attractive for drug delivery purposes, their degradation

sequences which facilitate the self-folding of these RNAs.

rate is very hard to control and the pharmacokinetics and

In addition to Watson-Crick base paring, non-Watson—Crick

biodistribution proﬁles of the nanocarriers can be elusive.

base pairs and coaxial stacking of helices also play important

Synthetic polymers usually are composed of repeated same

roles in promoting RNAs folding into complex 3D structures

subunit, while RNA is the repetition of four subunits A, U, G

[12,53]. This remarkable folding capability not only provides

and C in a speciﬁc sequence order. Therefore, RNA polymers

the structural basis for the diverse biochemical functions of

might be advantageous concerning controllable biodegrad-

different RNA molecules, but also provides huge opportuni-

ability [73,74] simply by tuning the ratio and location of

ties for designing novel RNA nanostructures. For example,

2 -modiﬁed nucleotides in the RNA sequence.

the subdomain IIa-1 RNA of the IRES from HCV has a unique

◦

90 bend L-shaped structure. Molecular modeling using this

L-shaped motif showed that a square shaped nanoparti-

The versatile and plastic properties of RNA

cle can be formed with four repeating L-shaped motifs, as

veriﬁed by gel shift assay and FRET assay [93] (Fig. 1I).

The versatility of RNA is highly evident given the diversity

Moreover, it has been found that proteins, small molecule

of structural repertoires available in nature, which include

ligands as well as monovalent and/or divalent metal ions

simple structures such as helical stems and single stranded

are also important mediators of RNA folding, which could

hairpin loops to more complicated structures such as multi-

add another layer of complexity to the assembly of RNA

way junctions and pseudoknots [75—84]. Persistence length

nanostructures [53].

is a basic mechanical property used in polymer science to

measure the ﬂexibility and stiffness of a polymer. If a poly-

mer is shorter than the persistence length, the molecule will Toolkits and methodologies for the

basically be a rigid rod. From a mechanical perspective, the

construction of RNA nanostructures

persistence length of dsRNA (62—63 nm) is slightly longer

than dsDNA (45—50 nm) in an aqueous solution [85,86]. The

The diverse function of RNA molecules in cells such as

stretch modulus of short dsRNA (<150 bp) is ∼100 pN, which

ribozymes, riboswitches, microRNAs, long non-coding RNAs

is 3 times lower than dsDNA of similar length, is based on AFM

and aptamers had attracted increasing attention in the sci-

and tweezer studies [87]. Interestingly, RNA was observed to

entiﬁc community. All these properties of RNA originated

shorten upon twisting with two-orders of magnitude slower

from nature that RNA can adapt different structures and

timescale compared to DNA, which in contrast lengthened

spatial conformations. The laws of how nature manipulates

with a faster timescale. More recently, single molecule force

RNA structures can also be explored to construct artiﬁcial

spectroscopy were used to study ∼10 nm long dsDNA con-

RNA nanostructures. Besides the template-guided folding of

taining ribonucleoside monophosphates (rNMPs) located at

RNA nanostructures that is similar to the tactics in DNA nano-

speciﬁc positions within the DNA strands [88]. The results

technology, self-assembly of RNA building blocks is another

demonstrated that the perturbation of stiffness and the

leading approach for bottom-up RNA nanotechnology. A

plastic properties by rG intrusions is location and sequence

wide variety of RNA nanostructures have been success-

dependent, and can either soften or stiffen the DNA com-

fully constructed based on different assembly principles and

plex. MD simulations indicated that the perturbations are

approaches (Fig. 1). In this section, we will review several

from local structural distortions arising from hydrogen bond-

toolkits and methodologies that have shown great success

ing between the OH group of rG and electronegative sites of

for constructing multifunctional RNA nanostructures.

either the phosphate backbone or vicinal base. However,

many factors such as sequence, local structural environ-

ment, and metal ion concentrations [89,90] can signiﬁcantly Toolkit I: Hand-in-hand interactions involving RNA

inﬂuence RNA versatility, stiffness and plasticity at the loops

molecular level [91]. The balance of the ion-mediated elec-

trostatic force and the non-electrostatic (chemical) forces Taking the construction of a wooden table as an analogy,

determine the equilibrium structure of the RNA. It is com- external dowels are used to link variety of wood blocks into

mon to ﬁnd that one RNA molecule can exist in more than a structure. One of the unique properties of RNA is its folding

one conformation, and one kind of RNA molecule can dis- into loops and hairpin structures. RNA loops can serve as

play multiple bands in native gel electrophoresis due to the inter-RNA mounting dovetails, thus external dowels are not

redistribution of modular motif and local thermal energy. necessary for self-assembly into unique structure.

The versatile and plastic properties of RNA as polymer have The packaging RNA (pRNA) in phi29 DNA packaging motor

been applied to the construction of RNA polygons by stretch- [94—102] forms a hexameric ring structure through inter-

ing the internal angle of the three-way junction (3WJ) of locking of two looped regions in each pRNA molecule, named

RNA as a stable polymer 635

Figure 1 Design and construction of RNA nanostructures. (A) Phi 29 DNA packaging motor pRNA hexamer [110]. Reprinted with

National Academy of Sciences of the United States of America. (J) RNA microsponges for siRNA delivery [147]. Reprinted with

Science. (N) Boiling-resistant RNA triangles and the array based on RNA triangles [36]. Reprinted with permission from Ref. [36].

2014 John Wiley and Sons.

the right- and left-hand loops (Fig. 1A). The left-hand and for constructing stable RNA polygonal nanoparticles. Loop

right-hand were brought together from interlocking inter- extended RNA pairs with conﬁrmed dimer formation capa-

action among four nucleotide sequences in the loops. Single bility were used as building blocks for higher order RNA

pRNA molecule with self-complimentary sequences in the oligomer nanoparticles (Fig. 1B). Using a reengineered loop

loops can be used to construct homodimer nanoparticles, extended pRNA toolkit, pRNA dimer, trimer, tetramer, pen-

or several pRNAs with the right-hand loop matching the tamer, hexamer, and heptamer were constructed with highly

sequence of the left-hand loop in another pRNA can be used efﬁcient self-assembly, as shown by gel shift assays and AFM

to construct heterodimer nanoparticles. Through the inter- imaging [113].

locking loop interactions between different pRNA, dimer, Other loop—loop interactions have also been reported

trimer, and hexamer RNA nanoparticles have been created to build various RNA nanostructures. For example, nonco-

[103—112]. The interlocking loop sequences were further valent loop—loop contacts based on RNAI/IIi kissing-loop

extended to increase the thermodynamic stability of gen- complex have been used to build RNA nanorings which

erated RNA nanoparticles [113]. A toolkit with a set of are thermostable, ribonuclease resistant and capable of

hand-in-hand loop sequences has been designed and tested delivering RNA interference modules [85,114,115] (Fig. 1E).

636 H. Li et al.

Moreover, RNA kiss-loops were also used to build square- able to bind MG to emit ﬂuorescence as shown in the ﬂuores-

shaped tetrameric RNA nanoparticles and three-dimensional cent spectra (Fig. 2A and B). This property makes pRNA 3WJ

polyhedrons based on rationally designed RNAs [116—118] structure an extraordinary nanocarrier for targeted gene

(Fig. 1D and F). In addition, micrometer-scale RNA ﬁlaments delivery [123]. Moreover, an X-shaped motif from the cen-

have also been constructed by the rational design of tectoR- tral domain of the pRNA was discovered through extending

NAs incorporating 4-way junction (4WJ) motifs, hairpin loops the right-hand loop with a double-stranded sequence [73].

and their cognate loop—receptors [119,120]. The X-shaped pRNA motif can be self-assembled from four

RNA oligos and it creates a scaffold that allows for the

conjugation of four RNA functional moieties at the same

Toolkit II: Foot-to-foot interactions involving

time. Functional RNA modules such as survivin siRNA pre-

palindrome sequence and sticky ends

served their target gene knock-down effect after being fused

to the X-shaped structure (Fig. 2C). Branched hexavalent

pRNA nanoparticles can also be constructed through the foot

RNA nanoparticles were further constructed from three 3WJ

to foot interaction between pRNA monomers. While hand-in-

motifs, integrated by one 3WJ core motif structure or by

hand interaction involve ‘‘left’’ and ‘‘right’’ hand and the

palindrome sequence mediated linking of two pRNA-X cores

interaction is ‘‘multiple’’ and ‘‘inter’’ heterosexual, foot-

[113].

to-foot interaction includes only one foot that is the one

Recently, Khisamutdinov et al. reported using RNA as

unique palindrome sequence by self-homosexual interaction

an anionic polymer to build programmable self-assembling

of the identical RNA molecule. The foot domain is located in

boiling-resistant RNA nanostructures based on pRNA-3WJ

the helical 5 /3 open region of pRNA. Extending this region

[36] (Fig. 1N). A triangular shaped RNA nanoparticle was

with helical sequences does not compromise the entire pRNA

designed and prepared by carefully joining extended helices

molecule folding. A palindrome sequence reads the same

to the thermodynamically stable pRNA-3WJ motif of the

→

whether from 5 3 direction on one strand or from 5 → 3

bacteriophage phi29 DNA packaging motor. The step-wise

direction on the complementary strand. Extending the 3 end

self-assembly of the triangle RNA nanoparticles was con-

of pRNA monomer with a palindrome sequence can serve

ﬁrmed by native PAGE analysis, and AFM imaging revealed

as a sticky end for linking two pRNA monomers, and is

the triangular shape of the nanoparticles as expected.

denoted as a foot-to-foot interaction [112]. The foot-to-foot

The constructed nanoparticles are thermodynamically ultra-

interaction by palindrome sequences can also be utilized to

stable and robust. Functional motifs including siRNA,

bridge RNA nanostructures, motifs, or scaffolds for construc-

ribozyme, folate and ﬂuorogenic RNA aptamers retained

ting RNA hexamers, octamers, decamers, and dodecamers

their activities after conjugation to the RNA nanoparticles.

(Fig. 1K). A pRNA array was constructed by combining

Moreover, these RNA triangles could be used as building

hand in hand-loop-interactions and foot-to-foot palindrome

blocks to construct supramolecular complexes such as RNA

sequence interactions [112] (Fig. 1C). Recently, strategies

hexagons and patterned hexagonal arrays. The pRNA-3WJ

involving both loop—loop interaction and sticky-end cohe-

is also highly tunable [92], the naturally preserved angle

sion were also reported to assemble three-dimensional and ◦

between the helices H1 and H2 of pRNA-3WJ is ∼60 and

structurally well-deﬁned RNA nanoprisms by re-engineering ◦

this angle can be stretched to form square-shaped (90

pRNA [121]. ◦

angle) and pentagonal shape (108 angle) nanoparticles

with pre-designed sequences (Fig. 3A). The polygons formed

Toolkit III: Grafts of motifs from naturally-occurring by one-step self-assembly manner with efﬁciency >90%, as

RNA molecules demonstrated by gel shift assays. The different sizes and

shapes of the polygons were also conﬁrmed by dynamic

Synthetic RNA nanoarchitectures can be designed with the light scattering (DLS) and AFM. The equilibrium dissocia-

known naturally-occurring three-dimensional RNA motifs. tion constants (KD) for triangle, square and pentagon are

◦

Particularly, some RNA motifs in nature exhibit extraordi- 18.8 nM, 20.3 nM and 22.5 nM, and the Tm values are 56 C,

◦ ◦

nary stability, for example, phi29 motor pRNA [54,110,122], 53 C and 50 C, respectively, revealing structure and shape-

tRNA [118], 5S RNA[123], and others [115]. These motifs dependent thermodynamic features. In a recent paper by

have been used to create synthetic RNA nanostructures Jasinski et al, the authors showed that square-shaped RNA

[36,93,118,124,125]. The central domain of the pRNA nanoparticles with ﬂuorogenic and ribozymatic properties as

molecule contains a three way junction (3WJ) core struc- well as different sizes can be successfully constructed by uti-

ture [54]. This 3WJ can be assembled from three individual lizing the pRNA-3WJ at each corner and different length RNA

RNA fragments with high efﬁciency in the absence of metal duplexes at each edge of the square RNA nanoparticles [125]

ions [123]. The 3WJ motif itself creates a branched structure (Fig. 3B). DLS and AFM determined the sizes of the small,

that allows for constructing multifunctional RNA nanoparti- medium, and large square RNA nanoparticles are 4.0, 11.2,

cles with different functional moieties at each end of the and 24.9 nm, respectively. The physiochemical properties of

branches. RNA nanoparticles can also be designed and fab- the nanoparticles can be easily tuned, as the utilization of

ricated via branch extension tool kit [113] based on the 3WJ 2 -F RNA as the core strand signiﬁcantly increased the melt-

motif (Fig. 1L). 3WJ core retained its original folding and the ing temperature as well as the nanoparticle’s resistance to

conjugated RNA module on the branches can fold into their serum-mediated degradation.

authentic structure and remain functional [123,126]. For Naturally occurring stable RNA-protein complexes were

example, after incorporated into 3WJ motif, HBV ribozyme also explored as building blocks for nanostructures. For

retain its capacity to cleave its RNA substrate and generate example, the assembly of archaeal box C/D sRNPs com-

smaller cleavage products, and MG binding aptamer was also prising the L7Ae protein from Archaeoglobus fulgidus and

RNA as a stable polymer 637

Figure 2 Construction and functional assays of thermodynamically stable pRNA-(three-way junction) 3WJ or pRNA-X nanoparticles.

(A and B) HBV ribozyme assay and MG aptamer assay of pRNA-3WJ nanoparticles [123]. Adapted with permission from Ref. [123].

a box C/D RNA has been utilized to build an equilateral tri- [114], and the tetraloops GNRA and UNCG [138] were used to

angle shaped synthetic RNA protein complex [127] (Fig. 1H). mediate internal RNA tertiary interactions, resulting in orga-

The construction relies on the interaction between ribo- nized and scalable RNA crossover tiles. The designed RNA

somal protein L7Ae and the Kink-turn motif in box C/D origami nanostructures are also robust, which could form

RNA. L7Ae can bind to the box C/D K-turn motif through by either annealing or co-transcription on mica. In contrast

hydrogen bonding with high speciﬁcity and afﬁnity [128]. to DNA origami, this RNA origami approach does not need

It was reported that using RNA-protein complex (RNP) as short staple strands to facilitate the folding of the origami

building block for nanostructures enhanced its stability in structure, but using RNA modules such as kissing loops to

serum comparing to pure unmodiﬁed RNA based nanopar- replace the role of staple strands. Interestingly, a differ-

ticles [129]. The RNP nanostructure could be served as a ent RNA origami approach which is a direct extension of

scaffold for protein or RNA functionalities. For example, an the DNA origami to RNA was also reported in 2014 [139]

afﬁbody peptide, which can recognize HER2 receptor, was (Fig. 1P). In this study, a single-stranded RNA scaffold and

connected to RNP triangle through fusing to L7Ae protein, multiple staple RNA strands were used to assemble deﬁned

and the functionality of afﬁbody peptide was still retained. RNA nanostructures including a 7-helix bundled RNA tile and

siRNA targeting to GFP gene can be conjugated to the RNP a 6-helix bundled RNA tube. The authors also showed that

triangle through extending the RNA strand and the GFP siRNA functional modules such as biotin could be introduced into

on RNP nanoparticles still can be processed by dicer and the RNA origami structures by chemical modiﬁcations of the

knock down GFP expression in cells. scaffold strands.

Toolkit IV: RNA origami Toolkit V: RNA/DNA hybrid nanostructures

DNA origami, which involves the folding of a long template RNA/DNA hybrids can also be used to construct func-

single stranded DNA aided by multiple short staple DNA tional nanoarchitectures. Similar to split-protein systems,

strands, was reported in 2006 [130]. A wide range of 2D RNA/DNA hybrids can be computationally designed for acti-

and 3D nanostructures have been successfully constructed vating different split functionalities in the presence of

using the DNA origami technique [131—135]. In 2014, a sim- respective equivalent strands [140—143] (Fig. 5A). Toe-

ilar approach for RNA origami was reported to fold single hold interactions were used to trigger disassociation of

stranded RNA into RNA tiles and further assembled into RNA/DNA hybrids and re-association of the double-stranded

hexagonal lattices [136] (Fig. 1M). A variety of RNA tertiary RNA and DNA. The thermodynamics and kinetics of the

◦

motifs, including the 180 kissing loop motif of the HIV-1 DIS toehold interactions can be tuned to control the dissocia-

◦

[137], the 120 loop—loop complex of RNA i/ii inverse loop tion and re-association processes. RNA interference modules

638 H. Li et al.

Figure 3 Construction of RNA polygons based on pRNA-3WJ. (A) Construction of RNA triangle, square and pentagon by tuning the

as well as other functionalities such as ﬂuorophores and more enzymatically stable than RNA, this approach has the

RNA aptamers can be successfully triggered inside mam- potential to improve the pharmacological proﬁles of RNA-

malian cells. This RNA/DNA hybrids concept has also been based nanoparticles. Moreover, controlled release of the

applied to other nanostructures such as nanorings and cubes split functionalities opens a new avenue for the develop-

[114,115,144—146], and the spilt functionalities still retain ment of nucleic acid-based switches to modulate cellular

their authentic functionalities. Because RNA/DNA hybrid is functions in vivo.

RNA as a stable polymer 639

Methodology I: Rolling circle transcription large-area of RNA membrane. After the cRCT process, the

water in the RNA membrane was evaporated and the dried

Typically, the design and production of RNA nanoparticles RNA membrane was concentrated by EISA process. The self-

have relied on the production of discrete RNA strands that assembled RNA membrane was fabricated densely on the

can be assembled in a controlled and predictive fashion to tube wall during the evaporation process. The durability

generate nanoparticles with a deﬁned structure and stoi- of the fabricated RNA membrane was tested under various

chiometry. Hammond and colleagues have departed from harsh conditions, including RNase and DNase-rich environ-

this approach with an innovative strategy that generates ments. In addition, they conﬁrmed the application of the

monodisperse spherulitic RNA particles from extremely high RNA membrane as an enzyme-responsive drug-release sys-

molecular weight RNA strands by using a rolling circle tran- tem with doxorubicin and siRNA. The results showed that

scription (RCT) approach [147,148] (Fig. 1J). They created the RNA membrane can provide a high drug-loading efﬁ-

a circular DNA construct containing a siRNA gene without ciency and can be a great candidate for future membrane

any terminator sequences and proceeded by a T7 pro- industry.

moter. As a result, T7 RNA polymerase can continuously

transcribe the circular DNA hundreds of times to gener-

ate a tremendous number of copies of the tandem RNA Methodology II: Chemical modiﬁcation of RNA

unit. As the continuous RNA strand is transcribed, magne-

sium pyrophosphate crystals are simultaneously generated, A great deal of work on chemical modiﬁcations aiming to

and the RNA strands attach to the crystallite surfaces to improve the chemical stability and in vivo properties of RNA

form composite structures; each structure grows in length have been reported [153—156]. Common chemical modi-

to become fiber-like, then, forms sheet-like lamellae. The fications of RNA can be categorized into five classes: (1)

lamellae, typically ∼10 nm thick, ﬁnally condense into Modiﬁcation of inter-nucleotide phosphodiester backbone.

spherulites with diameters varying from a few micrometers This type of modiﬁcation is the most classic and simplest

to hundreds of micrometers, referred by the author as RNAi method to improve the performance of RNA in the biological

microsponges. To achieve efﬁcient cellular uptake, PEI was environment. For example, creation of a phosphorothioate

introduced to condense the RNAi-microsponge from 2 ␮m to (PS) linkage by replacing one non-bridging oxygen atom on

200 nm, and to protect the RNA from degradation by RNase. the phosphate backbone with a sulfur atom substantially

The PEI introduced a positively charged outer layer to the increases the stability of RNA in vitro and in vivo [157]. Other

microsponges to facilitate cell binding and entry. It was backbone modiﬁcations such as boranophosphate, phospho-

also demonstrated that the spherulitic RNAi-microsponges ramidate and methylphosphonate have also been explored

produced ∼21 nt siRNA fragments after incubation with to enhance the resistance to nuclease-mediated degrada-

Dicer and could transfect a cancer cell line and silence tion [158—160]. However, cytotoxic side-effects were also

firefly luciferase expression. Gene expression knockdown observed if extensive modifications were applied [161].

in vivo by intratumoral injection of the PEI-condensed RNAi- (2) Substitution of 2 -OH group. This is the most widely

microsponges was also reported. This innovative approach used approach since it is well-tolerated, and can enhance

opens new directions to RNA nanoparticle self-assembly nuclease resistance as well as reduce immunogenicity.

and siRNA delivery. However, the therapeutic potential and For example, the naturally occurring 2 -O-methyl (OMe)

in vivo safety of this approach requires further evaluation, modiﬁcation is nontoxic and able to prevent immune acti-

as PEI has been shown to cause well-known adverse side vation while conferring biological stability simultaneously

effects, such as high cytotoxicity [149,150]. In addition, fur- [161,162]. Guo’s lab also reported that incorporation of

ther reduction of the size of the particles is recommended 2 -Fluoro nucleotides to pRNA scaffold allows the creation

to avoid non-speciﬁc healthy organ accumulation. of stable and RNase-resistant RNA nanoparticles with cor-

In material sciences, polymeric membranes are at rect folding and authentic biological activities. The melting

the forefront in the chemical and biotechnology indus- temperature of 2 -F RNA was further enhanced compared

try because of their versatile applications such as water to unmodiﬁed RNA [163]. The chemically modiﬁed pRNA-

puriﬁcation, dehydrogenation of natural gas, dialysis of based nanoparticles have shown promising applications in

blood, removal of cell particles, and others [151]. Recently, cancer therapy [36,66,164—167]. Recent study also showed

Lee’s group demonstrated rolling circle transcription can that 2 -F modiﬁed pRNA nanoparticles are resistant to I-125

also be applied to the synthesis of macroscopic RNA mem- and Cs-131 radiation with clinically relevant doses, which

branes that has the potential as a controlled drug-release is a required property for applying these RNA nanopar-

system [152] (Fig. 1O). The RNA membrane was fabri- ticles as delivery vehicles for targeted radiation therapy

␤

cated by T7 RNA polymerase transcription system with a [168]. Other options for 2 -modiﬁcation like 2 -ﬂuoro- -D-

combination of complementary rolling circle transcription arabinonucleotide (FANA) have also shown promise in gene

(cRCT) and evaporation-induced self-assembly (EISA). The silencing applications [169]. (3) Locked nucleic acids (LNA)

circular DNA template and complementary circular DNA and unlocked nucleic acid (UNA). LNA is another class of

template were prepared with long linear DNA and the pro- 2 -modiﬁcation in which 2 -O and 4 -C is linked via a methy-

moter DNA for starting the RCT, respectively. Upon addition lene bridge. This locked linkage constrains the ribose ring

of T7 polymerase to these two circular DNA templates, into C3 -endo conformation which confers both signiﬁcant

thousands of copies of single stranded RNA and single increase in thermostability and enhancement in nuclease

stranded complementary RNA were continuously generated. resistance [170]. Successful applications of LNA such as 2 -

The two complementary RNA strands hybridized to each amino-LNA have been studied by Astakhova et al. to show

other thereby forming the double stranded RNA for the great promise in the development of biosensor, aptamer

640 H. Li et al.

and other nanomaterials [171]. Antisense oligonucleotides provides useful RNA structures for designing RNA nanostruc-

(ASO) containing LNA also has been reported to have higher tures [185].

potency and speciﬁcity [172,173], but would also cause

liver toxicity in mouse models if LNAs were extensively

incorporated into the sequence[174]. In the other hand, Application of RNA as a polymer in biomedical

UNA is an acyclic and structurally ﬂexible-RNA analogue in

sciences

which the C2 —C3 bond is absent. As a result, the bind-

ing afﬁnity of UNA towards its complementary strand is

RNA as a natural and biocompatible polymer has many

decreased [175]. (4) Modiﬁcation of ribonucleotide base.

advantages for biomedical applications. It carries a neg-

This approach is less commonly used than other modiﬁca-

ative charge at physiological conditions, which disallows

tion approaches described thus far. However, recently RNA

nonspeciﬁc passing through negatively charged cell mem-

tube nanostructures have been reported to be constructed

brane. With the conjugation of chemical ligands and/or

successfully by using RNA scaffolds with 5-biotinylated mod-

RNA aptamers, RNA nanoparticles can be designed for spe-

iﬁed and 5-aminoallyl modiﬁed uracil [139]. Other common

ciﬁc cell targeting. It is less toxic compared with protein

modiﬁed bases such as 2-thio-, 4-thio, 5-iodo-, 5-bromo-,

based nanoparticles since it can avoid antibody induction

dihydro-, pseudo-uracil and diamino-purine also have been

(protein-free nanoparticle), allowing repeated treatment

demonstrated to confer enhancement in stability as well

of chronic diseases. It also does not induce an interferon

as speciﬁcity of base-pairing interactions [155,176,177].

response nor cytokine production [92,186]. RNA nanoparti-

(5) Modiﬁcation of ribose moiety. This modiﬁcation strat-

cles designed with a size range 10—40 nm display favorable

egy, for example, altritol nucleic acid (ANA) and hexitol

pharmacokinetic properties [186], such as extended half-

nucleic acid (HNA), is mainly applied in siRNA design to

life in vivo (5—12 h compared to 0.25—0.75 h for siRNA),

enhance potency and nuclease resistance [178]. Besides

clearance: < 0.13 L/kg/h, volume of distribution: 1.2 L/kg.

these classes, other novel methods such as photocaging

Furthermore, RNA nanoparticles are classiﬁed as chemical

modiﬁcation have been used to control RNAi induction [179].

drugs rather than biological entities. This classiﬁcation will

The particular effect of various chemical modiﬁcations on

facilitate drug approval.

RNA are summarized in Table 1. It is expected that the ratio-

As a building block for nanoparticles, RNA can be synthe-

nal choice of precise chemical modiﬁcations will greatly

sized with deﬁned structure and stoichiometry. Multivalent

contribute to the development of RNA nanostructures for

RNA nanoparticles can be constructed using special RNA

applications in biomedical sciences and materials sciences.

motifs as building blocks that combine therapy, targeting,

Moreover, the strategy of combining different modiﬁcation

and detection, all functionalities in one particle.

approaches also has the potential to improve the properties

of RNA more dramatically.

RNA as a biocompatible nanomaterial for tissue

Methodology III: Computational approach engineering

Tissue engineering is a new area with lots of active research

The ﬁrst step in RNA nanoparticle construction is the

and some recent success. Biocompatible nanomaterials are

consideration of the blueprint [180]. This requires an under-

needed for various applications in tissue engineering, such

standing of the assembly and folding mechanism in the

as scaffolds or arrays that can function as temporary matri-

bottom-up assembly. Designing the sequence of the building

ces and/or niches for the controlled deposition, inﬁltration,

block is critical for successful RNA nanostructure assembly,

proliferation, and differentiation of cells. It would also be

which can be achieved by experience and brainstorming

advantageous if these nanomaterials could be highly bio-

taking into consideration of RNA folding, complementa-

compatible and mimic the natural tissue microenvironment

tion, hand-in-hand interaction, foot-to-foot interaction, and

in vivo.

the use of thermostable motifs, kissing loops, sticky ends,

Shu et al. reported that nanometer scale 3D RNA arrays

helices, stem loops, etc. All RNA nanoparticles constructed

can be assembled by using pRNA as building blocks [112]

based on phi29 motor pRNA was achieved via experience and

(Fig. 1C). By rational design, the authors incorporated

brainstorming without computer algorithm besides the tra-

palindrome sequences (nucleotide sequences that read the

ditional RNA folding program developed by Zuker 30 years

same 5 → 3 on one strand and 3 → 5 on the complemen-

ago [181]. It is expected that computer algorithms will facil-

tary strand) into the 3 -end of the pRNA. The palindrome

itate RNA nanoparticle construction. A variety of computer

sequences served as links for bridging two pRNAs via foot-on-

programs such as NanoFolder, NanoTiler and RNA2D3D are

foot interactions. Loop—loop interactions were further used

available to facilitate the in silico design of RNA sequences

to link pRNA molecules into a chain. The formation of pRNA

(these sequences may contain inter-strand and intra-strand

arrays was conﬁrmed with both polyacrylamide gel elec-

pseudoknot-like interactions) capable of self-assembly into

trophoresis (PAGE) and AFM. These RNA arrays are unusually

multi-sequence RNA nanostructures and the 3D modeling

stable and resistant to a wide range of temperatures, salt

of such structures [182—184]. For example, by utilizing

concentrations, and pH environments. The microstructures

computational modeling and sequence optimization, three-

of the RNA assays are also tailorable by changing the

dimensional cubic RNA-based scaffolds can be successfully

RNA nucleotide sequences. Since RNA molecules are highly

designed and engineered with precise control over their

biocompatible and not toxic, these RNA arrays have the

shape, size and composition [144]. Moreover, online RNA

potential to be good tissue engineering scaffolds.

structure databases such as RNAJunction database also

RNA as a stable polymer 641

Table 1 Summary of chemical modiﬁcations of RNA.

Chemical modiﬁcations Advantages Disadvantages References

Phosphate backbone

• •

Phosphorothioate (PS) • Improve nuclease resistance Destabilize siRNA duplexes [157—162]

• • ◦

Boranophosphate (BO) Combine with other (e.g. decreases Tm by 0.5 C

•

Phosphonoacetate (PACE) modiﬁcations to dramatically per PS linkage)

• •

Phosphoramidate improve RNA property Extensive modiﬁcation causes

•

Methylphosphonate cytotoxic effect

2 -OH group

•

Small 2 -substituents (e.g. • Signiﬁcantly improve • Extensive or full modiﬁcation [36,66,161—169,

2 -O-methyl (2 -OMe), nuclease resistance will reduce or fully deactivate 173]

•

2 -ﬂuoro (2 -F), Greatly thermo-stabilizes siRNA potency

•

2 -aminoethyl, 2 -deoxy-2 - dsRNA duplex (e.g. increase Tm Bulky 2 -modiﬁcations are

◦ ◦

ﬂuoroarabinonu-cleic acid 0.5—0.7 C per 2 -OMe and 1 C only tolerated at limited

(2 -F-ANA)) per 2 -F) position owing to their

• •

Bulky 2 -modiﬁcations (e.g. Particularly, 2 -OMe is distortion of RNA helix

2 -O-MOE, 2 -O-allyl) nontoxic and prevents immune structure activation

•

Bulky 2 -modiﬁcations can

modulate thermo-stability and

duplex asymmetry, and also

give higher binding afﬁnity

Locked nucleic acid (LNA) • LNA enhances the • LNA would probably cause [170—175]

Unlock nucleic acid (UNA) complementary binding afﬁnity liver toxicity

•

and greatly improves Extensive modiﬁcation with

◦

thermostability by 2—10 C per LNA and UNA generally results

incorporation, as well as in decreased activity of siRNA

improves nuclease resistance and failure in annealing of

and reduces RNA dsRNA, respectively immunogenicity

•

Each UNA destabilizes duplex

◦

by 5—8 C to improve local

destabilization of siRNA

duplex, and enhance

biostability in vivo

Ribose moiety

•

Altritol nucleic acid (ANA) • Enhance thermostability (e.g. • Modiﬁcation at seed region [178]

•

Hexitol nucleic acid (HNA) 2 -F-ANA increases Tm of RNA would slightly reduce siRNA

• ◦

2 -deoxy-2 -ﬂuoroarabino- duplex by 0.5—0.8 C per potency

nucleic acid (2 -F-ANA) modiﬁcation)

• •

Cyclohexenyl nucleic acid Improve nuclease resistance

Ribonucleotide base

•

(5-bromo-, 5-iodo-, 2-thio-, Stabilize base-pairing and to • Some base modiﬁcations (e.g. [139,177]

4-thio-uracil, dihydro-, enhance binding speciﬁcity 5-bromo- and 5-iodo-uracil)

•

pseudo, 5-biotinylated, Particularly, 2-thio- and will affect siRNA potency

5-aminoallyl-uracil, pseudo-uracil reduce cellular

diamino-purine) immune response

RNA nanoparticles for targeted therapeutic system. In 2003, Hoeprich et al. constructed a pRNA-based

delivery carrier to deliver hammerhead ribozymes [187]. Upon conju-

gation to the pRNA 5 /3 ends, the HBV ribozymes were able

to fold correctly and almost completely cleaved the polyA

Targeted delivery is a major challenge that nucleic acid ther-

signal of HBV mRNA in vitro. Targeted therapeutics delivery

apeutics is facing. RNA nanoparticles with chemical ligand

to speciﬁc cancer cells can be achieved by using ligand-

and nucleic acid aptamers have shown great promise in this

conjugated RNA nanoparticles as carriers. Furthermore,

regard. The utilization of RNA nanostructures as a platform

the cargo in vivo release proﬁle from RNA nanoparticles

for targeted therapeutics delivery has been successfully

can also be controlled through the rational design of RNA

demonstrated by a series of studies using the phi29 pRNA

642 H. Li et al.

nanoparticles. In 2005, Guo et al. reported the construction effects [186]. The regression of gastric cancer and breast

of chimeric pRNA dimer with one subunit harboring folate cancer by RNA nanoparticles harboring siRNA [73,123,167]

and the other subunit harboring a gene silencing siRNA tar- or anti-miRNA [173] has also been reported recently.

geting surviving [108]. Incubation of these pRNA dimers with A recent paper also reported the development of novel

cancer cells resulted in receptor-mediated binding and entry immunomodulators by engineering rationally designed RNA

to cells and induced efﬁcient gene silencing. Animal trials polygonal nanoparticles [92]. When immunological adju-

showed that ex vivo delivery of the pRNA dimer harboring vants CpG DNA were incorporated into the RNA polygons,

both folate and survivin siRNA could suppress tumor develop- potent immunostimulation (cytokine TNF-␣ and IL-6 induc-

ment. Khaled et al. studied the fabrication of a protein-free tion) was observed both in vitro and in vivo, compared to

20—40 nm pRNA trimer which could harbor three functional controls. Moreover, the RNA nanoparticles could deliver CpG

modules including siRNA, CD4 aptamer or folate ligands DNA to macrophages speciﬁcally and the degree of immuno-

and a ﬂuorescent dye per nanoparticle [111]. They showed stimulation signiﬁcantly depended on the size, shape, and

that the pRNA trimer could also bind and enter into cells the number of payload per RNA nanoparticle. This ﬁnding

and modulate gene expression and apoptosis both in vitro demonstrates that RNA nanotechnology, such as developing

and in vivo. In 2006, Guo et al., reported incorporating a RNA nanoparticles based on pRNA, has great potential to

folate-AMP into the 5 -end of phi29 pRNA and showed that develop novel immunomodulators.

folate conjugated pRNA dimer nanoparticles were able to

deliver survivin siRNA into nasopharyngeal epidermal car-

RNA nanoparticles for controlled drug delivery

cinoma cells and silence the target gene [188]. The pRNA

system can also be used to deliver anti-virus siRNAs. Zhang

Many pre-clinical studies are evaluating RNAi as novel thera-

et al., developed a folate-linked pRNA conjugated with the

peutics for different diseases. However, one concern is that

siRNA targeting the Coxsackievirus B3 (CVB3) protease 2A

single gene targeted therapy might eventually fail due to

(siRNA/2A) [189]. They observed that the modiﬁed pRNA

mutations over time or development of alternative signaling

could achieve a similar antiviral effect to that of siRNA/2A

cascades or escape pathways. Multivalent RNA nanoparti-

alone and also strongly inhibited CVB3 replication.

cles can be a very promising vehicle for delivery of multiple

The latest advance in the utilization of phi29 pRNA

siRNAs to suppress multiple genes simultaneously. A recent

for therapeutics delivery is the discovery of a phi29 pRNA

publication by Afonin et al. showed that several RNA and

three-way junction (3WJ) motif with unusual thermody-

RNA/DNA nanocubes could be functioned with multiple dou-

namic stability. The slope of the melting temperature curve

◦ ble stranded RNAs and the siRNAs can be conditionally

of the three-fragment RNA complex is close to 90 , indicat-

released through ssDNA toehold-mediated interactions. Fur-

ing extremely low G of the phi29 pRNA-3WJ complex [123].

thermore the RNA nanocubes can be tracked intracellularly

The pRNA 3WJ motif was used as a RNA scaffold to construct

through FRET (Förster resonance energy transfer) stud-

bi-, tri-, and tetra-valent RNA nanoparticles with very high

ies using ﬂuorophores [190]. In addition, spherical nucleic

chemical and thermodynamic stability [73,123,180]. The

acid (SNA) nanoparticle conjugates developed by Mirkin

resulting RNA nanoparticles are resistant to denaturation

group have also shown promise for systemic siRNA deliv-

in 8 M urea and do not dissociate at ultra-low concen-

ery for treating diseases such as glioblastoma [191,192]

trations both in vitro and in vivo. Each arm of the 3WJ

(Fig. 5D). In vivo studies on glioma-bearing mice showed

or X-motif can harbor one siRNA, ribozyme, miRNA, or

that SNA nanoparticle conjugates targeting the oncoprotein

aptamer without affecting the folding of the central core,

Bcl2Like12 effectively increased intratumoral apoptosis,

and each daughter RNA molecule within the nanoparticle

and reduced tumor burden and progression without adverse

folds into respective correct structure with authentic bio-

side effects [192].

logical function. The effects of gene silencing progressively

DNA origamis have been exploited as a carrier for chem-

increased with increasing number of siRNA modules in the

ical drug doxorubicin through noncovalent intercalation

RNA nanoparticle. More importantly, systemic delivery of

interactions. The DNA origami/doxorubicin nanoparticles

targeting ligand containing RNA nanoparticles into tumor-

were able to enter into doxorubicin-resistant cancer cells

bearing mice revealed that the RNA nanoparticles remained

and circumvent their drug resistance [193]. Furthermore,

intact in vivo without showing any sign of dissociation or

the DNA origami/doxorubicin nanoparticles showed a slow

degradation. These RNA nanoparticles can speciﬁcally tar-

drug release proﬁle at pH 7.4 under physiological condi-

get subcutaneous tumor xenografts [73,123,167], as well

tions but showed enhanced release capability in pH 5.5

as orthotopic breast cancer [173] and intracranial glioma

corresponding to tumor subcellular organelles [194]. As

tumors [164] without detectable accumulation in liver, lung

we discussed in previous section, RNA origami structures

or other healthy organs or tissues (Fig. 4A—C). A recent

have been reported [136]. With favorable thermodynamic

study also showed that the pRNA-3WJ nanoparticle conju-

stability and excellent serum stability after chemical modi-

gated with folate can speciﬁcally target colorectal cancer

ﬁcations, RNA origami is expected to be more favorable than

metastasis in the liver, lungs and lymph node simulta-

its counterpart DNA origami as a drug carrier for achieving

neously in vivo, without accumulation in normal liver or

controlled drug release.

lung parenchyma [165] (Fig. 4D). Pharmacological analy-

sis in mice indicated that the pRNA nanoparticles display

favorable pharmacokinetic (PK) and pharmacodynamic (PD) RNA nanoparticles for vascular targeting

proﬁles, with in vivo half-life extended 10-fold compared to

siRNA alone and did not induce adverse immune response A critical feature to drug delivery is the transportation of

including cytokine, interferon, antibody, and other toxic therapeutics to their intended target sites. Arguably, as

RNA as a stable polymer 643

Figure 4 pRNA nanoparticles for cancer targeting. (A) pRNA-3WJ nanoparticles target folate-receptor positive tumor xenografts

∼ ␮

almost all cells in the body are within 100 m of the vas- [197,198], inﬂammatory markers (ICAM-1, VCAM-1)

culature, and by way a vascular endothelial cell, it stands to [199—204], and thrombomodulin [205]. Targeting of

reason that the vascular delivery route represents the most CD31, ICAM1 and VCAM represents a unique opportunity

promising mean of any site speciﬁc delivery strategy [195]. as their binding of nanoparticles to their sites highlights

Owing to the signiﬁcant heterogeneity in endothelial cells, a novel endocytic mechanism, CAM mediated endocytosis

both temporally and regionally, it has been observed that [198,206]. The use of antibodies as targeting agents is

the vascular endothelium has unique expression of surface technically challenging owing to their size, cost of produc-

markers that are highly regionally variant. In other words, it tion and potential immune responses. RNA aptamers have

is potentially possible to target unique endothelial zip codes already shown exciting advantages in vasculature targeting.

to locally accumulate nanocarriers [196]. High afﬁnity aptamers (56 pM) against P-selectin have been

Current strategies for vascular targeting of nanocarriers developed [207]. Microbubbles coated with a low density

have primarily focused on antibody and peptide based (1000 copie/␮m ) of P-selectin aptamers demonstrated high

strategies, targeting to surface markers CD31 (PECAM-1) adhesion even under shear rates as high as 1700 L/s. The

644 H. Li et al.

Figure 5 Functional RNA nanostructures for therapeutics delivery and medical detection. (A) Triggered siRNA delivery based on

QD-aptamer-doxorubicin conjugate nanoparticles as a cancer-targeted imaging, sensing and treatment platform [210]. Reprinted

American Chemical Society. (D) Spherical nucleic acids scaffold for loading RNA therapeutics [191]. Reprinted with permission from

real promise of vascular-targeted RNA nanoparticles comes RNA, RNA molecules with high binding afﬁnity to their tar-

from the combination of the high binding afﬁnity to carrier gets hold great potential as a platform to construct imaging

sizes below 70 nm. The capillary bed represents the greatest probes which can speciﬁcally detect their targets in vitro

promise of vascular targeting, where there already exists a and in vivo.

highly active transcytosis mechanism, with approximately Fluorescent RNA nanoparticles conjugated with aptamer

ten thousand times more caveolae than other endothelial have been widely explored for cancer detection. For

cells [208]. These 70 nm endocytic vesicles are an exciting example, Bagalkot et al. [210] developed a QD-aptamer-

target for drug delivery [209] yet require a carrier system doxorubicin conjugate nanoparticle as a cancer-targeted

like RNA nanoparticles that are simultaneously small and imaging, sensing and treatment platform (Fig. 5B). A10

have high afﬁnity to permit transcytosis and tissue bed RNA aptamer, which speciﬁcally binds to the extracellular

delivery. domain of the prostate speciﬁc membrane antigen (PSMA),

was conjugated to the surface of ﬂuorescent quantum

dots and the anticancer drug doxorubicin was intercalated

RNA nanoparticles as non-invasive medical into the double-stranded stem of the A10 aptamer. In the

detection reagents conjugate nanoparticle, ﬂuorescence of doxorubicin was

quenched by the aptamer through donor-quencher FRET,

Functional RNA structures such as aptamers can be used and the ﬂuorescence of QD was quenched by DOX through

for developing non-invasive molecular and cellular imaging donor-acceptor FRET mechanism. When the nanoparticle

reagents which may have applications in the diagnosis of enters the prostate cancer cell and doxorubicin is released,

various diseases including cancer, cardiovascular diseases, the ﬂuorescence of QD and DOX is re-activated. Other RNA

and infectious diseases. Considering it is relatively easy to aptamers conjugated with ﬂuorescent dye chemicals have

conjugate a ﬂuorophore, radionuclide, quantum dot (QD), also been utilized for breast cancer diagnosis, such as EpCAM

gold nanoparticle, or other imaging functional groups to aptamer labeled with DY647, TYE665 or FITC [211].

RNA as a stable polymer 645

Conjugating RNA aptamer with radioactive material can this novel design, the authors determined the half-life (t1/2)

also be applied as diagnostic radiopharmaceuticals to detect of the MG aptamer containing pRNA-3WJ inside living cells

cancer cell markers. In a recent study reported by Gomes to be ∼4.3 h.

et al. [212] a 36 nucleotide long truncated RNA aptamer

with 2 F pyrimidine and 2 -O-methyl purine modiﬁcation was

constructed for targeting to the human matrix metallopro- RNA as a polymer for biosensor systems

tease 9 (hMMP-9), which promotes tumor metastasis and The biomolecular-based detection system has been widely

is an important marker of malignant tumors. The 5 -end investigated in many applications including clinical diag-

amine modiﬁed RNA aptamer was conjugated to MAG3-NHS, nosis, food industry, environmental monitoring industry,

99m

which served as a chelator for Technetium-99m ( Tc). The and security industry [217,218]. Conventionally, biosensors

99m

Tc labeled modiﬁed RNA aptamer was able to speciﬁcally can detect proteins, nucleic acids such as DNA or RNA

detect its target in human brain tumors with autoradiogra- sequences, small organic molecules, and others [218—220].

phy. RNA molecules are well suited to serve as detection tools

With the conjugation of the PSMA RNA aptamer to the for small molecules, for example, antibiotics, peptides,

surface of gold nanoparticles (GNPs) [213], a targeted metal ions, ligands, etc. [221,222] because of their unique

computed tomography (CT) imaging and therapy system folding structures, functional conformation, predictable

for prostate cancer can also be established (Fig. 5C). base-pairing, and high ﬁdelity. These characteristics of RNA

The CT imaging study revealed that the PSMA aptamer- allow for the development of ribozyme or aptamer, and

conjugated GNPs could generate more than four-fold greater aptazyme-based biosensors [223—225]. Also, as the target,

CT intensity for a PSMA-expressing cell line than a non-PSMA- RNA can be used to develop the biosensor for the detection

expressing cell line, suggesting good speciﬁcity in detection of messenger RNA or micro RNA [226—229]. Various detec-

of the targeted cancer cells. tion methods of RNA-based biosensors were proposed such as

electrochemical type, ﬂuorescence type, optical type, and

electrical type. Also, there are various detection platforms

RNA nanoparticles for intracellular imaging and for RNA-based biosensors (Fig. 7) [230—235]. In this section,

detection we brieﬂy introduce RNA-based biosensors for biomedical

and environmental monitoring applications.

An interesting example of applying RNA in intracellular The ﬁrst proof of concept of using RNA in a biosen-

imaging is the Spinach RNA aptamer, which is the RNA sor was developed by Breaker Group [221]. Self-cleaving

mimic of Green ﬂuorescent protein (GFP). GFP has been hammerhead-ribozymes were created with seven differ-

extensively utilized as a reporter protein in cell and molec- ent RNA switches and these immobilized each pixel that

ular biology. Similarly, the RNA mimics of GFP should also responded allosterically to six types of analytes (Co ,

have remarkable applications in biomedical research, espe- cGMP, cCMP, cAMP, FMN and theophylline). The platform

cially for intracellular imaging of RNA. By using the method of biosensor array was prepared on a polystyrene cell cul-

of systematic evolution of ligands by exponential enrich- ture plate which was coated with gold by physical vapor

ment (SELEX), Paige et al. developed a novel class of RNA deposition. The ribozyme was immobilized onto gold sur-

aptamers, termed Spinach that bind to ﬂuorophores resem- face via a 5 -thiotriphosphate-terminated RNA moiety. Then,

bling the ﬂuorophore in GFP [214] (Fig. 6A). Upon binding each hammerhead ribozymes cleaved off their 3 frag-

of the targeted ﬂuorophores, these RNA aptamers were ment according to addtion of individual analytes. They also

capable of emitting tunable ﬂuorescence with comparable showed the quantitave and qualitive measurement of cAMP

brightness to enhanced GFP (EGFP) and many other ﬂuo- in culture media from E. coli strand.

rescent proteins. Moreover, the RNA-ﬂuorophore complex is RNA can also serve as the detection analytes to detect

resistant to photobleaching. Interestingly, the Spinach RNA viable E. coli as an indicator organism in drinking water,

aptamer can be expressed in vivo by fusing to other cellular which was proposed by Baeumner group [228,229]. This

RNAs, for example, 5S RNA, to enable live-cell imaging and RNA-based biosensor was generated with the extraction and

monitoring of these cellular RNAs. Furthermore, ﬂuorescent ampliﬁcation of mRNA molecules from E. coli in 20 min.

sensors for detecting a variety of small molecules and cel- Viable E. coli in the water were identiﬁed and quantiﬁed

lular metabolites, including adenosine 5 -diphosphate (ADP) using a 200 nt target sequence from mRNA. The detec-

and S-adenosylmethionine (SAM), in vitro and in living cells tion limit of the biosensor system could detect around

could also be generated by combining the Spinach aptamer, 40 viable E. coli in water (5 fmol per sample) using the

a transducer, and a target-binding aptamer [215] (Fig. 6B). electrochemiluminescence (ECL) detection method. They

Reif et al. [216] recently reported another novel method introduced the isothermal ampliﬁcation technique, a nucleic

to enable real-time monitoring of RNA folding and degra- acid sequence-based ampliﬁcation (NASBA) for mRNA ampli-

dation in living cells based on the pRNA-3WJ. The authors ﬁcation.

designed and constructed RNA nanoparticles by incorporat- RNA was also applied to develop medical diagnosis

ing an RNA aptamer capable of binding to malachite green biosensors. Theophylline (1,3-dimethylxanthine) is a com-

(MG), the hepatitis B virus ribozyme and the luciferase siRNA mon agent in the bronchodilators and used for acute

into the pRNA 3WJ. When MG aptamer binds to MG dye, it and chronic asthma. Gothelf group developed a RNA-

will emit ﬂuorescent light only if the aptamer folds correctly. based electrochemical biosensor for theophylline in serum

The MG aptamer system can then be used to monitor the with a ferrocence (Fc) redox probe [225,233]. In this

degradation of the constructed RNA nanoparticles by fluo- study, the thiol group-modified and amino group-modified

rescent microscopy and ﬂuorescence spectroscopy. By using RNA aptamer was immobilized onto gold substrate via

646 H. Li et al.

Figure 6 Spinach RNA aptamer for intracellular imaging and sensing. (A) Live-cell imaging of Spinach-tagged 5S RNA[214].

cellular metabolites in E. coli with sensor RNA[215]. The sensor RNA comprises Spinach (black), a transducer (orange), and a target-

of Science.

Figure 7 The basic constitution of RNA-based biosensor.

covalent bonding between thiol group and gold substrate. the theophylline added to RNA apatmer-modiﬁed electrode,

Then, the Fc redox probe was covalently attached to the the aptamer folds into the conformationally restricted hair-

3 -amino group of RNA aptamer through Fc-caroxylic acid pin strucutre. As a result, this phenomenon changed the

NHS reaction. Usually, the RNA aptamer stays on the open electron transfer that was monitored by cyclic and differ-

conformation in absence of theophylline. However, when ential pulse voltammetry. Moreover, speciﬁc recognition of

RNA as a stable polymer 647

Table 2 Classiﬁcation of RNA polymer-based bioelectronics.

RNA-based bioelectronics References

Biocomputation devices Biologic gate [238,243]

Biomemory [238,243,266]

Bioinformation processor [242,243,245,247,248,250]

Biosensors Biomedical Sensor [215,217,221,222,225—227]

Environmental sensor [228,229]

dopamine by the RNA aptamer allows selective amperomet- [237—239]. As a result, the ﬁeld of bioelectronics has

ric detection of dopamine from 100 nM to 5 ␮M range in the been created [240,241]. Bioelectronics has led to the

presence of competitive neurotransmitters such as catechol, development of the nanoscale biochip, biosensor, and bio-

epinephrine, norepinephrine, and others [232]. computation devices such as information storage devices,

In an alternative approach, Hammond group used a RNA- logic gates, ﬁeld-effect transistors and computation sys-

based ﬂuorescent biosensors for detecting the cyclic di-GMP tems (Table 2) [242—245]. Typically, bioelectronic devices

and cyclic AMP-GMP [226]. The c-di-GMP was generated are composed of a biological actuation portion, and an

by a GEMM-I riboswitch aptamer and the riboswitch ligand electronic signal transduction portion. So far, the biologi-

mutation can recognize the c-AMP-GMP, c-di-GMP. The ﬂu- cal component is usually composed of biomolecules, such

orescence turn-on was activated by the DFHBI ﬂuorescent as protein and DNA [239,242,244]. Recently, some pioneer-

molecule for cell imaging. This system provided selective ing groups have suggested that the RNA-based bioelectronic

and sensitive detection for each cyclic dinucleotide. Thus, devices also exhibited the logic gate behavior, processing

there are many examples demostrating that RNA molecules functions, and biocomputations [180,246—250]. The advan-

are promising candidates for biosensor applications. tages of RNA-based computation are summarized in Fig. 8.

Biocomputation approaches for information control, stor-

age, and processing using RNA-based devices brings new

Application of RNA as a polymer in material

possibilities that embody multiple functions in biological sys-

sciences tems. The development of biological computers composed

of artiﬁcial RNA molecules to operate inside living cells

or tissues would provide a new avenue. A combination of

RNA as a polymer for biocomputation systems

these results will pave the way to develop the new-concept

of biocomputer development in the future such as tissue-

Silicon-based computer systems are ubiquitous [236] but

mimicked computations.

new processors to implement computation, communication,

and controls are needed to meet the demands of vari-

ous applications. Gordon Moore’s Law suggested that due

to the increasing demand of the memory, computer elec- RNA as a polymer for logic gates

tronic devices on the microprocessor will be doubled every

18 months. Scientists and engineers are wondering whether In computer science, the ‘logic’ term is deﬁned as cir-

current computer technology growth can be continued in the cuits which gives an output corresponding to a set of two

next ten years. They raised the concern that silicon micro- input values (True ‘1’, High voltage and False ‘0’, Low volt-

processor speed and miniaturization will eventually reach age). The logic gate is required to have two input values

limits by current technology. Thus, molecular-scale com- for performing Boolean function [241]. Thereby, it gives

puting has been explored since 1994 due to the predictable the logical output to perform the next function. Usually,

ending of Moore’s Law for silicon-based computer devices. man-made computers consist of millions of combinational

In the last few decades, biotechnology has been inte- logic gates. Thus, logic gates are essential components of

grated with nanotechnology and electrical engineering the computer. As aforementioned, in the last few decades,

Figure 8 Advantages of RNA-based biocomputation.

648 H. Li et al.

the concept of a biomolecule-based computation system information processing devices that operate logic gates, sig-

has been proposed or explored to solve the current lim- nal ﬁltering, and cooperativity functions have been reported

itation of silicon-based computations [246,251—255]. The [250,259,265]. RNA devices composed of ribozymes and RNA

ﬁrst attempt of molecular-scale computation was devel- aptamers received, processed and transmitted the molecu-

oped by Adleman’s group in 1994 [251]. They introduced lar inputs to express the green ﬂuorescent protein as output.

the encoded DNA molecule to solve the Hamiltonian path As a sensor part, the RNA aptamer was introduced and a

problem. This research established a milestone for the hammerhead ribozyme was used to process the cleaving of

ﬁeld of biomolecule-based computations. Since then, sev- the aptamer. Also, the transmitter part was composed of

eral groups proposed the DNA polymer-based logic systems sequences that bind to the aptamer and ribozymes. It was

[252,254,255]. The basic operation mechanism of DNA-based suggested that the RNA aptamer and ribozyme combina-

logic gate is combinational DNA hybridization that undergoes tion can be used as actuating elements for multi-functional

conformational change or chemical reaction and includes information processor development [250,265].

pH, temperature, light, electrical, or electrochemical sig- An intracellular RNA computation device in a single mam-

nals [251—255]. The function of logic gate gives the discrete malian cell has also been reported [259]. Trigger-controlled

state change such as structure, ﬂuorescence, absorbance, transcription factors were used to regulate the gene expres-

and electrochemical or electrical current as the output. sion individually and the RNA-binding protein was used to

Like this, the various types of DNA-based computations have inhibit the translation of transcripts on target RNA motifs.

been proposed. DNA, however, is hard to use to perform the This computation biosynthetic circuit can provide the NOT,

complex functions because of its simple functionality. To AND, NAND, and N-IMPLY expression functions in individual

develop the new-concept of biocomputation systems, RNA cells. Moreover, two N-IMPLY expression functions can com-

is a candidate owing to its intriguing characteristics. The bine with other cells to operate XOR gate functions and three

folding and assembly of RNA molecules drive themselves logic gates can perform the half-adder and half-subtractor

into secondary and tertiary structures using the formation of functions for arithmetic calculation.

hairpin loops, dovetails, bulges, and internal loops, etc [10].

Thereby, RNA has functionality such as aptamer, ribozyme,

RNA as a polymer for resistive biomolecular

siRNA, non-coding RNA, circular RNA, etc. [180] These var-

ious functional groups can be easily applied to the new memory

concept of logic gate behavior, information storage, infor-

mation processing, and computations as the novel elements RNA can also provide several advantages such as ease of

[256—264]. construction, well deﬁned structure and thermodynamic

An autonomous biomolecular-based computer has been stability for the development of biomolecular memory

proposed to regulate the gene expression ‘‘logically’’ [248]. devices. Based on the discovery of the ultra-high ther-

The basic computation rule is governed by ‘diagnostic rule’ mostable pRNA-3WJ, a pRNA-3WJ/QD hybrid nanoparticle

for prostate cancer state detection. If the speciﬁc genes for resistive biomolecular memory application has been con-

relating to prostate cancer are overexpressed, then ssDNA structed [266]. The QD was used as a semiconducting part for

will bind to their mRNA and inhibits the protein synthesis. storage of the electrons and pRNA-3WJ was used as an insu-

The level of speciﬁc RNA and the concentration of spe- lating part and serves as the bridge between QD and metal

ciﬁc molecule which regulates the point mutations were substrate. The electrical measurement (I—V) was conducted

regarded as an input. The release of short ssDNA modu- to this hybrid structure for resistive memory performances.

lated the levels of gene expression for anticancer activity, This study showed the RNA polymer conjugates can be used

as the output logically. The computation module has two directly for molecular memory device fabrication.

states, ‘‘positive’’ and ‘‘negative’’, in response to the level

of speciﬁc gene expression. This biocomputation concept is

Hybrid RNA polymers as semiconductors

related with the identiﬁcation of mRNAs of disease-related

genes. Thereby, it can be applied to cancer diagnosis systems

based on logic analysis. Biomolecular hybrids of a conducting polymer [poly(O-

A RNAi-based logic evaluator has also been reported to methoxy aniline) (POMA)] and dsRNA have been reported

perform Boolean logic corresponding to input molecules in as semiconductors [267]. Fourier transform infrared spec-

the human kidney cells [249]. The reported biological circuit troscopy (FTIR) indicated that these hybrid polymers are

is composed of several mRNA species that encode the same held together by electrostatic, H-bonding, and pi—pi inter-

protein but have different non-coding regions. This protein actions. The circular dichroism spectra of the hybrid

served as the output. As an input, siRNAs were used to con- polymers indicated that the dsRNA underwent a small dis-

trol the degradation of the target mRNA and the expression tortion in conformation from the canonical A-form towards

of the output protein. Expressions with up to ﬁve logic vari- the B-form during the formation of the hybrid polymers. TEM

ables were directly evaluated by this system. micrographs revealed that these polymers create a ﬁbrillar

network structure. The conductivity values of the POMA-RNA

hybrids were three orders of magnitude higher than that of

RNA as a polymer for information processor RNA alone. The I—V curves of the POMA-RNA hybrid poly-

mers also demostrated a semiconducting nature. Based on

these physical characteristics, the POMA-RNA hybrids have

In computer sciences, the information processor is a unit

the potential to be candidates for fabricating biosensors for

that receives the input information and processes it into

other applications.

another form based on programmed functions. RNA-based

RNA as a stable polymer 649

Conjugation of RNA to graphene polymeric building block [36] into a new horizon. The con-

cern of yield and production costs has been and will be

Graphene is a one-atom thick, two-dimensional honeycomb addressed continually by industry scale production and fer-

lattice composed of sp -bonded carbon atoms. It can be mentation. It is expected that applications of RNA as a

used as a fundamental building block for constructing other polymer and as building blocks will appear more and more

graphite-based structures such as fullerenes and nanotubes in therapeutics, detection, sensing, nanoelectronic devices,

[268,269]. This newly discovered carbon-based material has and other polymer industries. The anion nature, the thermo-

been a popular subject in material science and nanotechnol- dynamic stability, the insulating property, the self-assembly

ogy. It has been used for the sensing of a toxin Microcystin-LR capability and other novel features such as versatility,

in water [270—273]. RNA aptamer have been covalently molecular-level plasticity, as well as the potential semicon-

immobilized on graphene oxide and a polydispersed sta- ductor behavior will make RNA unique for exploring new

ble RNA-graphene oxide nanosheet have been constructed scientiﬁc territories. RNA has been shown to be major com-

[274]. The RNA attached to these nanosheets was resistant ponents of cells and leading functionality of life, and it is

to nuclease degradation and the nanosheets competitively expected that RNA will also be the momentous material of

absorbed trace amounts of the peptide toxin microcystin- daily life in the future.

LR from drinking water. PEI-grafted graphene oxide was also

used to deliver short interfering RNA (siRNA) as well as anti- Acknowledgements

cancer drug doxorubicin to cancer cells [275]. RNA has also

been used as a surfactant to exfoliate ﬂakes of graphene

This work was supported by grants EB019036, EB003730,

from nanocrystalline graphite to produce transparent and

EB012135 from NIH/NIBIB and CA151648 from NIH/NCI to

conducting RNA-graphene-based thin ﬁlms [276], which has

P.G., Leading Foreign Research Institute Recruitment Pro-

a potential for a variety of electronic applications. Further

gram through the National Research Foundation of Korea

research in RNA-graphene nanocomposites may open a new

(NRF) funded by the Ministry of Science, ICT & Future

avenue towards many applications of graphene-based con-

Planning (MSIP) (2013K1A4A3055268) to T.L., National Dis-

ductive materials.

tinguished Young Scholars grant (NSFC No: 31225009) and

the ‘‘Strategic Priority Research Program’’ of the Chinese

Conjugation of RNA with other nanoparticles Academy of Sciences Grant no. XDA09030301 to X.L. The

funding to P.G.’s Endowed Chair in Nanobiotechnology posi-

The conjugation of a RNA with nanoparticles such as quan- tion is from the William Fairish Endowment Fund. P.G. is

tum dot, iron oxide nanoparticle or gold nanoparticle has the cofounder of Biomotor and RNA Nanotechnology Devel-

been successfully demonstrated by a series of studies for opment Corp. Ltd. We would like to thank Dr. Richard N.

nanosized imaging, therapy and diagnosis [277—282]. For Greenberg, Dr. Daniel W. Pack, Dr. Yi Shu and Dr. Ashwani

example, siRNA and tumor-homing peptides (F3) were conju- Sharma for their constructive comments.

gated to the PEGylated QD core as a scaffold [278,279]. The

complex was efﬁciently delivered to HeLa cells, released

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654 H. Li et al.

Hui Li, M.S., is currently a Ph.D. candidate Sijin Guo, B.S., received his B.S. in chem-

in College of Pharmacy, University of Ken- istry from Wuhan University, China in 2014.

tucky. He graduated from Shanghai Jiao Tong He is currently a Ph.D. candidate in Prof.

University in 2011 with a M.S. degree in phar- Peixuan Guo’s lab at University of Kentucky.

maceutical sciences and then joined in Prof. His research interests include the study of

Peixuan Guo’s lab in 2012 spring. He has a function, mechanism and application of bac-

broad training in pharmaceutics, molecular teriophage phi29 DNA-packaging nanomotor

biology, cancer biology and nanobiotechnol- as well as applications of RNA nanoparticles

ogy. His current research is focusing on RNA derived from phi29 packaging RNA (pRNA) in

nanotechnology and the development of new therapeutics delivery, targeting and disease

therapeutics and drug delivery systems based treatment (RNA nanotechnology).

on RNA.

Dr. Taek Lee, Ph.D., is currently a post-

doctoral fellow at College of Pharmacy,

Dr. Jing Xu, Ph.D., is an associate professor

University of Kentucky. He obtained her B.S.

at the National Center for Nanoscience and

from Sogang University, Department of Chem-

Technology, Beijing, China. She received her

ical and Biomolecular Engineering, South

B.S. in Chemistry in 2004 and M.S. in Polymer

Korea (2008), and Ph.D. from Department

Science in 2007 from Nankai University, Tian-

of Chemical and Biomolecular Engineering,

jin, China. In 2012, she received her Ph.D. in

Sogang University (2013). He has a broad

Chemistry from the University of North Car-

training in chemical engineering, biology, and

olina at Chapel Hill with Professor Joseph M.

nanobiotechnology. Dr. Lee’s scholarly inter-

DeSimone. Her work focuses on development

est broadly focuses on bioelectronics, biochip

of drug delivery systems for treatment of can-

and medicine. These include, RNA nanoparticle construction and

cers and prevention of infectious diseases.

conjugation for bioelectronics application, therapeutic and diag-

nostic applications.

Dr. Thomas Dziubla, Ph.D., is the Asso-

Dr. Chan Li, Ph.D., is currently an assistant

ciate Gill Professor and Director of Graduate

professor in CAS Key Laboratory for Biomedi-

Studies in the Department of Chemical and

cal Effects of Nanomaterials and Nanosafety,

Materials Engineering at the University of

National Center for Nanoscience and Tech-

Kentucky. He received his B.S. and Ph.D

nology of China. She received her Ph.D.

in Chemical Engineering from Purdue Uni-

degree in chemistry from University of Sci-

versity (1998) and Drexel University (2002),

ence and Technology of China in 2012. She

respectively. In 2002—2004, he was an NRSA

worked as a postdoc at Osaka University

postdoctoral fellow in the Institute for Envi-

in Japan before joining National Center for

ronmental Medicine at the University of

Nanoscience and Technology of China in

Pennsylvania School of Medicine under the

2014. Dr. Li’s research is focused on ligand-

guidance of Dr. Vladimir Muzykantov, where he worked on the

modiﬁed nanocarrier loading anticancer drugs to circumvent drug

design of degradable polymeric nanocarriers for the delivery of

resistance, and the formulation and process development of

antioxidants. His research group is interested in the design of new

nanomedicines.

functional polymeric biomaterials which can actively control local

cellular oxidative stress for improved biomaterial integration and

disease treatment. He holds 8 patents, has authored over 50 peer

reviewed publications and has started several companies that are

currently commercializing technologies that have originated from Dr. Farzin Haque, Ph.D., is a Research Assis-

his laboratory. tant Professor in the University of Kentucky

College of Pharmacy, Department of Pharma-

Fengmei Pi, M.S., is currently a Ph.D. can- ceutical Sciences. He received his B.A. degree

didate at College of Pharmacy, University of in Biochemistry and Mathematics (2004) from

Kentucky. She obtained her B.S. and M.S. Lawrence University and a Ph.D. degree in

from China Pharmaceutical University (2007). Chemistry (2008) from Purdue University. He

She worked as a research scientist at Dea- held a postdoctoral appointment (2009—2011)

woong Pharmaceuticals Co. Korea; and senior at the University of Cincinnati, with Pro-

formulation scientist in China GSK Consumer fessor Peixuan Guo. Dr. Haque’s scholarly

Healthcare before joining the University of interest broadly focuses on Nanoscience and

Kentucky (2012). She has broad training and Nanotechnology in Biology and Medicine. These include, nanopore-

working experience in pharmaceutical sci- based technology for single molecule detection and sensing of

ence. Her recent research focused on RNA chemicals and biopolymers; and RNA Nanotechnology - con-

nanotechnology, developing RNA nanoparticles based aptamers for struction of RNA nanoparticles for therapeutic and diagnostic

targeted drug delivery and cancer therapy. applications.

RNA as a stable polymer 655

Dr. Xing-Jie Liang, Ph.D., got his Ph.D. Dr. Peixuan Guo, Ph.D., is William Farish

at National Key Laboratory of Biomacro- Endowed Chair in Nanobiotechnology, direc-

molecules, Institute of Biophysics at CAS. tor of University of Kentucky Nanobiotechnol-

He ﬁnished his postdoc at Center for Can- ogy Center and director of NIH/NCI Cancer

cer Research, NCI, NIH, and worked as a Nanotechnology Platform Partnership Pro-

Research Fellow at Surgical Neurology Branch, gram: ‘‘RNA Nanotechnology for Cancer

NINDS. He worked on Molecular imaging Therapy’’. He obtained his Ph.D from Univer-

at School of Medicine, Howard University sity of Minnesota, and postdoctoral training

before he became deputy director of CAS at NIH, joined Purdue University in 1990,

Key Laboratory for Biomedical Effects of was tenured in 1993, became a full Profes-

Nanomaterials and Nanosafety, National Cen- sor in 1997, and was honored as a Purdue

ter for Nanoscience and Technology of China. He is a founder Faculty Scholar in 1998. He constructed phi29 DNA-packaging

member of International Society of Nanomedicine, member of motor, discovered phi29 motor pRNA, pioneered RNA nanotechnol-

American Association for Cancer Research, and member of Amer- ogy, incorporated phi29 motor channel into lipid membranes for

ican Society of Cell Biology. He is current Associate Editors of single-molecule sensing with potential for high-throughput dsDNA

‘Biomaterials’ and ‘Biophysics Report’; Advisory editorial board sequencing. He is a member of two prominent national nanotech

member of ‘ACS Nano’; Editorial member of ‘Advances in Nano initiatives sponsored by NIH, NSF, NIST, and National Council of

Research’, ‘Current Nanoscience’, ‘Biomaterials Research’, ‘Ther- Nanotechnology, director of one NIH Nanomedicine Development

anostics’ and guest editor of ‘Biotechnology Advances’. Developing Center from 2006—2011. His work was featured hundreds of times

drug delivery strategies for prevention/treatment of AIDS and can- over radio, TV such as ABC, NBC, newsletters NIH, NSF, MSNBC, NCI,

cers are current program ongoing in Dr. Liang’s lab based on and ScienceNow. He was a member of NIH/NCI intramural site-visit

understanding of basic physiochemical and biological processes of Review Panel at 2010 and 2014, and a member of the Examina-

nanomedicine. tion and Review Panel (Oversea Expert) of the Chinese Academy of

Sciences since 2014.