sorah yoon , dvm, phd and john j. rossi , phd...new biomarkers in clinical tissue specimens. rna...

In: Advances in Medicine and Biology ISBN: 978-1-53615-842-7

Editor: Leon V. Berhardt © 2019 Nova Science Publishers, Inc.

Chapter 3

APTAMERS IN MOLECULAR IMAGING:

FROM PRINCIPLES TO

BIOLOGICAL APPLICATIONS

Sorah Yoon*, DVM, PhD

and John J. Rossi†, PhD Department of Molecular and Cellular Biology,

Beckman Research Institute of City of Hope,

Duarte, CA, US

ABSTRACT

Molecular imaging is based on the specific recognition of targets of

interest by molecular probes. As the power of molecular imaging for

research and medical diagnosis increases, the advent of molecular probes

with exquisite targeting properties such as aptamers is inevitable.

Aptamers are nucleic acid ligands that bind to their targets with high

specificity and affinity, due to their unique three-dimensional structures.

Their compelling features, such as slow off-rate and smaller size, have

* Corresponding Author’s E-mail: [email protected]. † Corresponding Author’s E-mail: [email protected].

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Sorah Yoon and John J. Rossi 46

led to their use for quantitative cellular imaging at the nanoscale.

Therefore, in this chapter, we will discuss the principles of aptamer

selection and in vitro applications for bench scientists, including

aptahistochemistry, tissue imaging, enzyme-linked aptamer assay

(ELAA), aptablot, aptamer-mediated flow cytometry and mass cytometry,

and super-resolution microscopy. In addition, the remarkable discovery of

fluorogenic aptamers allows investigation of the dynamics of a gene of

interest using cellular imaging. To facilitate the translation of aptamers

for molecular imaging, a series of fluorogenic aptamers and the advanced

application of target-specific aptamers conjugated to various imaging

modalities in living subjects will be presented in an array of imaging

techniques such as magnetic resonance imaging (MRI), single-photon

emission computerized tomography (SPECT), positron emission

tomography (PET), computerized tomography (CT), and ultrasound scan

(US) for physicians.

Keywords: molecular imaging, molecular probes, aptamers, in vitro

application, fluorogenic aptamers, imaging modalities

1. INTRODUCTION

Aptamers are single-stranded oligonucleotides that adopt two- or three-

dimensional structures by way of well-defined sequences. Their unique

structures enable exceptional binding to their targets with high affinity and

specificity, mimicking antibodies. Therefore, they are often recognized as

“chemical antibodies.” Reportedly, some target-specific aptamers are

naturally existing, but most of them are artificially isolated in vitro for

specific purposes. After isolation, they can be tailored to serve unique

biological purposes in vitro. In this way, aptamers have great advantages

over antibodies for use as probing molecules, including a relatively short

time period for development, low cost of manufacturing, structural

flexibility and stability, low batch-to-batch variation, and high affinity and

specificity [1]. Aptamers (~3 nm) are smaller than antibodies (10–15 nm)

in size, enabling them to detect epitopes that antibodies cannot recognize.

The fine targeting of aptamers holds their true value for use as molecular

probes. These compelling features quickly led them to emerge as

Aptamers in Molecular Imaging 47

promising targeted molecular imaging probes to detect cancers [2]. In this

chapter, we will highlight successful studies of molecular and cellular

imaging applications for laboratory research and clinical in vivo imaging,

and discuss chemical modifications that can be applied to overcome

shortcomings.

2. IN VITRO SELECTION: SELEX

The in vitro aptamer selection method, termed Systematic Evolution of

Ligands by EXponential enrichment (SELEX), was developed in 1990 by

three independent groups [3-5]. Since the advent of SELEX, there have

been numerous advances in the technology of aptamer development [6].

Initially, aptamers were isolated against proteins in vitro. But, due to

technological advances, aptamers can now be isolated against any target,

including small molecules, nucleic acids, cultured cells, ex vivo organs, and

even in vivo targets [7-13]. Depending on the target, the detailed

techniques can be changed, but the core technology remains the same,

consisting of three steps as depicted in Figure 1. The first step is to

generate a single-stranded aptamer library pool comprised of 1014–1015

random sequences. To ensure a complex aptamer pool, single-stranded

DNA templates that contain randomized sequences (30–50 nucleotides)

between two conserved sequences are chemically synthesized and

amplified via polymerase chain reaction (PCR). The second step, called

negative selection or counter-selection, is to remove non-specific-binding

aptamers. In this step, aptamers are incubated with immobilizers or target-

negative cells. The third step, called positive selection, is to amplify the

remaining target-specific aptamers using PCR. This cycle is repeated until

the initial library becomes enriched with target-specific aptamers. In

general, enriched target-specific aptamers are obtained after 8–15 rounds

of selection. The individual aptamer sequences are then defined using

classical Sanger or high-throughput sequencing methods.


Figure 1. The SELEX procedure consists of three steps: 1) generation of an aptamer library,

2) negative selection to remove non-specific binders to targets, and 3) positive selection to

isolate specific binders to targets.

3. IN VITRO APPLICATIONS FOR IMAGING

Aptamers developed for research and clinical applications to recognize

specific epitopes for biological applications have mainly been DNA-based.

The logic behind this is their relative stability against nucleases, low cost,

and convenience of chemical synthesis compared to RNA-based aptamers.

For tissue imaging, fluorophore-labeled aptamers are used for visual

detection to targets. For other type of in vitro imaging techniques used in

laboratory, a hybridization method using both aptamers and antibodies,

herein called sandwich methods, are mainly developed. In sandwich

method, aptamers are used as capturing agents. Currently developed

laboratory techniques for in vitro imaging are hybridized methods using

both aptamers and antibodies, herein called sandwich method. Antibodies

conjugated to an enzyme, most commonly alkaline phosphatase or

horseradish peroxidase are used as detecting agents to targets. In this

section, we will present successful studies that proved that aptamers are

excellent molecular probes for detecting specific epitopes for research

purposes.


3.1. Aptahistochemistry

Immunohistochemical staining is a very powerful and versatile

technique for determining the status of pathophysiological conditions in

both research and clinical samples [14]. However, standard

immunohistochemistry using antibodies imposes limitations, such as slow

diffusion into tissues for target recognition and additional steps required to

amplify signals. As detecting probes, aptamers that are directly labelled

with fluorophores improve target specificity and do not require additional

steps for visualization. Therefore, aptamers have slowly begun to substitute

for the role of antibodies in immunohistochemistry; accordingly, this

approach is called aptahistochemistry. The DNA and RNA aptamers

currently used as histochemical probes are listed in Table 1. Although

many successful studies of aptahistochemistry have been reported,

aptahistochemistry is still in the very early stages. The optimization of

buffer conditions and antigen retrieval for recognition of epitopes are the

main bottlenecks. The most successful outcomes in aptahistochemistry

have been shown with the use of Slow Off-rate Modified Aptamers

(SOMAmers), which contain base modifications that mediate hydrophobic

interactions at protein–aptamer sites to achieve high binding affinity.

In this section, we describe representative studies that show how

aptahistochemistry may be applicable in the clinic. Currently, the main

issue in application of aptahistochemistry is non-specific binding to cell

nuclei in sliced sections of formalin-fixed or frozen tissues. This could be

due to the electrostatic attraction of polyanionic aptamers to positively

charged histones in the nuclei. To circumvent this issue, SOMAmers were

identified based on the extracellular domains of epidermal growth factor

receptor (EGFR) and human epidermal growth factor receptor 2 (HER2)

[15] in the presence of dextran sulfate (DS), which is a polyanionic

competitor. This approach allowed isolation of SOMAmers that favor slow

target dissociation rates and minimized non-specific ionic contributions to

binding. Cy3-labeled SOMAmers against HER2 and EGFR and HER2

were used as molecular probes in frozen breast cancer tissue sections in the

presence or absence of DS. HER2 SOMAmers in the presence of DS

showed a HER2-specific tissue staining pattern, which was not observed in


HER2-negative tissues or non-breast negative control tissues [15]. In the

absence of DS, the HER2 SOMAmer showed non-specific binding in the

nuclei, cytoplasm, and stroma of various tissues. The EGFR-specific

SOMAmer showed similar results. This indicates that DS can be applied as

an anionic competitor to reduce non-specific binding of SOMAmers.

Table 1. A list of aptamers currently used as histochemical probes

Target/Disease Aptamer type Tissue fixation Staining

methods

Refs

Pancreatic cancer

cells/Pancreatic cancer

RNA Frozen Fluorescent [17]

Matrix metallopeptidase 2

(MMP2)/Atherosclerosis and

gastric cancer

DNA Frozen Chromogenic [84]

Human epidermal growth

factor receptor 2

(HER2)/Breast cancer

SOMAmer Frozen Fluorescent [15]

Thrombospondin-2 (TSP-2),

Mannose receptor C-type 1

(MRC1), and Endothelial cell

adhesion molecule

(ESAM)/Non-small cell lung

cancer

SOMAmer Frozen Fluorescent and

chromogenic

[16]

Lamin, vimentin, tubulin α,

neutrophil defensin1, CD31,

and actin/Lung cancer

DNA Frozen Fluorescent [85]

Epithelial cell adhesion

molecule (EpCAM)/Colorectal

cancer

DNA Frozen/FFFPE Fluorescent [86]

CD30/Lymphomas DNA FFPE Chromogenic [87]

Heparanase/Ovarian cancer DNA FFPE Fluorescent and

chromogenic

[88]

Understanding differences in protein expression between tumor and

non-tumor tissues is critical for biomarker discovery. Three SOMAmers

against thrombospondin-2 (TSP-2), macrophage mannose receptor 1

(MRC1), and endothelial cell-selective adhesion molecule (ESAM) were

used as fluorophore-labeled histochemical probes in frozen tissue sections

of non-small cell lung cancer or normal lung [16]. The TSP-2-specific

SOMAmer showed predominant staining in areas of fibrous stroma


surrounding tumors, but the staining was largely absent in normal tissues.

In contrast, MRC1- or ESAM-specific SOMAmers showed predominant

staining in normal lung tissues, with a lack of staining in lung cancer

regions. These aptahistochemistry results were consistent with expression

profiling of tissue homogenates, in which TSP-2 was increased and MRC1

and ESAM were decreased in tumor vs. healthy tissue [16]. This suggests

that profiling protein expression using SOMAmers may allow discovery of

new biomarkers in clinical tissue specimens.

RNA aptamers isolated using naïve cell SELEX in pancreatic cancer

cells showed pancreatic cancer-specific recognition [17]. Cy3-labeled

pancreatic cancer-specific aptamers, P19 and P1, were used as

histochemical probes in human pancreatic cancer frozen tissues.

Interestingly, the staining intensity of Cy3-P19 or Cy3-P1 showed a close

relationship with survival rates in cancer patients. This suggests that the

target of P19 and P1 may have potential as a biomarker for predicting

prognosis.

In summary, these illustrative studies of aptahistochemistry clearly

demonstrate that aptamers have great potential as molecular probes in

clinical histology. They also provide very meaningful indications that

aptamers show promise as detecting molecules for the discovery of

disease-specific biomarkers. Currently, Base Pair Biotechnologies, Inc.

provides customized aptahistochemistry services for researchers.

3.2. Mass Spectrometry Tissue Imaging

Mass spectrometry tissue imaging (MSI) is a very attractive imaging

technique for analysis of multiple biomolecules simultaneously and

biomarker discovery from tissue samples [18]. Recently, laser

desorption/ionization MS (LDI-MS) techniques have been developed to

analyze the distribution of nanomaterials in tissue specimens [19].

Generally, antibodies were used to capture targets of interest in tissue

samples. However, current limitation using antibodies for tissue imaging is

lack of cancer specificity. As a proof-of-concept study for tissue LDI-MS,


the anti-nucleolin aptamer AS1411, cancer specific aptamers, was

conjugated with gold (Au) nanoparticles [20]. Under laser

desorption/ionization, gold cluster ions were disassociated from AS1411-

Au nanoparticles and acted as signal amplifiers to detect nucleolin-positive

tumor tissues. This aptamer-guided LDI-MS imaging was tested on human

breast tumor and normal breast tissues. The MS images generated through

monitoring of Au signal intensity indicated that the tumor tissues had much

stronger signal intensities than normal tissues. Furthermore, the expression

and distribution of nucleolin in breast tissue by immunohistochemistry

showed consistent results with those obtained through LDI-MS. This

successful imaging acquisition using aptamers has major implications for

aptamers as suitable imaging probes in an MSI platform for the diagnosis

of malignancies. The schematic work flow is depicted in Figure 2.

However, a disadvantage of LDI-MS imaging is that it can only detect

highly abundant targets on tissue samples. Therefore, it provides diagnostic

information of greater value when combined with in vivo imaging.

Figure 2. Mass spectrometry tissue imaging. Anti-nucleolin aptamers conjugated with

gold nanoparticles are used to stain tumor tissues. Under laser desorption/ionization,

gold cluster ion-amplified signal reporters are collected for laser mass spectrometry

tissue imaging (MSI). The image is adopted from “Targeted molecular imaging using

aptamers in cancer,” Pharmaceuticals 2018; 11(3):71; doi:10.3390/ph11030071.


3.3. Enzyme-Linked Aptamer Assay

The first conceptualization of an enzyme-linked aptamer assay

(ELAA), in which aptamers replace antibodies in enzyme-linked

immunosorbent assays (ELISA), was reported in 1996 to detect human

vascular endothelial growth factors [21]. Since the first development of

ELAA, the majority of ELAA development has been for detection of

infectious diseases, replacing antibodies in ELISAs [22]. Due to their

convenient chemical synthesis and relative serum stability, DNA aptamers

were used to develop ELAA. The principle of ELAA is based on using a

target-specific aptamer as a capture molecule or as a detection molecule.

Because sandwich ELAAs show better specificity and sensitivity compared

with direct or indirect ELAAs, most studies have adopted the sandwich

format. The configuration of a sandwich ELAA is depicted in Figure 3A. A

list of aptamers currently used in ELAA is given in Table 2. Current trends

in the development of ELAA are being led by biotechnology companies

such as Aptatek Biosciences and Aptasol for diagnostic tools.

Figure 3. In vitro imaging by aptamers: (A) Enzyme-linked aptamer assay (ELAA) and

(B) aptablot. (A) In a sandwich ELAA assay performed in wells of microplates,

aptamers are used to capture target molecules, then enzyme-conjugated antibodies and

chromogenic substrate are added for visualization. (B) In an aptablot, aptamers are

cross-linked on a polyester membrane and target molecules are detected with enzyme-

conjugated antibodies and chromogenic substrate.


Table 2. A list of aptamers used in ELAA

Targets Aptamer

type

Type of ELAA Detection

limits

Refs

Aflatoxin B DNA Sandwich, capturing agent 1pg/mL [89]

Cholera toxin DNA Sandwich, capturing agent 2.1ng/mL [90]

Acinetobacter

baumannii

DNA Sandwich, detecting agent N/A [91]

Staphylococcus

aureus

DNA Direct, sandwich, and

competitive/capturing or detecting agent

NA [92]

Zika NS1 DNA Sandwich, capturing 0.1 ng/mL [93]

Hepatitis C virus

particles

DNA Sandwich, capturing N/A [94]

Leishmania infantum DNA Direct N/A [95]

3.4. Aptablot

The specific detection of macromolecules with chromogenic

substrates, such as Western blotting, still remains a major research tool.

However, new techniques have been developed that apply aptamers to

detect macromolecules, called “aptablot” [23]. In the aptablot system, a

sandwich approach has been popularized. In one study, anti-thrombin

DNA aptamers were immobilized on polyester cloth by UV cross-linking

as a capturing agent. Thrombin was used as a model protein analyte and

target molecules were detected by reaction of antibodies linked to a marker

enzyme (peroxidase) and developed with TMB peroxidase substrate [23].

A schematic of this detection method is depicted in Figure 3B. Because the

principle of aptablot is simple and broadly applicable, its biological

application is expected to extend into multiple research situations.

3.5. Aptamer-Mediated Flow Cytometry and Mass Cytometry

Antibodies are still the most commonly used molecular probes for

determining cell characteristics and obtaining highly specific information

about individual cells for researchers. However, in contrast to antibodies,


aptamers can be isolated against whole cells. Therefore, aptamers can

recognize native forms of epitopes, providing greater specificity. This

compelling characteristic has led to their use as probes for flow cytometry,

especially for assessing cell characteristics, cell phenotypes, virus-infected

cells, and pathogens [24]. The range of applications using aptamers as

probing agents for flow cytometry are very wide, from surface markers of

cancer cells to pathogens [24], showing high potential for

commercialization.

An emerging type of cytometry is mass cytometry, which uses metal

tags instead of fluorophores. The major benefit of mass cytometry is the

capability to measure at least 34 cellular parameters simultaneously [25].

As proof of concept for using aptamers as molecular probes for mass

cytometry, 165Ho-conjugated sgc8 DNA aptamers that bind to the human

protein tyrosine kinase 7 (PTK7) receptor were used to characterize PTK7-

positive cells (human acute lymphoblastic leukemia) and PTK7-negative

cells (human Burkitt lymphoma) using mass cytometry [26]. The study

successfully discriminated between the cell types, suggesting that metal-

tagged aptamers can be multiplexed for mass cytometry in the near future.

3.6. Super-Resolution Microscopy Imaging

Optical imaging is a critical research tool for biologists. The advent of

super-resolution microscopy has transformed biomedical research by

allowing researchers to observe nano-structures. Traditional staining

methods rely on antibodies against the target of interest. Due to their large

size, antibody-based staining has limitations for high resolution and low

density labeling due to steric hindrances. An ideal labeling probe should be

the smallest size possible to achieve maximal labeling efficacy. In this

respect, aptamers fall into this highly desirable category. One group

performed a direct comparison between aptamers and antibodies on live

cells, targeting three membrane receptors: EGFR, epidermal growth factor

receptor 2 (ErbB2), and ephrin type-A receptor 2 (Epha2) [27]. They

showed that the fluorescence intensity of single antibodies was at least two


times higher than single aptamers. However, antibodies showed fewer

cellular epitopes than aptamers. Aptamers also provided dense labeling of

epitopes, resulting in better structure recognition. This may be due to the

smaller size of aptamers [27]. This study clearly underscores the

advantages and disadvantages of the different types of probes in imaging

work.

Super-resolution microscopy combined with DNA points accumulation

for imaging in nanoscale topography (DNA-PAINT) is a newly developed

imaging technique that visualizes the transient binding of short

fluorescently labeled oligonucleotides to their complementary target

strands, which “blink” due to stochastic activation of the fluorophores [28].

To perform DNA-PAINT imaging, one group employed seven different

SOMAmers against EGFR, Nup107, catalase, ErbB2, Hsp90, LIMP-2, and

HSP60 which are highly expressed in cancers. They showed that

SOMAmers have a twofold smaller apparent size in single distribution

pattern than antibodies, indicative of precision detection. This study also

showed the potential for multiplexed labeling and imaging. Such an

application has the potential to differentiate single proteins with nanoscale

imaging.

4. CELL IMAGING

Cell imaging is considered essential for understanding cellular

physiology. Historically, green fluorescent protein (GFP) has been central

to cellular imaging applications by way of being tagged onto endogenously

coding genes to investigate the expression, interaction, localization, and

role of target proteins in cells [29, 30]. However, the vast amount of

research in the RNA field has begun expanding the roles of RNAs, leading

to development of innovative tools for intracellular imaging. This includes

fluorogenic aptamers that activate fluorescence upon binding of their target

fluorophores: Spinach [31], Broccoli [32], Mango [33], and Corn [34]. For

cell imaging, these fluorogenic aptamers can be engineered as biosensors

to detect intracellular metabolites or imaging tags to investigate the


dynamics of intracellular RNAs in living cells by microscopy. To date,

although fluorogenic aptamers have great potential for in vivo imaging,

they have not yet been applied in this manner. Thus, in this section, we will

discuss fluorogenic aptamers and their applications in cells.

4.1. Green Fluorogenic Aptamers: Spinach and Broccoli

The “Spinach” fluorogenic RNA aptamer activates green fluorescence

upon the binding of the small-molecule fluorophore 3,5-difluoro-4-

hydroxybenzylidene imidazolinone (DFHBI) and its variants (e.g., DFHBI

1T and DFHBI 2T) [31]. To investigate the precise working mechanism of

fluorescence activation, the Spinach-DFHBI complex was determined by

analyzing the crystal structure. The co-crystal structure of the Spinach-

DFHBI complex revealed an unexpected quadruplex core connected with

two G-tetrads and a mixed-base tetrad [35]. DFHBI was inserted by a base

triple and confined around an unpaired guanine residue (G31). In

comparison with a fluorophore-free condition, the collapsed fluorophore

binding site was rearranged with the base tier of the quadruplex, extruding

G31 from the helical stack [35]. The fluorogenic Spinach was a novel

proof of concept, but weaknesses of Spinach such as thermal instability,

requirement for a high magnesium concentration, misfolding, and reduced

brightness [36] have limited its application for imaging. In the following

studies, Spinach was mutated to multiple variants such as Spinach2 (36)

and Baby Spinach [37] to increase thermal stability and improve folding

structure. Because Spinach had some drawbacks, the microfluidic-assisted

in vitro compartmentalization (μIVC) method was employed to screen for

aptamers able to work in low bivalent concentrations; the selected

aptamers were minimized for in vitro application, which was named

iSpinach [38]. In a comparison of iSpinach with Spinach2, iSpinach

showed 1.4 times more fluorescence in the presence of potassium, with

better thermostability and binding affinity than Spinach2. However, live-

cell imaging was not evaluated. For live-cell imaging, Spinach and

Spinach2 were engineered as biosensors to detect cellular metabolites (S-


adenosylmethionine [SAM], adenosine 5-diphosphate [ADP], and second

messenger Cyclic di-AMP [cdiA]) in Escherichia coli (E. coli) and Listeria

monocytogenes (L. monocytogenes) [39, 40]. Spinach engineered with

SAM-binding RNA aptamers via a stem sequence that acted like a

transducer for a biosensor showed 6-fold increased fluorescence over 3 h,

when the SAM precursor methionine was given to DFHBI-treated E. coli

(40). A Spinach-ADP sensor showed a 20-fold increase of fluorescence in

DFHBI-treated E. coli [40]. For imaging of intracellular cdiA levels in L.

monocytogenes, the ligand-sensing domain of cdiA riboswitch (yuaA

riboswitch) was combined with Spinach2, termed YuaA-Spinach2, to act

as a fluorescent biosensor [39]. When a plasmid encoding the biosensor

was transformed into live L. monocytogenes, robust intracellular

fluorescence was observed under fluorescence microscopy in the presence

of DFHBI [39]. This clearly demonstrates that the yuaA-Spinach2

biosensor works in living cells.

To overcome the limitations of Spinach, the green fluorogenic RNA

aptamer “Broccoli,” which also binds to the DFHBI fluorophore, was

isolated using a different approach by the same group that developed

Spinach [32]. The method of Broccoli isolation was designed to improve

the specificity and functionality of the fluorogenic aptamer. Fluorescent

RNA aptamers were treated with fluorophores after the first round of

conventional aptamer selection method, then inserted into plasmids. After

transformation of the cloned plasmids into E. coli, cells were treated with

DFHBI and fluorescent cells were isolated using fluorescence-activated

cell sorting (FACS). This approach successfully isolated the fluorogenic

aptamer Broccoli, which showed robust folding in low salt concentrations,

with increased thermostability and stronger fluorescence relative to

Spinach2 [32]. For additional applications in in vitro imaging, Broccoli

was truncated to tBroccoli and then dimerized to tdBroccoli. The

dimerized tdBroccoli showed twice the fluorescence intensity compared to

Broccoli in living E. coli [32]. To make a biosensor, Broccoli or dBroccoli

was inserted to the 3´ end of a 5S plasmid without the pAV5S tRNA

scaffold. It showed higher fluorescence intensity in transfected HEK293T

cells (human embryonic kidney) using microscopy [32]. The Broccoli

study suggests that the strong thermostability and folding structure of


aptamers do not require a tRNA scaffold system to support the appropriate

folding of aptamers intracellularly, which will be a valuable advantage for

future in vivo imaging.

4.2. Orange Fluorogenic Aptamer: Mango

The “Mango” RNA aptamer binds to derivatives of the fluorophore

thiazole orange (TO1) and shows high levels of orange fluorescence,

which increases by up to 1,100-fold upon the binding of TO1 [33]. The

structure of Mango was predicted to be folded as two-tiered, all-parallel G-

quadruplexes in an A-form duplex [33]. Crystal structure analysis of the

Mango-TO1 complex showed three antiparallel quinines forming G-

quadruples [41]. The binding of TO1 on one of the flat faces of the G-

quadruplex stabilizes the Mango structure and activates orange

fluorescence. For imaging applications, TO1 was directly injected into

Caenorhabditis elegans (C. elegans) syncytial gonads with equimolar

amounts of RNA Mango. Strong fluorescence composition was observed

in the nuclei of gonadal cells [33].

A new orange fluorogenic RNA Mango aptamer was isolated by the

group that isolated iSpinach using microfluidics-based selection methods

(μIVC) [42]. This “New Mango” was brighter than enhanced green

fluorescent protein (EGFP) when bound to TO1, which is a major

advantage for imaging applications. For imaging sensors, New Mango was

tagged by incorporating human 5S ribosomal RNA or U6 small nuclear

RNA (snRNA) into F30 folding scaffold plasmids. RNAs of 5S-F30-New

Mango or U6-F30-New Mango were generated by in vitro transcription

and transfected into HEK293T cells [42]. Subcellular co-localization and

fluorescence were investigated using fluorescence microscopy. The 5S-

F30-New Mango RNAs showed up to 10 bright RNA foci per cell in the

cytoplasm, mainly co-localized with mitochondria [42]. The U6-F30-New

Mango RNA showed a different localization; it was mainly co-localized

with the small nuclear ribonuclear protein (snRNP) Lsm3 [42]. To develop

a genetically encoded imaging tag expressed in mammalian cells, New

Mango was inserted into the pSLQ plasmid to express 5S rRNA under the


RNA pol III promoter (pSLQ-5S-F30-New Mango) [42]. The engineered

pSLQ-5S-F30-New Mango plasmid was transfected into cells.

Fluorescence was observed in both nucleolar and cytoplasmic

compartments using fluorescence microscopy. pSLQ-5S-F30-New Mango

was also observed to co-localize with ribosomal protein L7 [42]. These

results clearly demonstrate that New Mango has the capability to be used

as an imaging tag to visualize the subcellular location of target genes.

4.3. Yellow Fluorogenic Aptamer: Corn

The RNA aptamer that binds to 3, 5-difluoro-4-hydroxynenzylidene

imidazolinone-2-oxime (DFHO), named “Corn,” activates yellow

fluorescence [34]. Compared with Broccoli, Corn showed strong

photostability; its fluorescence was detected at 320-ms imaging times vs.

160 ms with Broccoli [34]. In irradiation experiments, the Corn–DFHO

complex exhibited minimal loss of fluorescence after as long as 10 s; in

contrast, >50% of fluorescence was lost after 200 ms with the Broccoli–

DFHBI complex. Therefore, Corn is considered an emerging new

fluorogenic aptamer for biosensors. To test the feasibility of Corn as a

genetically encoded fluorescent RNA tag, three main subclasses of Pol III

promoters (5S RNA, tRNA, and U6) with a tRNA Lys scaffold plasmid

were constructed with the Corn aptamer. When these reporter plasmids

were transfected into HEK293T cells, yellow fluorescence was observed

by fluorescence microscopy after treatment with DFHO [34].

4.4. Rainbow Fluorogenic Aptamer: SRB-2

The SRB-2 aptamer, originally isolated against the fluorophore

sulforhodamine B, binds to various dyes with xanthene-like cores, such as

pyrosin B, pyrosin Y, acridine orange, SR-DN, and TMR-DN, and yields a

“rainbow” of differently colored bright fluorescence [43]. To confirm

various fluorescent signals given off by the SRB-2 aptamer upon binding


of different fluorophores, a single copy of the SRB-2 aptamer sequence

was inserted into a tRNA scaffold plasmid. After transformation of the

SRB-2 inserted plasmid into E. coli, fluorescence activation was

investigated using microscopy in the presence of a variety of fluorophores.

SRB2-expressing bacteria showed various colors after incubation with

each fluorophore: pyrosin B (yellow color), pyrosin Y (yellow color),

acridine orange (green color), SR-DN (red color), and TMR-DN

(yellowish-orange color) [43]. Because the SRB-2 aptamer showed various

colors of fluorescence, selection of the most appropriate fluorophore was

investigated for subsequent no-wash and live-cell imaging methods. Even

though pyrosin B, pyrosin Y, and acridine orange showed differently

colored fluorescence, the three fluorophores showed high signal-to-

background ratio, compared with SR-DN and TMR-DN. SR-DN was

previously developed as a live-cell imaging fluorophore for the SRB-2

aptamer, but it showed low binding affinity (Kd = 1.3 μM). In contrast,

TMR-DN showed higher binding affinity (Kd = 35 nM) for the SRB-2

aptamer. In a comparison of SR-DN and TMR-DN, SRB-2-expressing

bacteria displayed 11-fold higher fluorescence intensity after incubation

with TMR-DN than with SR-DN [43]. Therefore, TMR-DN was used for

subsequent live-cell imaging. For live-cell imaging of rRNA and mRNA in

mammalian cells, 5S rRNA embedded with a single repeat of SRB-2 in a

tRNA scaffold was applied in mammalian cell imaging [43]. The nuclear

and cytoplasmic distribution of fluorescent 5S rRNA were clearly

visualized using a 5S-SRB-2 construct in HeLa cells (human cervical

cancer) by microscopy [43]. For mRNA imaging in live prokaryotic cells,

6 or 15 tandem repeats of SRB-2 without a tRNA scaffold were fused to

the 3´-UTR GFP mRNA. Bacteria expressing GFP-6xSRB-2 or GFP-

15xSRB-2 showed significantly higher fluorescence than bacteria

expressing only GFP after incubation with TMR-DN. For mRNA imaging

in live eukaryotic cells, 15 repeats of SRB-2 without a tRNA scaffold were

fused to 3´-UTR CFP-TM (cyan fluorescent protein). Microscopy revealed

that HeLa cells transfected with CFP-TM-15xSBR-2 plasmid showed

significantly higher fluorescence in TMR-DN than control cells, with

mostly cytoplasmic distribution of the CFP-TM mRNAs [43].


Taken together, fluorogenic RNA aptamers such as Spinach, Broccoli,

Mango, Corn, and Rainbow aptamers can be successfully applied for

intracellular imaging in live cells. Thus, these fluorogenic RNA aptamers

are great tools for investigating the dynamics of intracellular RNAs, as

depicted in Figure 4. Theoretically, RNA aptamers can be developed to

any fluorophores, and because fluorogenic RNA aptamer tags have diverse

applications, we expect a variety of fluorogenic aptamers suitable for

mammalian in vivo imaging will be developed in the near future.

Figure 4. Live-cell imaging with bioengineered aptamer tags. Fluorogenic RNA

aptamers such as Spinach, Broccoli, Mango, Corn, and Rainbow are inserted after a

gene of interest, bind to their target fluorophores, and turn on the fluorescence in the

presence of their target fluorophores. The fluorogenic RNA aptamer tags can be used

to for tracking and for investigating the dynamics of the gene of interest in live cells.

The image is adopted from “Targeted molecular imaging using aptamers in cancer,”

Pharmaceuticals 2018; 11(3):71; doi:10.3390/ph11030071.

5. IN VIVO IMAGING

Identifying target-specific probes is critical to conferring specificity for

in vivo imaging. In this respect, the majority of aptamers that have already

been developed target cancer-specific surface antigens [44]. Therefore,


these cancer-specific aptamers are effective probes for a variety of imaging

techniques in cancer diagnostics, including fluorescence optical imaging,

MRI, PET, SPECT, CT, US, and photoacoustic imaging. In this section,

we will discuss cancer-specific aptamers labeled with multiple imaging

dyes for targeted in vivo imaging.

5.1. Fluorescence Imaging

Fluorescence is the most adapted tool in aptamer-guided imaging

because of its low cost and high sensitivity. The most commonly used

fluorescent dyes are cyanine 5 (Cy 5), quantum dots (QDs), and near-

infrared (NIR) dye. Cancer cell-type-specific DNA aptamers against

Ramos cells (B-cell lymphoma) were labeled with a Cy5 probe (Cy5-

TD05) and the efficacy of in vivo imaging was determined in

subcutaneously xenografted nude mice via tail vein injection [45]. This

demonstrated that Cy5-TD05 specifically accumulated in engrafted tumor

regions by 3.5 h or later, compared with control aptamers, Cy5 dye only, or

in non-targeted cells. Cy5-TD05 also showed a high signal-to-background

ratio at 115:50. In biodistribution assays, high fluorescence was observed

in the small intestine [45]. In another cancer cell-type-specific aptamer

study, DNA aptamers specific to A549 cells (lung carcinoma cells) were

labeled with a Cy5 probe (Cy5-S6) for in vivo imaging [46]. In

subcutaneously xenografted nude mice, Cy5-S6 showed tumor-specific

accumulation between 3 to 5 h after intravenous injection. In contrast, no

fluorescence was observed with a Cy5-labeled library or in non-targeted

cells. Using the same approach, two other cancer cell-type-specific DNA

aptamers against Bel-7404 and SMMC-7721 (human hepatocellular

carcinomas) were tested in vivo. Both aptamers showed tumor-specific

fluorescence accumulation by 3 h post-injection.

QDs have been studied as another fluorescence imaging reporter in

various cancer models. QDs are fluorescent nanoparticles that have great

advantages for imaging, such as low photobleaching, high fluorescence

yield, and chemical stability [47]. Fluorescence imaging using QDs in vivo


has been investigated in the form of chemical conjugation with aptamers

specific to mucin-1 (MUC-1) or variant of epidermal growth factor

receptor (EGFRvIII). Because MUC-1 is highly expressed on the cancer

plasma membrane, the cancer specificity of an anti-MUC-1 DNA aptamer

conjugated with QDs was evaluated in A549 cells [48]. Anti-MUC-1 DNA

aptamers labeled with QDs were injected into subcutaneously xenografted

nude mice via the tail vein. Strong fluorescence was observed in the tumor

specifically, but not with control QDs. EGFRvIII is selectively expressed

on cancer cells, and enhances cancer cell proliferation, invasion, and

resistance to chemotherapy and radiotherapy [49]. Therefore, it has been

popularized as a target for therapeutics and imaging. The anti-EGFRvIII

DNA aptamer A32 was conjugated with streptavidin-PEG-CdSe/ZnS QDs

(QD-A32 Apt). For in vivo imaging, U87 (glioma) cells and U87-

EGFRvIII overexpressing cells were orthotopically xenografted into nude

mice [50]. After tail-vein injection of QD-A32 Apt, fluorescence imaging

was performed 6 h post-injection. Mice harboring U87-EGFRvIII

overexpressing cells, but not U87 tumors, showed strong fluorescence

signals in tumor areas. No significant fluorescence was observed in mice

injected with unconjugated QDs. In a biodistribution assay, QD-A32 Apt

showed high accumulation in the tumor region, liver, and kidneys, and low

accumulation in the tumor pararegion, normal brain, spleen, heart, and

lungs. In another study, anti-EGFR aptamers targeting wild-type EGFR

were conjugated with lipid QDs (Apt-QLs) [51]. The feasibility of using

Apt-QLs for in vivo imaging was tested in an MDA-MB-231 breast in vivo

tumor model. By 4 h post-intravenous injection, it showed breast cancer-

specific accumulation in engrafted mice [51].

Because NIR dyes enhance sensitivity due to low background

autofluorescence and high signal-to-noise ratios, they are another suitable

fluorescent dye for in vivo imaging. In a study of prostate cancer (PC), an

anti-prostate-specific membrane antigen (PSMA) RNA aptamer (A9g) was

conjugated with NIR dye (IRDye 800CW) [52]. The NIR-labeled aptamer

(NIR-A9g) was injected into the tail veins of mice bearing PC-3 (PSMA-

positive) tumor cells. It showed intense cancer-specific fluorescence,

beginning at 24 h and up to 72 h after administration [52]. In another study,


a new NIR dye, which is a derivative of indocyanine green (MPA;

absorbance peak at 780 nm and emission peak at 810 nm), was tested in

breast cancer and liver cancer animal models. Anti-MUC-1 DNA aptamers

were conjugated with the MPA dye (APT-MPA) or with polyethylene

glycol-NIR (APT-PEG-MPA) [53]. MCF-7 cells (human breast cancer) or

HepG2 cells (human liver cancer) were subcutaneously xenografted into

nude mice. MPA, APT-MPA, or APT-PEG-MPA were injected into mice

via tail-vein injection. Strong tumor-specific fluorescence resulting from

targeting of APT-MPA or APT-PEG-MPA was observed starting at 2 h up

to 72 h on both breast and liver cancers in vivo. In contrast, control MPA

did not show accumulation of fluorescence in the tumor-engrafted region

[53]. In a biodistribution assay, the PEG-modified probe (APT-PEG-MPA)

showed much quicker clearance than that of APT-MPA [53].

5.2. Magnetic Resonance Imaging (MRI)

MRI is a very powerful imaging technique for non-invasive

visualization of internal organ structures and soft tissue morphology by

using a powerful magnet and radiofrequency energy [54]. Introducing MRI

contrast agents by way of specific targeting moieties like aptamers has

significantly improved the quality of imaging. The αvβ3 integrin subunit is

involved in cell migration and invasion during neovascularization and/or

angiogenesis in cancer. Therefore, it was targeted for molecular imaging

[55]. An anti-αvβ3 aptamer (Apt αvβ3) was conjugated with magnetic

nanoparticles (MNPs) for cancer-specific detection via MRI [56]. Apt αvβ3-

conjugated magnetic nanoparticles (Apt αvβ3-MNPs) were injected into

A431 (human epidermoid carcinoma) xenografted nude mice

intravenously. The evaluated MRI signals showed maximum intensity at

24 h post-injection. The accumulated amount of Apt αvβ3-MNPs was 129 ±

34.3% in the tumor compared to short peptide-conjugated MNPs as control

[56].

Vascular endothelial growth factor receptor 2 (VEGFR2) is commonly

overexpressed in the tumor vasculature, and could be another target-of-


interest for cancer imaging. To recognize glioblastoma vasculature, anti-

VEGFR2 aptamers were immobilized on the surface of carboxylated

magnetic nanocrystals (MNCs) [57]. The molecular imaging potential of

the VEGFR2 aptamer-MNCs (VEGFR2 Apt-MNCs) were assessed in an

orthotopic glioblastoma nude mouse model. MRI was performed on mice

after intravenous tail veil injection of VEGFR2 Apt-MNCs or

unconjugated MNCs using 3.0-T MRI. At 4 h post-injection, MRI was

darker in tumor sites treated with VEGFR2 Apt-MNC than in those treated

with unconjugated MNC, confirming that VEGFR2 Apt-MNC enabled

precise in vivo detection [57].

A common feature of solid cancer is hypoxia, which induces the

activation of hypoxia-inducible factor-1α (HIF-1α) [58]. HIF-1α is also

involved in the maintenance of cancer stem cells. For non-invasive

imaging to identify hypoxia-induced cancer stem cells, HIF-1α aptamer-

PEG-modified manganese magnetic nanoparticles, D-Fe3O4@Mn, were

generated [59]. For in vivo imaging, PANC-1 cells (human pancreatic

cancer) were subcutaneously xenografted into nude mice. D-Fe3O4@Mn

was injected into the mice intravenously and MRI was performed before

and after injection at 2 h. Compared with images of mice without NP, the

D-Fe3O4@Mn-injected mice showed significantly bright enhanced effects

and 3.5-fold higher signal intensity in T1-weighted MRI imaging (T1W1).

Upon histopathological examination, no signs of toxicity were observed in

the major organs at day 10 after injection of D-Fe3O4@ Mn [59].

5.3. Single-Photon Emission Computed Tomography

(SPECT) Imaging

Nuclear imaging techniques like SPECT and PET can monitor

biological events deep within the body and provide longitudinal

assessment of a patient with high detection sensitivity [60]. Therefore,

development of imaging modalities for SPECT and PET with high

specificity is critical. In this respect, cancer-specific aptamers are excellent

detecting probes. SPECT imaging detects γ-ray emissions from


radioisotopes using a gamma camera. The most commonly used isotopes

for SPECT imaging are 99mTc (Technetium, t1/2: 6 h), 111In (Indium, t1/2:

2.8 d), 123I (Iodine, t1/2: 13.2 h), and 131I (Iodine, t1/2: 8.0 d) [61]. For

SPECT imaging, an anti-tenascin-C aptamer (TTA1) was conjugated to the

succinimidyl ester MAG2 and radiolabeled with 99mTc (MAG2-TTA1-99mTc) [62]. To assess in vivo tumor targeting, MAG2-TTA1-99mTc was

intravenously injected into mice bearing U251 (human glioblastoma) or

MDA-MB-435 (human breast cancer) tumors. After aptamer-based γ-

camera imaging of the tumors, the U251 glioblastoma was prominently

visible at 3 h, but not after application of control aptamers. The MDA-MB-

435 breast tumor showed cancer-specific imaging at 20 h. Biodistribution

assays showed that bladder and liver (10 min) and bladder and intestines (3

h) were predominant clearance pathways [62]. When the radiometal

chelator was switched to diethylenetriaminepentaacetic acid (DTPA;

DTPA (111In)-TTA1), the DTPA (111In)-TTA1 aptamer showed

persistent accumulation in the liver and kidneys at 3 h.

EGFR is overexpressed in 90% of head and neck cancers [63]. For

comparison studies of aptamers with antibodies, an anti-EGFR aptamer

(apt) and antibody (C225) were conjugated with Hollow gold nanospheres

(HAuNS), followed by radiolabeling with 111In-DTPA. For in vivo

imaging, the apt-HAuNS-111In-DTPA, C225-HAuNS-111In-DTPA, and

control-HAuNS-111In-DTPA were injected intravenously into OSC-19

(human tongue squamous cell carcinoma) orthotopic xenografted nude

mice. SPECT imaging was performed at 4 h and 24 h post-injection [64].

The imaging revealed that at 24 h post-injection, more apt-HAuNS-111In-

DTPA accumulated in the tumor than C225-HAuNS-111In-DTPA. These

results demonstrate that aptamers are easily taken up into tumors, likely

due to their relatively smaller size compared to antibodies, suggesting that

aptamers might be better tumor-homing ligands. In a biodistribution assay,

apt-HAuNS-111In-DTPA showed greater retention in blood, kidneys, and

lymph nodes. In contrast, C225-HAuNS-111In-DTPA showed greater

retention in the liver and spleen. Human matrix metalloprotease-9 (hMMP-

9) is overexpressed in cancers, particularly in cutaneous melanomas [65].

An RNA aptamer against hMMP9 (F3B) was conjugated with 111In-DOTA


(111In-DOTA-F3B). After intravenous injection of 111In-DOTA-F3B into

mice bearing human melanoma tumors, SPECT images were acquired at 1

h. Micro-imaging analysis showed that 111In-DOTA-F3B induced a

stronger accumulated signal than 111In-DOTA-control. Signal activity was

increased in a tumor grade-dependent manner in ex vivo images [66]. The

biodistribution of 111In-DOTA-F3B was determined at 30 min, 1 h, and 2 h

post-injection. The average half-life was 11 min, which represents a rapid

clearance from the blood. The aptamer also showed high uptake in kidneys

and liver, but not other normal organs. The uptake of 111In-DOTA-F3B in

the tumor region was significantly higher at 1 h post-injection, compared

with 111In-DOTA-control-aptamer. A variant of the anti-EGFRvIII DNA

aptamer (U2) was labeled with rhenium radioisotope (188Re) [67]. SPECT

molecular imaging of 188Re-U2 was performed in U87MG (human

glioblastoma) xenografted mice at 1 h post-injection. The 188Re-U2

aptamer, but not control aptamer, was retained in tumors specifically. Its

main route of excretion was through the urinary tract [67].

5.4. Positron-Emission Tomography (PET)

PET has at least ten-fold more sensitivity than SPECT [54]. Therefore,

it has quickly emerged as a robust molecular imaging technique. For PET

imaging probes, four chelators (DOTA, CB-TE2A, S-2-(4-

Isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-tetraacetic acid

[DOTA-Bn], and S-2-(4-Isothiocyanatobenzyl)-1,4,7-triazacyclononane-

1,4,7-triacetic acid [NOTA-Bn]) were selected and used to label the anti-

nucleolin AS1411 aptamer, along with 64Cu [68]. In comparison studies of

these chelators, 64Cu-DOTA-AS1411 and 64Cu-CB-TE2A-AS1411 were

internalized into cells at significantly greater levels than 64Cu-DOTA-Bn-

AS1411 and 64Cu-NOTA-Bn-AS1411. Thus, 64Cu-DOTA-AS1411 and 64Cu-CB-TE2A-AS1411 were chosen for further in vivo imaging. 64Cu-

DOTA-AS1411 or 64Cu-CB-TE2A-AS1411 was injected into H460

(human lung cancer)-bearing mice via tail-vein injection and mice were

imaged at multiple time-points up to 24 h post-injection. In mice injected


with 64Cu-CB-TE2A-AS1411, tumors were visible from 1–24 h. However,

the tumor was not detectable in mice injected with 64Cu-DOTA-AS1411.

This suggests that CB-TE2A is a more desirable chelator for in vivo

kinetics. In biodistribution assays, 64Cu-CB-TE2A-AS1411 also had faster

clearance and less accumulation in the liver and kidney at 1 h post-

injection than 64Cu-DOTA-AS1411 [68]. Tenascin C shows elevated

expression in tumor stroma and plays a pivotal role in brain and breast

cancer initiation and progression [69, 70]. Anti-tenascin C DNA aptamers

were conjugated with 64Cu-NOTA to construct 64Cu-NOTA-tenascin C

[71]. For targeted molecular imaging of cancer, U86 MG cells (human

glioma) and H460 cells were subcutaneously xenografted into mice and

MDA-MB-435 cells were orthotopically xenografted into the mammary

glands of mice. The U86 MG and MDA-MB-435 tumors were positive for

tenascin C, whereas H460 tumors were negative. After injection of 64Cu-

NOTA-tenascin C, PET images were taken at 1, 2, 6, and 24 h in the three

cancer cell-engrafted mouse models. Tumor accumulation was clearly

evident up to 24 h post-injection in U86 MG- and MDA-MB-435-

engrafted mice, but not in H460-engrafted mice [71].

Conventionally, aptamers were directly conjugated with a radioactive

tracer isotope for PET imaging. As a more versatile platform for PET

imaging probes, a hybridization strategy between two complementary

oligodeoxynucleotides (ODN) has recently been used to label aptamers.

The advantage of this platform is that the radioactive tracer isotope can be

easily switched. As a proof of concept, AS1411 aptamers were hybridized

with fluorine radioisotope (18F)-labeled complementary ODN9 (cODN)

[72]. The feasibility of hybridization-based aptamer labeling for PET

imaging was tested in vivo. C6 (human glioma)-bearing mice were injected

with 18F-hyAS1411 and 18F-hy control C-rich aptamer (18F-hyCRO) via

tail-vein injection and PET imaging was performed 60 min post-injection. 18F-hyAS1411 clearly showed tumor-specific accumulation, compared

with 18F-hyCRO. sgc8 is a 41-nt DNA aptamer that binds to protein

tyrosine kinase 7 (PTK-7) [73], which is overexpressed in several human

malignancies. sgc8 was conjugated with 18F using click chemistry [74].

The 18F-tagged DNA aptamer sgc8 was injected into nude mice bearing


HCT116 (human colon cancer) tumors, which are positive for PTK-7. 18F-

sgc8 clearly visualized the engrafted tumor region at 2 h post-injection.

The main clearance routes were reported as urinary and hepatic.

5.5. Computed Tomography (CT) Imaging

CT imaging is a commonly used technique to examine anatomical

structures. However, contrast agents/probes for this modality are very

limited. Recently, gold nanoparticles have emerged as new nanoplatforms

for CT imaging. As a proof of concept for CT imaging, anti-PSMA RNA

aptamers were conjugated to gold nanoparticles and used as a CT contrast

agent in PC [75]. The PSMA aptamer-conjugated gold nanoparticles

showed more than 4-fold greater CT intensity for targeted LNCaP cells

(human prostate adenocarcinoma, PSMA positive cells) than that of non-

targeted PC3 cells (human prostate carcinoma, PSMA negative cells), but

no in vivo imaging was carried out. As another CT imaging approach,

fluorescent gold nanoparticles were conjugated with diatrizoic acid and the

anti-nucleolin AS1411 aptamer (AS1411-DA-AuNPs) [76]. The

fluorescence spectrum of AS1411-DA-AuNPs showed a clear orange-red

emission, with a maximum at 620 nm. In vivo molecular imaging was

investigated in CLI-5 (lung adenocarcinoma)-bearing NOD-SCID mice via

tail-vein injection of AS1411-DA-AuNPs. CT imaging was performed 30

min post-injection and showed 106% greater contrast enhancement in CLI-

5 tumor-bearing mice than before-injection. In parallel, the orange-red

fluorescence emitting from AS1411-DA-AuNPs in the CLI-5 tumor could

also be visualized by the naked eye [76].

5.6. Ultrasound (US) Imaging

The use of gas-filled nanobubbles (NBs) constructed from poly

(lactide-co-glycolic acid) (PLGA), which is a biodegradable copolymer,

improves US signals [77]. Therefore, it has been popularized as a contrast


agent for CT imaging. For targeted molecular imaging, the anti-PSMA

A10-3.2 aptamer was conjugated with PLGA NBs (A10-3.2-PLGA NBs)

[78]. For in vivo imaging, the A10-3.2-PLGA NBs were injected into

LNCaP PC-engrafted nude mice intravenously. Compared with the saline

control group, the distribution of contrast agent echo signals was rich and

enhancement of contrast was observed in mice treated with A10-3.2-PLGA

NBs [78]. The A10-3.2-PLGA NBs showed strong signal intensity,

peaking at approximately 25 dB.

5.7. Photoacoustic (PA) Imaging

PA imaging has recently emerged as an alternative to MRI and X-ray

tomography in the biomedical field, due to the ability to capture high-

resolution images at depth. However, a lack of selective probes limits its

application. To perform PA imaging using aptamers, a thrombin binding

aptamer (TBA) was hybridized with IRDye 800CW-labeled (FDNA) and

IRDye QC-1 quencher-labeled (QDNA) single-stranded DNA to form a

DNA duplex structure for imaging probes [79]. Upon binding targets, the

labelled aptamers triggered the release of the quencher strand and induced

relief of the quenching strand. Thus, it induced the PA signal to change at

780/725 nm. In other words, the binding of thrombin (TBA) loosens the

hybridization of the TBA–QDNA complexes and results in release of

FDNA, with increase of both fluorescence and PA signals. Without

thrombin, IRDye is placed next to quencher, inducing low fluorescence

and PA signals. In an imaging study in experimental mice, the DNA probes

were injected into each flank of BALB/c mice. Next, thrombin or PBS

were injected into each flank of the mice. The PA images recorded at 45

min post-injection in thrombin-injected mice showed a higher PA signal

ratio at 780/725 nm than PBS- or untreated mice.


5.8. Multimodal Imaging

Every molecular imaging modality has unique advantages and

disadvantages. Therefore, the current trend in imaging is to develop

multimodal imaging that will increase diagnostic accuracy. As a proof of

concept for a multimodal imaging system, multimodal nanoparticles have

been developed for optical, MR, and PET imaging. To act as a single

multimodal nanoparticle imaging agent, cobalt ferrite magnetic

nanoparticles surrounded by fluorescent rhodamine (MF) within a silica

shell matrix were conjugated to an anti-underglycosylated mucin-1

(uMUC-1) aptamer (MF-uMUC-1). Next, MF-uMUC-1 was labeled with

the Gallium isotype 68Ga (MFR-uMUC-1) with a p-SCN-bn-NOTA

chelating agent [80]. For in vivo aptamer-based multimodal imaging,

MFR-uMUC-1 nanoparticles or MFR-uMUC-1 mutant (uMUC-1 mt,

control) nanoparticles were injected into BT-20 (human breast cancer)-

implanted nude mice through the tail vein [80]. Three imaging

modalities—PET, fluorescence, and MRI—were used at multiple time-

points (2, 12, 24 h) after IV injection. PET imaging showed significant

accumulation of 68Ga radioactivity in the tumor region of the MFR-uMUC-

1-injected group, but not in the MFR-uMUC-1 mt-injected group.

Fluorescence imaging also showed significantly elevated tumor-specific

fluorescence and MRI showed significantly enhanced dark signals in

MFR-uMUC-1-injected mice compared to controls [80]. These results

demonstrate a high specificity for targeting cancers using the uMUC-1

aptamer and suggest the aptamer-guided conjugate is feasible as a single

multimodal probe in vivo.

Taken together, targeted molecular in vivo imaging using aptamers, as

depicted in Figure 5, allows cancer-specific recognition in multiple

imaging modalities. Each modality has various advantages and

disadvantages. Fluorescence imaging is very cost effective and shows high

sensitivity, but high autofluorescence and poor tissue penetration prevent

the translation of fluorescence imaging into clinics. Nuclear imaging such

as SPECT and PET provide improved resolution of imaging and

longitudinal assessment of deep tissues in the same patient with high


sensitivity, but the expensive cost and handling of isotypes are

disadvantages. MRI and CT are excellent techniques to examine

anatomical body structures, so enhancement of contrast with imaging

agents is a key factor. Currently, aptamer-guided molecular imaging

remains in the preclinical stage, but we expect that aptamer-guided

molecular imaging can be used in clinics in the near future.

Figure 5. Targeted in vivo imaging using aptamers. Mice possessing xenografted

cancer cells are injected with aptamers labeled with imaging dyes. Non-invasive

imaging modalities such as fluorescence, magnetic resonance (MR), single-photon-

emission computed tomography (SPECT), position-emission tomography (PET), or

computed tomography (CT) are used for imaging from a lateral or sagittal view. The

image is adopted from “Targeted molecular imaging using aptamers in cancer,”

Pharmaceuticals 2018; 11(3):71; doi:10.3390/ph11030071.

6. CHEMICAL MODIFICATION

To facilitate the translation of aptamer-guided molecular imaging,

chemical modifications should be considered to increase resistance to

cellular nucleases and to improve structural stability and binding affinity

[81]. The most common strategies employed are depicted in Figure 6. This

includes: phosphodiester backbone modifications such as

phosphorothioate, methylphosphonate, and triazole linkages; base

modifications such as 5-(N-benzycarboxyamide)-2´-deoxyuridine (5-

BndU), 5-naphthylmethylaminocarbonyl-dU (NapdU), 5-tryptamino-

carbonyl-dU (TrpdU), and 5-isobutylaminocarbonyl-dU (iBudU) in


SOMAmers; sugar ring modifications such as 2´-fluoro (2´-F), 2´-amino

(2´-NH2), 2´-O-methyl (2´-OMe), locked nucleic acid (LNA), unlocked

nucleic acid (UNA), 2´-deoxy-2´-fluoro-D-arabinonucleic acid (2´-FANA);

and the mirror image of L-deoxynucleotides in Spiegelmers® [81]. Such

chemical modifications are mainly developed for therapeutic purposes to

avoid immune responses and to increase serum resistance and potency.

Figure 6. Strategies for chemical modification of nucleic acid aptamers to increase

structural stability and nuclease resistance. The most commonly used strategies in

DNA/RNA aptamers are modifications of the phosphodiester linkage, the sugar ring,

and the bases. The image is adopted from “Targeted molecular imaging using aptamers

in cancer,” Pharmaceuticals 2018; 11(3):71; doi:10.3390/ph11030071.

To date, DNA aptamers and 2´-F RNA aptamers have been proven as

targeted imaging probes in tissues and in vivo imaging. We believe that

introducing these chemical modifications improves the function of

aptamers as targeted imaging probes, because improvement of nuclease

resistance and structural stability will increase the retention time of

aptamers in the body and target specificity. Indeed, the efficacy of anti-

epithelial cell adhesion molecule (EpCAM) and nucleolin LNA-modified

aptamers conjugated with Fe3O4 nanoparticles was tested for multimodal

imaging using NIR, MRI, and CT imaging in vivo [82]. After feeding mice

the formula for 48 h, imaging was performed using the three modalities.

Compared with untreated control, comparatively higher fluorescence using

modified aptamers was shown using NIR imaging. Comparatively higher


contrast in tumor regions was also observed using NIR, CT, and MRI. This

suggests that chemical modifications of aptamers are well tolerated as

imaging probes. However, this benefit must be balanced with increased

manufacturing costs.

CONCLUSION

Currently, targeted molecular imaging using aptamers is an actively

growing research field. Aptamers have proven target-specific recognition

for use in in vitro imaging for research, tissue imaging, and cellular

imaging, as well as for in vivo preclinical imaging in multiple cancer

models. To date, DNA aptamers have mainly been developed as molecular

probes, whereas RNA aptamers are less common. One explanation for this

may be that although RNA aptamers show high affinity and specificity due

to structural flexibility, they are limited due to a lack of nuclease

resistance, a possibility of misfolded structures, and a high dependency on

the concentration of divalent cations. Use of modified nucleic acids may

overcome the drawbacks of RNA aptamers, but the feasibility of this

approach is not clear yet.

In vivo imaging using either DNA aptamers (e.g., anti-nucleolin, anti-

MUC-1, anti-EGFRvIII, anti-cancer cell-type-specific aptamers) or RNA

aptamers (e.g., anti-PSMA aptamer) show good performance for cancer-

specific detection in imaging techniques including fluorescence, PET,

SPECT, MRI, CT, and US. This suggests that aptamers have great

potential as targeted imaging agents to differentiate disease-specific

targets, which will be applicable in the clinic. Because every imaging

technique has its own detecting mechanism and unique pros and cons, a

multimodal imaging platform (e.g., PET-MRI, SPECT-MRI, fluorescence-

PET, and US-PET) may be the preferred option for filling these gaps in

developing novel, clinically relevant approaches. Regardless of what type

of imaging techniques will be used, targeted molecular imaging using

aptamers shows quick accumulation into target sites, which gives suitable


target-to-background signals. Thus, we believe that aptamer-guided

imaging will improve diagnostic accuracy.

The next promising aptamer-guided imaging application to be

developed is tissue MSI imaging and mass cytometry. Multiple target-

specific aptamers conjugated with various metals will provide very

powerful tools to characterize multiple targets simultaneously in clinical

specimens.

This book chapter was adopted and modified with permission from an

article published in Pharmaceuticals (Basel, Switzerland). 2018; 11(3):71;

doi:10.3390/ph11030071, entitled “Targeted molecular imaging using

aptamers in cancer” (83). Images were also adopted under permission.

ACKNOWLEDGMENT

We thank Sarah T. Wilkinson, scientific writer, at City of Hope, for

language editing.

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BIOGRAPHICAL SKETCHES

Sorah Yoon, DVM, MS, PhD

Affiliation: Department of Molecular and Cellular biology, Beckman

Research Institute of City of Hope.

Education:

2016, Post-doc, Beckman Research Institute of City of Hope.

2010, Ph.D., College of Veterinary medicine at Seoul National

University.


1998, M.S., College of Veterinary medicine at Cheonnam National

University.

1996, D.V.M., College of Veterinary medicine at Cheonnam National

University.

Research and Professional Experience:

2017 – present, Staff scientist in Beckman Research Institute of City of

Hope, Duarte, CA, USA.

2011 – 2016, Postdoctoral Fellow in Beckman Research Institute of

City of Hope, Duarte, CA, USA.

2004 – 2006, Visiting scientist in Torrey Pines Institute for Molecular

Studies (TPIMS), La Jolla, CA, USA.

1998 – 2011, Junior veterinary researcher in National Veterinary

Research and Quarantine Service (NVRQS), South Korea.

Professional Appointments: Staff scientist

Honors:

2016 Travel Award, 12th Annual Meeting of the OTS.

2016 Post-doctoral Fellow Travel Grant award at City of hope.

2016 Meritorious Abstract Travel Award, The ASGCT 18th Annual

Meeting.

2015 First place oral presentation award in 35th RSO advance meeting

at City of Hope.

2004 Korean Government fellowship for overseas training.

Publications from the Last 3 Years:

Yoon S, Wu X, Armstrong B, Habib N, Rossi JJ. An RNA Aptamer

Targeting the Receptor Tyrosine Kinase PDGFRα Induces Anti-tumor

Effects through STAT3 and p53 in Glioblastoma. Mol Ther Nucleic

Acids. 2018 Dec 1;14:131-141.


Yoon S, Rossi JJ. Targeted Molecular Imaging Using Aptamers in Cancer.

Pharmaceuticals (Basel). 2018 Jul 19;11(3)(selected Cover story, Sep

issue).

Yoon S, Rossi JJ. Aptamers: Uptake mechanisms and intracellular

applications. Adv Drug Deliv Rev. 2018 Jul 6.

Yoon S, Rossi JJ. Therapeutic Potential of Small Activating RNAs

(saRNAs) in Human cancers. Curr Pharm Biotechnol. 2018 May 27.

Yoon S, Rossi JJ. Emerging cancer-specific therapeutic aptamers. Curr

Opin Oncol. 2017. Sep;29(5):366-374.

Yoon S, Armstrong B, Habib N, Rossi JJ. Blind SELEX Approach

Identifies RNA Aptamers That Regulate EMT and Inhibit Metastasis,

Mol Cancer Res. 2017 Apr 10 (selected Highlights, July 2017, 15(7))

Yoon S and Rossi J, Future strategies for the discovery of therapeutic

aptamers, Expert Opinion and Drug discovery, 2017. Expert Opin

Drug Discov. 2017 Apr;12(4):317-319.

Yoon S, Huang KW, Reebye V, Spalding D, Przytycka TM, Wang Y,

Swiderski P, Li L, Armstrong B, Reccia I, Zacharoulis D, Dimas K,

Kusano T, Shively J, Habib N and Rossi J, Aptamer drug conjugates

(ApDCs) of active metabolites of nucleoside analogues and cytotoxic

agents inhibit pancreatic tumor cell growth. Molecular Therapy -

Nucleic Acids. (2017) 60:80- 88.

Yoon S, Huang KW, Reebye V, Mintz P, Tien YW, Lai SH, Sætrom P,

Reccia I, Swiderski P, Armstrong B, Jozwiak A, Spalding D, Jiao L,

Habib N, and Rossi J. Targeted Delivery of C/EBPα -saRNA by

Pancreatic Ductal Adenocarcinoma (PDAC)-specific RNA aptamers

Inhibits Tumor Growth in vivo. Molecular therapy. (2016) 24:1106-

1116.

John J. Rossi, PhD

Affiliation: Lidow Family Research Endowed Chair, Professor,

Department, Molecular and Cellular Biology, Beckman Research Institute

of City of Hope


Education:

12/1976 Ph.D., University of Connecticut, CT, Ph.D.

12/1979 Postdoc, Brown University, RI, Postdoc.

Research and Professional Experience: I am the leader of an established

research group and my research primary focuses on the functions and

therapeutic application of small RNAs, such as ribozyme, siRNA,

microRNA and aptamer, etc. Currently I am Chairman and Professor in

Department of Molecular and Cellular Biology, and Lidow Family

Research Endowed Chair and Morgan and Helen Chu Dean’s Chair of

Beckman Research Inst. of City of Hope. For the past 25 years I have been

exploring the mechanisms of action and therapeutic applications of small

RNAs for the treatment of HIV-1. Recent efforts have focused on

approaches for systemic applications of small interfering RNAs and

nucleic acid aptamers. I am particularly interested in combining multiple

RNA-based therapeutics into a combinatorial therapeutic regimen against

HIV. Indeed, my lab developed the first combinatorial RNA-based gene

therapy strategy to enter human clinical testing. My lab has developed a

broad variety of anti-HIV small RNA therapeutics, including anti-HIV

ribozymes, Dicer substrate siRNAs, and HIV-specific RNA aptamers, and

established NSG humanized mouse model for the research projects based

on HIV-1 latency and HIV-1 reservoir reactivation.

Professional Appointments:

1980-1984 Assist. Res. Scientist, Dept. of Mol. Genetics, Beckman

Res. Institute of City of Hope, Duarte, CA

1984-1989 Assoc. Res. Scientist, Dept. of Mol. Genetics, Beckman

Res. Institute of City of Hope, Duarte, CA

1988-1992 Assoc. Chairman, Div. of Biology, Beckman Res.

Institute of City of Hope, Duarte, CA

1990-1994 Res. Scientist, Dept. of Mol. Genetics, Beckman Res.

Institute of City of Hope, Duarte, CA


1990-1994 Director, Dept. of Mol. Genetics, Beckman Res. Institute

of City of Hope, Duarte, CA

1992-1994 Chairman, Div. of Biology, Beckman Res. Institute of

City of Hope, Duarte, CA

1994-1996 Assoc. Director, Ctr. for Molecular Biology and Gene

Therapy, Loma Linda University, Loma Linda, CA

1996-1996 Director, Ctr. for Mol. Biology and Gene Therapy,

Loma Linda University, Loma Linda, CA

1996-2001 Director, Dept. of Mol. Biology, Section Head, Gene

Regulation, Beckman Res. Institute of City of Hope,

Duarte, CA

1994- Adjunct Prof., Div. of Biology and Dept. of

Hematology, BMT, City of Hope, Duarte, CA

1996-2017 Adjunct Professor, Department of Basic Sciences, Loma

Linda University, Loma Linda, CA

1998- Dean, Irell & Manella Graduate School of Biological

Sciences, Beckman Research Institute of the City of

Hope, Duarte, CA

2001-2011 Associate Director for Basic Research, City of Hope

Comprehensive Cancer Center, City of Hope National

Medical Center, Duarte CA

2001-2011 Associate Chair, Shared Resources Advisory

Committee, City of Hope Comprehensive Cancer

Center, City of Hope National Medical Center, Duarte

CA

2001-2011 Co-Program Director, Cancer Biology Program, City of

Hope Comprehensive Cancer Center, City of Hope

National Medical Center, Duarte CA., USA

2001-2010 Adjunct Prof., Div. of Biomedical Sciences, Univ. of

California, Riverside, Riverside, CA

1996- Chair and Professor, Department of Molecular and

Cellular Biology, Beckman Res. Institute of City of

Hope, Duarte, CA


2008- Lidow Family Research Endowed Chair, Beckman

Research Inst. of City of Hope, Duarte, CA

2010- Morgan and Helen Chu Dean’s Chair, Beckman

Research Inst. of City of Hope, Duarte, CA

2014- Associate Director, Education & Training, City of Hope

Comprehensive Cancer Center, Duarte, CA

Honors:

1990 Walter MacPherson Society Research Award, Loma Linda

Univ. School of Medicine

1991 American Association for Clinical Chemistry Outstanding

Speaker Award

1993 Gallery of Scientific and Medical Achievement, City of Hope

National Medical Center

2002 Merit Award, Division of AIDS, National Institute of Allergy

and Infectious Diseases

2008 Cozzarelli Award, National Academy of Sciences

2011 Elected as AAAS Fellow in Medical Sciences

2013 Special Recognition Award, American Society of Gene and

Cell Therapy

Publications from the Last 3 Years:

Arizala, J., Ouellet, D., Burnett, J. and Rossi, J.J. (2018) Nucleolar

localization of HIV-1 Rev is required yet insufficient for production of

infectious viral particles. AIDS Res Hum Retroviruses., 34(1): 961-981

PMCID: PMC6238656.

Arizala, J.A.C., Chomchan, P., Li, H., Moore, R, Ge, H., Ouellet, D.L. and

Rossi, J.J. (2018) Identification of nucleolar factors during HIV-1

replication through Rev immunoprecipitation and mass spectrometry.

JOVE J. (In Press).

Castanotto, D., Zhang, X., Alluin, J, Zhang, X., Rüger, J., Armstrong, B.,

Rossi, J.J., Riggs, A, Stein, C.A. (2018) A stress-induced response


complex (SIRC) shuttles miRNAs, siRNAs and oligonucleotides to the

nucleus. Proc. Natl. Acad. Sci.,115(25): E5756–E5765 PMCID:

PMC6016802.

Chen, C., Posocco, Liu, X., Cheng, Q., Laurini, E., Zhou, J., Liu, C.,

Wang, Y., Tang, J., Dal Col, V., Yu, T., Giorgio, S., Fermeglia, M.,

Qu, F., Liang, Z., Rossi, J.J., Liu, M., Rocchi, P., Pricl, S. and Peng, L.

(2016) Mastering dendrimer self-assembly for efficient siRNA

delivery: from conceptual design to in vivo efficient gene silencing.

Small J. 12(27): 3667-76. PMCID: PMC5724382.

Cui, Q., Yang, S., Ye, P., Tian, E., Sun, G., Zhou, J., Sun, G., Liu, X.,

Chen, C., Murasi, K., Zhao, C., Azizian, K., Yang, L., Warden, C.,

We, X., D’Apuzzo, M., Brown, C., Badie, B., Peng, L., Riggs, A.,

Rossi, J.J., and Shi, Y. (2016) Downregulation of TLX induces TET3

expression and inhibits glioblastoma stem cell self-renewal and

tumorigenesis. Nature Comm. 7, 10637 PMCID: PMC4742843.

Dao, P., Hoinka, J., Takahashi, M., Zhou, J., Ho, M., Wang, Y., Costa, F.,

Rossi, J.J., Backofen, R., Burnett, J. and Przytycka, T.M. (2016)

AptaTRACE elucidates RNA sequence-structure motifs from selection

trends in HT-SELEX experiments. Cell Syst. 3(1): 62-70. PMCID:

PMC5042215.

Hori, S., Herrera, A., Rossi, J.J. and Zhou, J. (2017) Review - Current

advances in aptamers of cancer diagnosis and therapy. Cancers J. 10:

1-33 DOI:10.3390/cancers10020009 (review article; not peer-

reviewed).

Lightfoot H.L., Setten, R.L., Habib, N.A. and Rossi, J.J. (2018) Review -

Development of MTLCEPBA: Sa RNA drug for liver disease. Curr

Pharm Biotechnol., 19(8): 611-621, PMCID: PMC6204661.

Pang, K.M., Castanotto, D., Li, H., Scherer, S. and Rossi, J.J. (2017)

Incorporation of aptamers in the terminal loop of shRNAs yields an

effective and novel combinatorial targeting strategy. Nucl. Acid Res.

Oct. 25, 17 gkx980, https://DOI.org/10.1093/nar/gkx980 PMCID:

PMC5758892.

Reebye, V., Huang, K.-W., Lin, V., Jarvis, S., Cutilas, P., Dorman, S.,

Ciriello, S., Andrikakou, P., Voutila, J., Saetrom, P., Mintz, P.J.,

https://doi.org/10.1093/nar/gkx980


Reccia, I., Rossi, J.J., Huber, H., Habib, R., Kostomitsopoulos, N.,

Balkey, D.C., Habib, N.A. (2018) Gene activation of CEBPA using

saRNA: preclinical studies of the first in human saRNA drub candidate

for liver cancer. Oncogene, 37(24): 3216-3228, PMCID:

PMC6013054.

Satheesan, S., Li, H., Burnett, J.C., Takahashi, M., Li, S., Wu, S.X.,

Synold, T.W., Rossi, J.J. and Zhou, J.H. (2018) HIV replication and

latency in a humanized NSG mouse model during suppressive oral

combinational ART. J. Virology, 92(7): 2118-2117. PMCID

PMC5972864.

Setten, R.L, Rossi, J.J. and Han, S.P. (2019) Review – The current state and

future directions of RNAi-based therapeutics. Nat Rev.- Drug Discov.,

Mar 7. doi: 10.1038/s41573-019-0017-4. [Epub ahead of print], PMID:

30846871 PMCID: PMC Journal - In Process.

Song, M-N. and Rossi, J.J. (2017) Molecular mechanisms of Dicer:

endonuclease and enzymatic activity. Biochem. J. 424: 1603-1618

DOI: 10.1042/BCJ20160759 PMCID: PMC5415849.

Takahashi, M., Wu, X., Ho, M. Chomchan, P., Rossi, J.J. Burnett, J.A. and

Zhou, J. (2016) High throughput sequencing analysis of RNA libraries

reveals the influences of initial library and PCR methods on SELEX

efficiency. Nature-Scientific Reports DOI:10.1038/srep33697 PMCID:

PMC5031971.

Xia, X. and Rossi, J.J. (2018) Review - RAFTing towards the shore of

nanotherapeutic. Curr Drug Deliv., Feb 19. DOI: 10.2174/

1567201815666180220100733. [Epub ahead of print] PMID:

29468969.

Xia, X., Li, H., Satheesan, S., Zhou, J. and Rossi, J.J. (2019) Humanized

NOD/SCID/IL2rγnull (hu-NSG) mouse model for HIV replication and

latency studies. J Vis Exp., 2019 Jan 7;(143). doi: 10.3791/58255.

PMID: 30663638 PMCID: PMC Journal - In Process.

Yoon, S. and Rossi, J.J. (2018) Therapeutic potential of small activating

RNAs (saRNAs) in human diseases. Curr Pharm Biotechnol., 19(8):

604-610. PMCID: PMC6204660.


Yoon, S., and Rossi, J.J. (2018) Review – Targeted molecular imaging

using aptamers in cancer. Pharmaceuticals, 11 (3): 71, (review article;

not peer-reviewed) PMCID: PMC6160950.

Yoon, S., Armstrong, B., Habib, N., and Rossi, J.J. (2017) Blind SELEX

approach identifies RNA aptamer that regulate EMT and Inhibit

metastasis. Mol. Cancer Res. DOI 10.1158/1541- 7786.MCR-16-0462

Published April 2017 PMCID: PMC5555160.

Yoon, S., Huang, K.W., Reebye, V., Mintz, Tien, Y.U., Lai, H.S., Sætrom,

P., Reccia, I., Swiderski, P., Armstrong, B., Jozwiak, A., Spalding, D.,

Jiao, L., Habib, N. and Rossi J.J. (2016) Targeted Delivery of C/EBP -

saRNA by Pancreatic Ductal Adenocarcinoma (PDAC)-specific RNA

aptamers Inhibits Tumor Growth in vivo. Mol Ther. 24(6): 1101- 1116.

PMCID: PMC4923325.

Yoon, S., Wu, X., Armstrong, B., Habib, N. and Rossi, J.J. (2019) An

RNA aptamer targeting the receptor tyrosine kinase PDGFRα induces

anti-tumor effects through STAT3 and p53 in glioblastoma. Mol. Ther-

Nucleic Acids, 1(14):131-14, PMCID: PMC6307106.

Zhou, J. and Rossi, J.J. (2017) Aptamers as targeted therapeutics: current

potential and challenges. Nat Rev Drug Discov. Jun;16(6):440. DOI:

10.1038/nrd.2017.86. Epub 2017 Apr 28. PMCID: PMC5700751.

Zhou, J., Li, H., Xia, X., Herrera, A., Pollock, N., Reebye, V., Sodergren,

M., Dorman, S., Littman, B., Doogan, D. Huang, K-W, Habib, R.,

Habib, H., and Rossi, J.J. (2019) Anti-inflammatory activity of MTL-

CEBPA, a small activating RNA drug, in LPS-stimulated monocytes

and humanized mice. Mol. Ther., 2019 Feb 26. pii: S1525-

0016(19)30059-0. doi: 10.1016/j.ymthe.2019.02.018. [Epub ahead of

print, PMID: 30852139 PMCID: PMC Journal - In Process.

Zhou, J.H., Lazar, D., Li, H., Xia, X., Satheesan, S., Charlins, P., O’Mealy,

D., Akkina, R., Saayman, S., Weinberg, M.S., Rossi, J.J. and Morris,

K.V. (2018) Receptor-targeted aptamersiRNA conjugate-directed

transcriptional regulation of HIV-1. Theranostics, 8(6): 1575-1590.

PMCID: PMC5858168.

sorah yoon , dvm, phd and john j. rossi , phd...new biomarkers in clinical tissue specimens. rna...

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