Rbfox RNA-binding proteins play important roles in the regulation of alternative pre-mRNA splicing, but their role in other gene regulatory mechanisms is not well understood. Here, we show that Rbfox2 is a novel constituent of cytoplasmic stress granules, the translational silencing machinery assembled in response to cellular stress. We also show that the RNA binding activity of the Rbfox family protein is crucial for its localization into stress granules. To investigate the role of Rbfox2 in stress granules we used RNA-immunoprecipitation sequencing to identify cytoplasmic transcriptome-wide targets of Rbfox2. We report that a subset of cell cycle-related genes including retinoblastoma 1 is the target of Rbfox2 in cytoplasmic stress granules, and Rbfox2 regulates the retinoblastoma 1 mRNA and protein expression levels during and following stress exposure. Our study proposes a novel function for Rbfox2 in cytoplasmic stress granules. RNA-binding proteins (RBPs) control various steps of nuclear and cytoplasmic RNA processing at the post-transcriptional level2. RBP-targeting to a RNA transcript affects RNA fate through various mechanisms that enable specific cellular functions to be established and provides a mechanism for post-transcriptional gene regulation during many biological processes. As RBPs have been implicated at various RNA processing steps and RNA metabolic events, it is therefore important to consider the cellular context in order to understand how RBPs exert effects on target RNA.Cells show significant gene-regulatory flexibility that allows them to adjust to external conditions. The ability to control gene expression plays an important role in the adaptation of cells to changes in the environment. Regulation of gene expression at the post-transcriptional level, including the control of pre-mRNA splicing, RNA transport, RNA stability and translation provides a rapid and efficient process by which the proteome of a cell can adapt to environmental changes4. The Rbfox family in vertebrates consists of three genes: Rbfox1, Rbfox2, and Rbfox3. Rbfox1 is specifically expressed in heart, skeletal muscle and neuronal tissues, whereas Rbfox2 is widely expressed in many tissues throughout life, from the early embryonic stage to the fully-grown adult stage6. Rbfox3 expression is only detectable in postmitotic neurons of neural tissues7. All Rbfox family proteins contain a single RNA-recognition motif (RRM) in the middle of the molecule that binds to the hexanucleotide (U)GCAUG with a high affinity in vitro, and binding of Rbfox proteins to this element has also been well demonstrated in intact cells and tissues14. Numerous reports involving depletion of Rbfox in cultured cells and animal models have shown that the regulation of tissue-specific-splicing by Rbfox is critical to a number of biological processes19. Recent reports show that the role of Rbfox is not limited to regulating alternative splicing in the nucleus but also reveal other nuclear and cytoplasmic functions for Rbfox proteins23. The enrichment of Rbfox binding sites in the 3′ UTR of many mRNAs was confirmed at a genome-wide level, and it has been shown that its targeting to the 3′ UTR stabilizes the target mRNA24. However, the precise function of Rbfox in the cytoplasm is largely unknown. Moreover recent studies have revealed other RNA binding sequences different from the consensus (U)GCAUG sequence in vitro and in intact cells22.The RNA-binding Rbfox family is highly conserved with established roles in alternative pre-mRNA splicing regulation26. Stress granules (SGs) are active assemblies of messenger ribonucleoprotein particles (mRNPs), which are formed in the cytoplasm of cells under many different types of environmental stress27. SGs are non-membranous structures formed through stress-induced phosphorylation of the eukaryotic initiation factor (eIF2). Phosphorylation of eIF2 results in impaired translational initiation and a consequent decrease in protein synthesis, and at the same time mRNAs selectively incorporated into SGs show increased stability30. SG assembly requires a subset of mRNA-associated RBPs such as T-cell internal antigen 1 (TIA1) and Ras-GTPase-activating protein SH3-domain-binding protein 1 (G3BP1). Moreover, multiple post-translational modifications of various SG components affect SG assembly31. Recent studies show that several components of SGs exist in the cytoplasmic granules of neuronal cells in the brains of patients with various neurodegenerative diseases including Alzheimer′s disease, Creutzfeldt-Jakob disease, and Huntington’s chorea, suggesting that the proper formation of SGs is important for neurological function33. While the precise mechanisms by which SGs influence various biological processes including human diseases are largely unknown, it is clear that the post-transcriptional mechanisms mediated by SG-associated proteins and RNAs play a major role in enabling cells to adapt to external stimuli.The interaction between RBPs and RNA granules regulates the stability and translational efficiency of mRNAs and plays an important role in the regulation of protein expression in response to external stimuliIn this study, we investigate the cytoplasmic function of Rbfox2 under stress conditions. Unexpectedly, we found that Rbfox2 is a component of SGs and thus set out to identify the endogenous target of Rbfox2 in the SGs at the transcriptome scale, using RNA-immunoprecipitation sequencing (RIP-seq). This analysis revealed that cell cycle-related mRNAs were over-represented as cytoplasmic Rbfox2 targets under cell stress.34. Although nuclear localization of the splicing factor prolineand glutamine-rich (SFPQ) was not affected by arsenite-induced cellular stress, arsenite treatment induced the formation of cytoplasmic Rbfox2 granules in HeLa cells  and the C-terminal half (RRM-C) tagged with myc were expressed at their expected size, as shown in Fig. 22. Surprisingly, the F142A mutant protein completely lost the ability to localize into SGs  of Rbfox2, which seems to be diffusely distributed in unstressed cells, might also be recruited to SGs, since the N-RRM deletion mutant of Rbfox3, which distributes to the cytoplasm to a significant extent, is also recruited to SGs. Although we have not formally tested Rbfox2 deletion mutants, it is likely that the N- and C-terminal domains of Rbfox2 are not essential for targeting to SGs, based on the data from Rbfox3 deletion mutants. It is also unlikely that a unique amino acid sequence of Rbfox2 is required for SG localization, because all exogenously expressed Rbfox1, 2, and 3 show recruitment into SGs to a similar extent. However, we cannot completely rule out the possibility that the Rbfox3 deletion mutant or full-length Rbfox3 and 1 forms a dimer (or multimer) with endogenous intact Rbfox2 through the RRM domain and is recruited to SGs together with endogenous Rbfox2. Of note, RNA binding activity is essential for Rbfox recruitment to SGs, whereas neither the N-terminal nor C-terminal domain is essential. This suggests that translocation of nuclear Rbfox to cytoplasmic SGs might be navigated by its target RNAs. This observation is in contrast to the fact that the C-terminal and/or N-terminal domains, in addition to the RNA binding activity, are required for regulation of alternative splicing in many cases6. However, understanding the detailed mechanism by which Rbfox undergoes SG localization will require precise kinetic probing of Rbfox binding to target RNAs and determination of the molecular mechanism underlying posttranslational modification of Rbfox during cellular stress. To date cytoplasmic Rbfox1 has been reported to increase mRNA stability and translation by binding to the 3′-UTRs of its target RNAs in unstressed conditions, in some cases by competing with miRNAs20. Our direct evidence for SG localization of Rbfox2 during cellular stress provides a new gene regulatory mechanism for Rbfox.Each member of the Rbfox family of genes is known to generate a number of isoforms through splicing of multiple alternative exons and alternative promoterset al. reported widespread inhibition of pre-mRNA splicing during heat stress and nuclear retention of intron-containing transcripts37. Interestingly, they observed splicing-inhibited transcripts were mostly spliced posttranscriptionally in the nucleoplasm and large intron-containing transcripts are more susceptible to splicing inhibition. The physiological significance of intron-containing RNAs associated with cytoplasmic Rbfox2 will be determined in a future work.In this study we provide comprehensive analysis of cytoplasmic Rbfox2-associated RNAs that are generated under oxidative stress. The fact that more than half of genic reads are mapped to introns suggests a leak of incompletely processed pre-mRNAs from the nucleus and/or a splicing inhibition under the stress condition. It is also possible that the number of intron-mapped reads is apparently large because intron length is generally much longer than exon length. Shalgi 26. SGs are transient and reversible structures. Upon removal of stress or adaptation of cells in a new environment, SGs are disassembled and then the protected mRNAs become readily available for translation. RB1 was initially identified as a tumor suppressor gene that is dysfunctional in several major cancers38. Since abnormal cells, including cancer cells, are under various cellular stresses such as oxidative stress, nutrient stress, and osmotic stress, we speculate that regulation of the RB1-mediated cell cycle by Rbfox2 could play a role in disease development and cancer progression.Importantly, we found that the target RNAs of cytoplasmic Rbfox2 are related to the cell cycle progression. Among Rbfox2-target RNAs in SGs, we demonstrated the significance of RB1 mRNA association with Rbfox2 to regulate RB1 mRNA stability and RB1 protein expression under oxidative stress. The generally accepted roles of SGs are to provide a pause in translation of incorporated mRNAs and to protect them from degradation39. Although SG localization of HIV-induced-cytoplasmic Rbfox3 has not been confirmed, this is consistent with our observation that the subcellular localization of Rbfox2 is altered during cellular stress. Since the Rbfox family proteins are highly expressed in neuronal cells and all three members of this family are capable of responding to cellular stress, Rbfox proteins might play a role in development or prevention of neurodegenerative diseases which are often triggered by cellular stresses.Cells are constantly encountering numerous stresses such as an excess in hormone secretion, viral infections, oxidative stress, exposure to an abnormal pH, heat shock, hypoxia, and high salt concentrations. Recently, it was reported that cytoplasmic Rbfox3 levels were significantly higher in brain sections obtained from patients infected with human immunodeficiency virus type I (HIV) compared to those obtained from controlsThe human cervix adenocarcinoma cell line HeLa was maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum and antibiotics. Cells were transfected with plasmids or siRNA by electroporation . Transfected cells were generally analyzed 36 hr later. Sodium arsenite and sodium chloride were purchased from Sigma-Aldrich. Stress conditions were induced by treating cells with different stress inducers such as 500 μM sodium arsenite, 150 mM sodium chloride, or 43 °C heat shock for 40 min.36. The N-RRM and RRM-C deletion constructs of Rbfox3 have been described previously6. The generation of F108A and F142A mutant clones of Rbfox3 has been described previously22. The generation of the siRNA designed for silencing the Rbfox2 (siRbfox2) has been described previously as a Fox2 siRNA6.The preparation of the expression plasmids for Rbfox1, Rbfox2, and Rbfox3 in the pCS3 + MT vector, which contains an N-terminal myc-tag, has been described previously6 for RIP-seq-B. The RNAs recovered from the complexes were subjected to cDNA-library preparation using a TruSeq Standard Total RNA library Prep Kit (Illumina). An Illumina Hiseq. 2000 platform was used to generate 100-bp and 50-bp paired-end reads for RIP-sequencing of Rbfox2 and control RNA-sequencing of total RNAs from untreated HeLa cells, respectively. For RIP-PCR, the RNAs recovered from the complexes of whole-cell extracts were subjected to RT-PCR using the primers 5′-CCA TGG AGA AGG CTG GGG-3′ and 5′-CAA AGT TGT CAT GGA TGA CC-3′ for Gapdh mRNA and 5′-GTT GGT CCT TCT CGG TCC TT-3′ and 5′-CAA AGC AGA AGG CAA CTT GA-3′ for RB1 mRNA.A cytosolic fraction of sodium arsenite-treated HeLa cells was isolated for SG enrichment using a Nuclear Extract Kit (Active Motif). Immunoprecipitation of RNA-Rbfox2 complexes was performed with an RNA ChIP-IT kit (Active Motif) using two different Rbfox2 antibodies; one obtained from Bethyl Laboratory, A-300-864A, for RIP-seq-A and a second generated in this laboratory (LMC)40 under the default parameter setting. Only uniquely map-able reads with a maximum of two mismatches when compared with the reference were considered. The mapped results were fed to Cufflinks41 for quantification of known human genes. Differential expression analysis was performed with Cuffdiff42. Genes with a false discovery rate (FDR) of less than 5% were marked as significant. Unless otherwise stated, all gene expression levels in our analyses are represented as FPKM (fragments per kilobase of exon per million sequences fragments) values on log 2 scales. The RNA-sequencing statistics on all four samples are listed in Supplementary Table After the sequencing run, the resulting raw reads in FASTQ format were processed using in-house Perl scripts. This enabled clean reads to be obtained by removing those containing adapter sequences, poly-N sequences, or low quality bases (below a mean Phred score of 20). The trimmed paired-end reads were mapped to the UCSC human reference genome (hg19) using Tophat 2Cells were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.5% Triton X-100 in phosphate-buffered saline (PBS) for 15 min, blocked with 5% goat serum for 1 h and incubated overnight at 4 °C with primary antibodies recognizing G3BP1 , TIA-1 , SFPQ , GW182 , LC3A/B , myc , and Rbfox2 . Alexa-488- and Alexa-594-conjugated goat antibodies against mouse and rabbit IgG  were used as secondary antibodies. Nuclei were co-stained with 4,6-diamidino-2-phenylindole . Images were acquired with a Zeiss LSM 510 Meta confocal laser-scanning microscope.Whole cell lysates were prepared with M-PER buffer (Thermo Scientific) supplemented with a protease-inhibitor cocktail (Roche). Protein samples, denatured and reduced by sodium dodecyl sulfate (SDS) and β-mercaptoethanol respectively, were separated on SDS-polyacrylamide gels and transferred onto a nitrocellulose membrane. The SuperSignal system (Pierce) or Odyssey system (LI-COR) was used to detect the binding of secondary antibodies. Immunoblot bands were quantified using NIH ImageJ software. The primary antibodies recognized myc , RB1 , Rbfox2 , and GAPDH .https://david.ncifcrf.gov)43.We analyzed enrichment or depletion in Gene Ontology (GO) categories for the differentially expressed genes using the DAVID tool  Sequence Read Archive under accession number SRP069155.Supplementary InformationSupplementary Table S1Supplementary Table S2


Depending on the classification of the virus and the type of RNA in question, viral RNAs must alternately be replicated, spliced, transcribed, transported from the nucleus into the cytoplasm, translated and/or packaged into nascent virions, and in most cases, provide the sequence and structural determinants to facilitate these processes. One consequence of this compact multifunctionality is that viral RNA structures can be exquisitely complex, often involving intermolecular interactions with RNA or protein, intramolecular interactions between sequence segments separated by several thousands of nucleotides, or specialized motifs such as pseudoknots or kissing loops. The fluidity of viral RNA structure can also present a challenge when attempting to characterize it, as genomic RNAs especially are likely to sample numerous conformations at various stages of the virus life cycle. Here we review advances in chemoenzymatic structure probing that have made it possible to address such challenges with respect to  Effective therapeutic targeting of these elements, which include highly specialized structural motifs such as 5′-3′ interactions, pseudoknots, riboswitches and triple helices, would render the virus or viruses in question incapable of replication and completely non-pathogenic.As a consequence of constant improvements in screening technology  and variations in structures and binding partners associated with localization to different biological compartments. These are especially intriguing challenges for retroviral RNA genomes, which are synthesized in the nucleus, transported to the cytoplasm where they provide the template for translation, and are packaged into the budding virion.Structural characterization of such complex RNAs requires highly robust and reproducible biochemical techniques, the current gold standard of which is selective 2′-hydroxyl acylation analyzed by primer extension, or SHAPE , the bands/peaks are matched with corresponding ribonucleotides, and their intensities are measured. Band/peak intensity measurements are subsequently transformed into reactivity values that reflect the frequency with which the corresponding ribonucleotides have been acylated, and thus, the likelihood that they are single stranded in the context of the folded RNA. The collection of reactivity values is then inputted into RNAstructure software , a highly structured RNA motif embedded within the envelope gene in HIV transcripts  that likewise makes use of cellular export machinery  of murine leukemia virus (MLV) and xenotropic murine leukemia virus-related virus (XMRV), which share 98% homology and can functionally substitute for HIV-1 Rev/RRE  using ND-PAGE to quantify conformers present under differing experimental conditions and then applying this quantification to mathematically deconvolute reactivity values obtained under these conditions into conformer-specific profiles. The former approach, also referred to as in-gel SHAPE, was used to distinguish among alternative conformations assumed by monomeric and dimeric HIV 5′UTR constructs as well as the HIV-1 RRE. The latter method permitted characterization of the HIV-2 RRE as it assumed progressively more stable structural intermediates over time.Interpretation of SHAPE-derived structural data assumes that RNAs under investigation are structurally homogeneous; unfortunately, this is not always the case. Whenever possible, SHAPE experiments should be preceded by an evaluation of structural uniformity by non-denaturing polyacrylamide gel electrophoresis (ND-PAGE), wherein different conformers typically migrate at different rates. When multiple conformers are detected, site-directed mutagenesis can be used to try to force an RNA to assume a single structure; however, this approach frequently produces unexpected conformational changes that can further confound interpretation of SHAPE data. Alternative means of resolving the different structures assumed by an RNA have been developed, including (i) fractionating the conformers by ND-PAGE, excising the respective gel slices and then probing them separately in vitro transcription for structural analysis using CE-SHAPE  strand RNA genome is central to HIV packaging  and p = fractional conformer contribution as determined by ND-PAGE , the % contribution of each conformer at 5 min , and the reactivity values of B and C (RB and RC) were substituted into this equation to calculate the reactivity of A at each nucleotide position (RA5). Values obtained at 10 min , together with the calculated values of RB and RC, were substituted into an equivalent equation to obtain a second set of reactivity values for A (RA10). Finally, RA5 and RA10 NMIA reactivity values were averaged to obtain RA values for determining the Conformer A structure. Combining extrapolated Conformer A, B, and C NMIA reactivity profiles with the folding algorithm RNAComposer , West Nile virus (WNV) and dengue virus (DENV)  experiments—an advantage that significantly simplifies detection and characterization of long-range, in vitro transcription   RNA template.The Dumbbells region is comprised of tandem dumbbell-shaped motifs and adjacent sequences  complementary to the respective apical loops. Mutational analysis of these motifs indicates that they are essential for both RNA replication and optimal translation  played by the mutated nucleotides in maintaining the structure of the motif in question. AiSHAPE was developed as a complement to the site-directed mutagenesis, where the sequence of the RNA of interest is not altered  RNA transport element  DNA synthesis. In a separate study, this element was mutationally verified and demonstrated essential for transposition  and ex vivo, where proteins have been removed by organic solvent extraction. SHAPE-MaP reactivity values obtained from the two RNA samples are subsequently compared, with reduced values in the presence of protein considered as regions of protection attributable to protein binding, and the inverse result indicating a local, protein-induced increase in RNA flexibility. Collectively, these techniques were used to characterize the overall secondary structure and structural homogeneity of KSHV polyadenylated nuclear (PAN) RNA, as well as to map the binding sites of select viral proteins (Sztuba-Solinska et al., In separate work, SHAPE-MaP has been used to identify protein binding sites on RNase MRP, SRP RNA, 5S rRNA (Smola et al., via interactions with transcriptional regulators, chromatin modifiers and other host and viral proteins (Rossetto and Pari, The double-stranded DNA genome of KSHV is ~165 kB in length, with an associated transcriptome comprised of ~90 protein-coding genes and numerous non-coding RNAs, including PAN. PAN RNA is ~1.1 kB in length, and although primarily localized to the nucleus (Sun et al., in vitro transcribed RNA, including at common sites at or near the MRE, within a large internal loop of Domain I and at the helical base of Domain III. Overlap among protein binding sites suggests that these motifs may individually or collectively seed formation of multi-protein RNA complexes in the context of the KSHV life cycle.One of the principle achievements of the PAN RNA structural study was using SHAPE-MaP to characterize PAN isolated from infected cell nuclei, cytoplasm, and KSHV virions Figure . Each ofSHAPE-MaP permits high-throughput secondary structure mapping of lengthy, sparse RNAs in their native contexts. In conjunction, ShapeKnots and Shannon entropy calculations allow researchers to identify otherwise hidden pseudoknots as well as structural motifs likely to be conserved during RNA conformational sampling. Acylating reagents used for SHAPE may also be used to map protein binding sites in the context of these experiments. For more routine analyses of short- and intermediate-length RNAs, and to avoid the costs associated with next generation sequencing, CE-SHAPE offers a practical alternative for identifying and characterizing RNA structural motifs that can be independently verified using the aiSHAPE antisense technology. Moreover, in-gel SHAPE and mathematical deconvolution of mixed reactivity profiles provide utility in examining heterogeneous and transitional RNA structures. Each of these approaches will continue to have value in the face of increasing demand for structural characterization of viral RNAs.JR, JS-S, and SL wrote and edited the manuscript and generated the figures.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer CR-L and handling Editor declared their shared affiliation.


We report the design of a luminescent sensor based upon the zinc finger protein TIS11d, that allows for the selective time-resolved detection of the UUAUUUAUU sequence of the 3′-untranslated region of messenger RNA. 3+ complex on one of the ZFs and a sensitizing antenna on the other. This work provides the proof of principle that an RNA binding protein can be re-engineered as an RNA sensor and, more generally, that tunable synthetic luminescent probes for biomolecules can be obtained by modifying biomolecule-binding domains.We report the design of a luminescent sensor based upon the zinc finger (ZF) protein TIS11d, that allows for the selective time-resolved detection of the UUAUUUAUU sequence of the 3′-untranslated region of messenger RNA. This sensor is composed of the tandem ZF RNA binding domain of TIS11d functionalized with a luminescent Tb Even though synthetic chemists have developed a large number of low-molecular-weight fluorescent probes that can detect various analytes  or that can respond to their microenvironment ,8 selective sensing of larger biomolecules such as proteins, DNA or RNA is more challenging with small molecular probes.16 Compared to low-molecular-weight analytes, the selective recognition of large biomolecules requires a high number of interactions between the probe and its target, which are difficult to implement and control with small synthetic molecular probes.Fluorescence spectroscopy is routinely utilized in laboratories to study biological systems because it is a sensitive, cheap and easy-to-use technique.‡The complementary strand may be a DNA oligonucleotide but alternate oligonucleotides such as locked nucleic acid (LNA) or peptide nucleic acid (PNA) can be used to create the probe strand. that incorporates a suitable fluorophore (and a quencher in the case of molecular beacons).18 The binding domains of proteins are also attractive for the design of probes for various analytes, including biomolecules. Here, the best examples are the genetically encoded probes that have been developed for various small bio-analytes,24 , Cl–, H2O2, cAMP) or larger biomolecules like DNA25 or RNA26 oligonucleotides. These probes were created by linking fluorescent proteins with a protein that binds the analyte of interest. One advantage of this approach is that the binding domains of proteins intrinsically feature the required selectivity and affinity as well as association and dissociation kinetics in a suitable range for reversible and dynamic exchange of the analyte in biological media. This makes them ideal recognition units to design probes for biological applications. However, there are several drawbacks to the use of genetically encoded sensors: they can only be used in organisms that can be genetically modified and control of the levels of the probe's expression, localization and optical properties is not straightforward. Alternative strategies, such as the development of synthetic luminescent probes of large biomolecules – and more generally bio-analytes – that can be tuned and utilized for applications in vitro or in vivo (in any organism) are needed. To meet this need, one approach would be to prepare synthetic probes that replicate the biomolecule-binding domains of proteins. However, these domains are typically large (>50 amino acids) and the entire domain must be synthesized in order for it to adopt the stable three-dimensional structure required for an optimal interaction with the target biomolecule. This approach is therefore synthetically challenging. A few examples of synthetic luminescent probes of biomolecules have been described based upon short peptide segments (<25–30 amino acids) derived from protein- or DNA- or RNA-binding proteins.30 These peptide-based probes are almost unfolded in the absence of the analyte and fold upon analyte binding. The energy cost associated with this folding event lowers the affinity of the probe for the analyte compared to the well-folded parent protein. Therefore, such probes are less sensitive. Recent progress in chemical protein synthesis33 allowed us to envision the design of synthetic luminescent probes based on biomolecule-binding domains of proteins comprising up to more than 100 amino acids and incorporating chromophores/fluorophores with optimized and tunable optical properties. Such probes should have superior sensitivity to short peptide based probes.An attractive strategy for the design of luminescent probes of biomolecules is to exploit the native recognition properties of certain biomolecules for other biomolecules. For instance, DNA/RNA or DNA/DNA recognition has been used successfully to design selective fluorescent probes for specific RNA or DNA sequences. The approach relies on the hybridization of the target RNA or DNA strand with a complementary DNA strandi.e. the change in light emission is due only to the binding of the receptor to its target. Trivalent lanthanide ions (Ln3+) are appealing emitting moieties for biological sensing applications because of their desirable luminescence properties.37 These properties include narrow emission bands that are not influenced by the environment, emission spectra covering the visible to NIR domain depending on Ln3+ and long luminescence lifetimes that allow for time-resolved detection to suppress background fluorescence contributions. Additionally, sensitization of Ln3+ luminescence requires the presence of a proximal chromophore, called an antenna. Once excited, the antenna transfers its energy to generate the excited state of the Ln3+ ion. Modulation of the lanthanide emission can be achieved by varying the antenna/Ln3+ distance, which alters the energy transfer.34 This approach has the potential to be generalizable as it only requires that the probe adopts different conformations in the bound and unbound states, affecting the antenna/Ln3+ distance.34The main challenge in designing a luminescent sensor is to couple the binding event to the emission event, e.g. TNFα) and also functions as a tumor suppressor.40 The probe was obtained by chemical synthesis, is selective for the target mRNA sequence and exhibits a similar binding affinity compared to that of the native TIS11d RBD with the same RNA target. These findings provide the proof-of-principle that fully synthetic biomolecule-based luminescent probes that are selective for a specific target can be prepared.In this article, we demonstrate that a biomolecule-binding domain of a protein can be re-engineered into a lanthanide-based turn-on luminescent probe that retains the intrinsic binding properties of the native protein. We describe the rational design, the synthesis and the characterization of a luminescent probe that detects the 9-nucleotide long UUAUUUAUU RNA sequence located in the adenylate-uracylate-rich elements (AU-rich elements) of the 3′-untranslated region of messenger RNA (mRNA). The probe utilizes lanthanide luminescence and it was designed based upon the tandem zinc finger RNA binding domain (RBD) of TIS11d , a protein of the tristetraprolin  family that plays critical roles in mRNA regulation by simultaneously regulating multiple cytokines associated with inflammation  between the antenna and the lanthanide was expected in the presence of the target UUAUUUAUU RNA sequence, resulting in a stronger Tb3+ emission (3+ acceptor pair rather than an organic donor/organic acceptor pair was motivated by both the luminescence properties of lanthanides and by the requirement of a short distance between the antenna and the lanthanide (below ca. 10–15 Å) for efficient sensitization of Ln3+ luminescence. Purely organic systems require distances up to 40–60 Å in order for efficient energy transfer to occur. This distance exceeds the size of the TIS11d RBD .Among the proteins of the TTP family, TTP/ZFP36, TIS11b/ZFP36L1, and TIS11d/ZFP36L2 are highly homologous and all bind to same AU-rich mRNA targets. We chose TIS11d as the basis for our designed luminescent probe because it is the only homolog for which there is an NMR structure of the two CCCH domains bound to RNA. This allowed us to identify amino acids that we could mutate without affecting the RNA binding activity. TIS11d is a e linker .42,43 RNS11d RBD  adopts aemission . The useTb, we used a 68 amino acid peptide with the sequence corresponding to residues 151 to 218 of human TIS11d, which is the RBD.§In order maximize the conformational changes arising upon RNA binding, we decided to exclude Asp219 and Glu220 that were proposed to limit the unfolding of the linker region in the absence of RNA see . For the antenna, we utilized Carbostyril 124 (Cs124) because it is excited in the 325–350 nm which is a wavelength range where RNA or DNA nucleobases do not absorb. Cs124 is an efficient sensitizer of Tb3+ luminescence45 that can be used in the vicinity of an oligonucleotide,46 without quenching the sensitizing process by nucleobases.47 Due to the short antenna/Ln3+ distance required for efficient energy transfer, we searched for ideal positions to introduce the Ln3+ complex and the antenna on the TIS11d RBD sequence. The amino acid residues had to be (i) in close proximity when TIS11d RBD binds RNA, (ii) solvent exposed, (iii) not conserved among TIS11d homologs so as not to be functionally important and (iv) not involved in hydrogen bonding or hydrophobic interactions with the RNA strand or with other amino acids of the protein. Inspection of the solution structure of TIS11d42 revealed that Arg160 and Pro210 fulfilled all of these criteria, with a ca. 5.5 Å gap between their side chains.209, just neighbouring Pro210, seemed an interesting candidate but it is conserved in many tristetraprolin homologs.¶Gly Therefore, positions 160 and 210 in the sequence of TIS11d were chosen to incorporate the Tb3+ complex and the Cs124 antenna, respectively 48 and SEA ligation (SEA = bis(sulfanylethyl)amido),49 taking advantage of the numerous zinc-binding cysteines present in TIS11d RBD  ester on the lysine side chain after removal of its alloc protecting group. After cleavage from the resin, the unprotected peptide displaying a SEA group at its C-terminus  at its C-terminus to prevent oligomerization or cyclization during NCL assembly of segments 1 and 2.50 Finally, the C-terminal segment 3 displays the Cs124 chromophore on the side chain of a glutamate. This chromophore was introduced using Fmoc–Glu(Cs124)–OH51 during solid phase peptide synthesis of the segment. The one-pot assembly of the three segments was performed as described by Melnyk et al.501 and 2 were assembled in a phosphate buffer (pH 7.2) containing mercaptophenylacetic acid (MPAA) to unmask the C-terminal cysteine of 2 and catalyze NCL. After completion of this first ligation step (24 h), 3 was added to the reaction mixture along with the reducing agent tris(carboxyethyl)phosphine (TCEP) to convert the SEAoff group at the C-end of the (1 + 2) segment into an active SEAon form and initiate the SEA ligation step.50 The overall process was completed within 48 h and apo-LTIS was obtained in 48% yield after purification. Inspired by recent achievements in the field of protein chemical synthesis for accessing small functional protein domains,S11d RBD . The N-tTb  with the RNA 11-mer UUUAUUUAUUU (11AU), which comprises the target sequence of proteins of the TTP family. In the absence of RNA, excitation at 330 nm promotes emission from the 5D4 excited state of Tb3+ with characteristic 5D4 → 7FJ  transitions at 490, 545, 585 and 623 nm, respectively (3+ luminescence (see ESI11AU to LTISTb (90 nM), the intensity of the Tb3+ emission increases progressively and plateaus after 1 equiv. RNA, with a 5.6-fold enhancement of Tb3+ emission , which indicates that the association and dissociation kinetics for 11AU·LTISTb are rapid, a property that is required for efficient real time monitoring of reversible biological processes. Another critical issue for biological probes is selectivity. The selectivity of the luminescence response of LTISTb was investigated by measuring the response of LTISTb with the 11-mer RNA sequence in which one or the two As were changed for U or G  as well as the DNA sequence TTTATTTATTT -2, -3, and -6, COX-2) and loss of TTP results in systemic hyperinflammation.59 A TTP-based probe could function to measure these cytokine levels in cells under different stimuli and therefore serve as a read-out of inflammation. Similarly, TTP recognizes and destabilizes a large number of tumor-related mRNAs (e.g. regulators of the cell cycle (e.g. cyclin D1), angiogenesis , and metastasis ),64 and we envision using the TTP-based probes to monitor mRNA levels in cancer cells. Our approach involved modifying the RBD of one of the TTP family proteins (TIS11d) with a lanthanide ion (Tb3+) and a suitable chromophore to sensitize lanthanide luminescence. By using TIS11d as the basis for the probe, we created a probe that kept the native RBD's selective RNA binding properties, i.e. reversible and dynamic binding with the Kd in the nM range.We have described the successful design and Tb is a rare example of a protein-based RNA sensor.30 Most RNA sensors described to date are based on hybridization with a nucleic acid oligomer-based fluorescent probe. With such a probe, the target RNA sequence must be longer than 15 nucleotides to form an RNA/probe hybrid with sufficient stability. LTISTb recognizes a shorter RNA sequence  showing that this protein-based approach may be a valuable alternative to hybridization probes for the sensing of short RNA sequences.LTISTb probe, the RNA binding event is transduced into an increased Tb3+ emission based upon the modulation of Tb3+ sensitization though a conformational change between the Cs124 antenna and Tb3+ ion. The use of a lanthanide emitter is critical to the design of LTISTb as small conformational changes are sensed due to the short distances required for efficient energy transfer from the antenna. The lanthanide complex and its antenna were introduced on the RBD of TIS11d based upon simple structural and functional considerations. Moreover, lanthanide luminescence offers unique properties to study biological systems. They have a long luminescence lifetime – exemplified here by the 1.9 ms lifetime for Tb3+ luminescence in LTISTb – that allows the time-resolved detection to suppress autofluorescence of the biological background and increase the signal/noise ratio. With this promising prototype probe in hand, we envision future studies in which modifications are made to the probe for different applications. For example, we could apply the convergent synthetic pathway for LTISTb in a combinatorial manner by varying the Ln3+ ion, its ligand , the antenna and the position of the DOTA and antenna on the RBD to optimize the probe's emission properties. Additionally, we envision appending functionalities for in vivo applications, e.g. addition of a cell penetrating peptide sequence or a second lanthanide complex to create a ratiometric sensor.In the LTISvia chemical ligation methods67 because they contain numerous zinc-binding cysteines . In addition, Nature often uses zinc fingers to fold biomolecule-binding sites and mediate protein/protein, protein/DNA, protein/RNA and protein/low-molecular-weight molecules interactions, suggesting that our approach can be expanded to target other biomolecules by functionalizing other zinc fingers. Additionally, this design strategy is not limited to zinc finger proteins or cysteine-containing proteins as progress in chemical ligation methods now allows straightforward synthesis of proteins that do not contain cysteines,68 thereby extending the scope of such probe design.The work we have described here demonstrates that a fully synthetic luminescent probe of a target analyte (RNA) based upon a biomolecule-binding domain of a protein can be engineered. By utilizing the robust chemical ligation methodologies that have been developed in the past decade for the chemical synthesis of proteins, the straightforward preparation of a biomolecule with a conjugated antenna and lanthanide complex from short peptide segments was achieved. The biomolecule utilized as the basis for the probe contains two zinc finger domains. These types of domains are amenable to chemical synthesis 3+ complex and a sensitizing antenna. The sensor was designed based on simple structural considerations and synthesized by the native chemical ligation methodology, which allows straightforward introduction of the Ln3+ emitter and antenna. Of note, the probe retains all of the RNA binding properties of the native TIS11d RBD, validating this approach. We believe that the design strategy has the potential to be expanded to other biomolecule-binding proteins, providing a general strategy for the development of luminescent probes.We have successfully engineered a luminescent sensor for the RNA sequence UUAUUUAUU by modifying the RNA binding domain of TIS11d, a member of the TTP family, such that it contains a Ln


Studies show that implicit and explicit attitudes influence food choice. However, precursors of food choice often are investigated using tasks offering a very limited number of options despite the comparably complex environment surrounding real life food choice. In the present study, we investigated how the assortment impacts the relationship between implicit and explicit attitudes and food choice (confectionery and fruit), assuming that a more complex choice architecture is more taxing on cognitive resources. Specifically, a binary and a multiple option choice task based on the same stimulus set (fake food items) were presented to ninety-seven participants. Path modeling revealed that both explicit and implicit attitudes were associated with relative food choice (confectionery vs. fruit) in both tasks. In the binary option choice task, both explicit and implicit attitudes were significant precursors of food choice, with explicit attitudes having a greater impact. Conversely, in the multiple option choice task, the additive impact of explicit and implicit attitudes was qualified by an interaction indicating that, even if explicit and implicit attitudes toward confectionery were inconsistent, more confectionery was chosen than fruit if either was positive. This compensatory ‘one is sufficient’-effect indicates that the structure of the choice environment modulates the relationship between attitudes and choice. The study highlights that environmental constraints, such as the number of choice options, are an important boundary condition that need to be included when investigating the relationship between psychological precursors and behavior. Food choices and eating behavior show great diversity across people, and, with people making more than 200 food decisions a day, they represent a core aspect of our everyday life , which represents a core part of the ‘choice architecture’  or ‘obesBuilding on previous work by Figure 1) , assuming three predictors in a linear multiple regression, yielded an N of 77 for 80% power. Ninety-seven participants  were recruited by e-mail using student mailing lists from the University of Konstanz, short notices distributed around the university and postings on Facebook groups. Potential participants were informed that omnivores  and people without health related dietary restrictions were eligible for the study. In addition, participants were asked on-site whether they were vegetarian, vegan or had any health related dietary restrictions. As none of the participants reported these limitations, all participants were included in the analyses. Participants were aged 19 to 46  and 97.9% were students, representing a wide range of subjects. As compensation, participants received either 12€ or 1.5 h of course credit.A power analysis using Gower 3.1  to detecThe participants were invited to the laboratory for individual sessions. Prior to participation, all participants gave written informed consent in accordance with the Declaration of Helsinki. They were seated in front of a 19 inch computer screen and were asked to fill in a questionnaire assessing explicit attitudes. Afterward, a single category implicit attitude test (SC-IAT) was conducted followed by the binary food choice task. Participants were then asked to step in front of the fake food buffet that was covered by a table cloth. The experimenter asked the participants to serve themselves a meal that they would typically have for lunch, provided them with tableware and removed the table cloth. Participants were free to choose the contents and size of their meal (multiple food choice task). When the participants were finished, they were asked to place the dishes on the serving tray. In a final step, they filled in a second computer-based questionnaire assessing demographic and anthropomorphic variables, as well as eating motives that will not be discussed in the present publication. Finally, participants were thanked and paid. During the study, participants wore SMI eye tracking glasses to monitor their eye movements; however, eye movements and selective attention are not the focus of the current paper and are reported only in the interest of full disclosure.Figure 1). The same 15 stimuli were used both as realistic food replicas on a buffet and as pictures of the replicas in the questionnaire and the two computerized tasks (SC-IAT and binary option choice task). The food pictures had a size of 400 × 300 pixels and depicted one standard serving of the respective food item according to German dietary guidelines  were used in the study  rated a total of 27 pictures of food replicas from the five food categories. Participants were asked to indicate what food item they saw in the picture and indicate to what extent the food item was identifiable and realistic on a four point Likert scale, with higher values representing a more positive evaluation. For each food category, three items that had been correctly identified by all participants and rated the most identifiable and realistic were selected  of the foods, but this item was not included in the scale because it reduced internal consistency . This concurs with previous research, which consistently finds a difference between palatability and health attitudes . The thr2) and conducted following the procedure described by 3 . They differed significantly regarding valence ratings  = 18.61, p < 0.001), but the valence ratings were equally extreme, as shown by the absolute z-standardized values .A SC-IAT was implemented in Presentation software  from the D-score for confectionery (α = 0.66), with a positive value reflecting a preference for confectionery over fruit.The first block consisted of 24 trials and served as a training phase where the participants were familiarized with the positive and negative IAPS pictures to ensure that they were able to correctly categorize images as ‘positive’ or ‘negative.’ Afterward, the food pictures and categories were added in the following 10 blocks, with two blocks per food category. The present publication focuses on the four blocks for the categories confectionery and fruit. The two target categories were coupled with the category ‘negative’ in the first block and ‘positive’ in the second block. Each block consisted of 42 trials with each picture appearing at least twice. Similar to the explicit attitudes measure, a final score for implicit attitudes was obtained by subtracting the 2). The two presented food items were centered vertically on a gray background on the computer screen with a 6 cm distance between the inner edges of the two images. The presentation procedure followed Figure 2).In order to assess food choices in a binary option context, participants were presented with two fake food pictures per trial using Presentation software . Participants were provided with a serving tray (55 cm × 35 cm) containing a large (27 cm diameter) and a small plate (21 cm diameter) as well as a small bowl (12 cm diameter) and were asked to serve themselves a meal which they would usually eat for lunch. Pictures showing examples for the meals self-served can be found in Supplementary Data Sheet To create a realistic, multiple option context to investigate food choice, a fake food buffet was composed according to Body Mass Index (BMI) was calculated based on self-reported weight and height.Participants were asked to indicate their hunger on a six point Likert scale ranging from (1) very hungry to (6) very full.To analyze the relationships between explicit and implicit attitudes and food choices in the binary and multiple option conditions, a path model analysis was conducted in Mplus 7.1 . For expTable 1.Descriptive statistics as well as zero-order correlations between study variables are depicted in M = -0.67, SD = 1.15). The SC-IAT also indicated a more positive implicit attitude toward fruit . Explicit and implicit attitudes were not significantly correlated .On average, participants held a more positive explicit attitude toward fruit than confectionery . While confectionery was chosen in 7.41 (SD = 3.33) out of 36 comparisons, fruit was chosen in 8.96 (SD = 2.38) out of 36 comparisons. In the multiple option context, participants served themselves on average 153.86 (SD = 93.93) grams of fruit and 53.32 grams (SD = 56.05) of confectionery. On average, they selected 107.0 grams more confectionery than fruit (SD = 216.83). Of the total meal, confectionery accounted for 8.43% (SD = 8.51) of the total real weight (grams) and 21.98% (SD = 19.99) of the energy , while fruit accounted for 25.20% (SD = 14.32) of the total real weight and 13.81% (SD = 10.60) of the energy. Together, they accounted for 33.64% (SD = 14.7) of the total real weight and 35.79% (SD = 18.94) of the energy of the meals self-served.In the binary option context, participants on average chose fruit more often than confectionery . Hunger was included as a control variable.4To test the relationship between implicit and explicit attitudes and food choice (confectionery versus fruit) in the two different choice tasks, a path analysis was performed . Also, the positive relation between implicit attitudes and confectionery choice reached statistical significance . The interaction between explicit and implicit attitudes was not significant . Furthermore, hunger was negatively associated with the amount of confectionery versus fruit chosen . Together these variables explained 51% of the variance in food choice in the binary option choice task.In the binary option choice task, the amount of confectionery versus fruit chosen significantly increased as a function of explicit attitudes  as well as implicit attitudes . The two main effects were qualified by a significant interaction between explicit and implicit attitudes on food choice . As Figure 4 depicts, a greater amount of confectionery was chosen on average when both attitudes showed a consistent preference for confectionery. However, a greater amount of confectionery was also chosen when explicit and implicit attitudes toward confectionery were inconsistent , indicating a compensatory ‘one is sufficient’-effect. Conversely, participants only chose more fruit on average when both attitude types consistently indicated a greater preference for fruit. Accordingly, simple effects revealed that food choice differed significantly between participants with low (-1 SD) and high (+1 SD) implicit attitudes  when explicit attitudes were 1 SD below the mean. However, food choice did not differ between participants with low or high implicit attitudes  when explicit attitudes were 1 SD above the mean. Moreover, hunger was negatively associated with the amount of confectionery versus fruit chosen . Together, the included variables explained 26% of the variance in food choice in the multiple option context5.Turning to the multiple option choice task shows that the amount of confectionery versus fruit chosen from the buffet increased as a function of explicit  = 15.74, p < 0.001], while the impact of implicit attitudes was comparable to the impact of explicit attitudes on food choice in the multiple option choice task . Explicit attitudes were more strongly associated with confectionery choices in the binary option choice task . Regarding implicit attitudes, the relationships in both choice tasks did not differ significantly .To further test the relative impact of implicit and explicit attitudes across the two choice tasks, we additionally compared the path coefficients. Comparing the two attitude-food choice path coefficients within the respective choice task shows that the impact of implicit attitudes was significantly weaker than the impact of explicit attitudes in the binary option choice task [χIn the present study, the relationships between explicit and implicit attitudes and food choice (confectionery vs. fruit) were investigated in two different choice tasks. In the binary option choice task, both types of attitudes were significant precursors of food choice. However, explicit attitudes clearly had a greater impact. Conversely, in the multiple option choice task, the additive impact of explicit and implicit attitudes was qualified by an interaction indicating that more confectionery was chosen even if explicit and implicit attitudes toward confectionaries were inconsistent and one of them was positive. Therefore, the results support the notion that the context of the target behavior is a significant boundary condition which shifts the balance between implicit and explicit attitudes as precursors within choice environments.Inherent in dual process models e.g.,  is the aWhile most studies that investigated the relationship between implicit and explicit attitudes and food choice did not include the interaction in their analysis, the studies that tested the multiplicative model have yet to report significant results . HoweverTo investigate shifting between implicit and explicit attitudes as precursors, one behavior is examined under different circumstances by taking dispositional or situational moderators into account. Thus, to investigate the relationship between explicit and implicit attitudes and food choice, one also needs to ask when they are associated in addition to whether they are associated in general c.f. . PreviouFrom the choice tasks compared in the present study, a parallel can be drawn to food choice contexts in daily life that may also differ in cognitive and self-control demands. In situations where only limited information is available or the number of available foods to choose from is limited, more cognitive resources may be available for deliberate decision making. In a recent point-of-purchase intervention study, Environmental influences on eating behavior have recently gained a lot of attention. These studies suggest an impact of the eating environment on the evaluation of food items or foods chosen . For exar = 0.77 to r = 0.93; The present study used two tasks to assess food choice. The two tasks differed in sensory stimulation and the number of options presented simultaneously. Whereas 2D pictures were displayed on a computer screen in the binary option choice task, the multiple option choice task presented 3D objects that had to be touched by the participants. The multiple option choice task thus increased cognitive load through the combination of a greater stimulus array. To disentangle the effects of choice assortment and sensory stimulation, fakes foods could be used in both choice tasks in future studies. However, realizing a sufficient number of trials for the binary option choice task might be challenging since an experimental set up needs to be realized where choice tasks are presented in a timely manner without participants glimpsing previous or upcoming tasks. Another limitation might be that the weight of food replica and real food diverges. However, a study which invited participants on two separate occasions showed a high agreement between self-served meals from a real food buffet and a fake food buffet (Regarding the study design, further limitations need to be acknowledged. The two choice tasks differed slightly in the instructions given. While participants were asked to choose which food they prefer in the binary option choice task, they were asked to choose what they typically eat for lunch in the multiple option choice task. Although the differences in instructions were subtler in the present study than in previous research examining task instruction differences on reaction times and eye movements during food choice e.g., , we cannThe sample of the present study mainly consisted of female undergraduate students without any dietary restrictions, thus it has to be acknowledge that the generalizability of the findings is limited. Although female students are a common population in previous studies on the relationship between reflective and impulsive influences and food choice e.g.,  and the The results of the present study demonstrate that different food choice tasks might lead to different conclusions about how food choices are made. Thus, choice environments and their ‘choice architecture’ offering different kinds of choice assortments might only be comparable to a limited extent. Both explicit and implicit attitudes were related to food choice in both tasks, with explicit attitudes being more strongly related to food choice in the ‘less demanding’ binary option choice task than implicit attitudes. Importantly, in the ‘more complex,’ multiple option choice task, an interaction between implicit and explicit attitudes emerged, indicating a compensatory ‘one-is-sufficient’ effect that triggers an ‘unhealthy’ choice pattern. Generally, the results suggest that both explicit and implicit attitudes independently contribute to shaping behavior in contexts providing a limited number of choice options, while explicit and implicit attitudes interact in more complex and demanding contexts. These findings underline that the environment in which food choice takes place is an important moderator that could be included as a boundary condition in existing dual process models c.f.  and furtLK, HG, BR, and HS designed the study. LK analyzed the data. LK and BR drafted the manuscript with critical comments from HG and HS. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Targeted nano-drug delivery systems conjugated with specific ligands to target selective cell-surface receptors or transporters could enhance the efficacy of drug delivery and therapy. Transporters are expressed differentially on the cell-surface of different cell types, and also specific transporters are expressed at higher than normal levels in selective cell types under pathological conditions. They also play a key role in intestinal absorption, delivery via non-oral routes , and transfer across biological barriers (e.g., blood–brain barrier and blood–retinal barrier. As such, the cell-surface transporters represent ideal targets for nano-drug delivery systems to facilitate drug delivery to selective cell types under normal or pathological conditions and also to avoid off-target adverse side effects of the drugs. There is increasing evidence in recent years supporting the utility of cell-surface transporters in the field of nano-drug delivery to increase oral bioavailability, to improve transfer across the blood–brain barrier, and to enhance delivery of therapeutics in a cell-type selective manner in disease states. Here we provide a comprehensive review of recent advancements in this interesting and important area. We also highlight certain key aspects that need to be taken into account for optimal development of transporter-assisted nano-drug delivery systems. ATP-binding cassette (ABC) transporters and solute carriers (SLC)  . The trars (SLC) , valgancrs (SLC) , and gabrs (SLC) .With the development of nanotechnology and nanomaterials, enormous progress on nano-drug delivery systems has been made. There is substantial and convincing evidence for enhanced efficacy of nanosized materials in oral drug delivery, cancer therapy, and site-specific drug delivery ,b. Some Figure 1A) or enhance permeation across biological barriers such as the blood–brain barrier . In the intestinal tract, specific nutrient transporters are highly expressed to mediate the absorption of diet-derived nutrients; these transporters have been exploited for oral drug delivery to enhance bioavailability of therapeutics (Table 1). Tumor cells have an increased demand for nutrients such as glucose, amino acids and vitamins to support their malignant proliferation. As most of the nutrients are water-soluble, they can’t simply diffuse into cells across the lipid bilayer that constitutes the plasma membrane; they need selective cell-surface transporters to enter the cells. Tumor cells upregulate certain selective transporters in the plasma membrane to meet their increased demands for nutrients. Such transporters have been used as targets as delivery systems for anticancer therapeutics to effectively mediate tumor-cell selective drug delivery, thereby increasing the drug efficacy against the tumors (Table 2). Tumors residing behind biological barriers present an additional problem in effective treatment with drugs; the chemotherapeutics need to cross the biological barrier before they encounter the tumor cells. Brain tumors represent such an example. Drugs targeted for the treatment of brain tumors have to cross the blood–brain barrier before getting into the target cells. As the common nutrients necessary for the growth and survival of normal brain cells face the same issue, selective transporters are expressed at high levels in endothelial cells that constitute the blood–brain barrier. Examples of these transporters include GLUT1 (glucose transporter 1 or SLC2A1) for glucose transport, LAT1  for amino acid transport, and ChT1  for choline transport. These transporters have been utilized as an effective means to facilitate the transfer of therapeutic drugs across this biological barrier and hence for optimal therapy of tumors such as glioma that reside enclosed within the barrier (Table 3).Targeted nano-drug delivery systems conjugated with specific ligands could enhance the efficacy of drug delivery. Most transporters have a site-specific expression, which provide ideal targets for drug delivery to increase uptake at specific site  The substrates of transporters are small molecules, which are stable, easily modifiable, and have no or little immunogenicity and steric hindrance whereas most of ligands for receptors are macromolecules , and (ii) Transporters usually have broad substrate selectivity whereas the ligands for the receptors are much more specific. These differences could actually offer certain advantages in selecting cell-surface transporters for nano-drug delivery systems as it provides multiple choices in terms of ligands for modification of the surface of the nanoparticles to target the transporters . Lack ofThere is very little information in published literature with regard to the effects of nanoparticles’ physicochemical properties on their interaction with transporters, but their effects on the cellular uptake of nanoparticles have been investigated . The smaIn the case of intravenous administration, the half-life of nanoparticles in circulation is an important consideration. The particle size should not be too small; otherwise, the nanoparticles would undergo filtration at the renal glomerulus and consequently would get eliminated. The particle size should not be too large either; otherwise, the nanoparticles would promote thrombosis. The surface potential of the nanoparticles should be negative in order to increase their half-life in circulation.L-carnitine to investigate the role of PEG linker in the interaction of the conjugated substrate with OCTN2; the uptake of L-carnitine-conjugated nanoparticles increased with the length of PEG to a certain degree, but then decreased with further increase in the length (Poly (ethyleneglycol) (PEG) is often used as a linker to decorate the ligand/substrate on the surface of the nanoparticles; the linker distances the ligand/substrate from the surface and provides flexibility for more efficient interaction with the transporter. We manipulated the length of PEG for conjugation of e length . A simile length .0,+  because the transporter does not tolerate negative charge on the side chains of its amino acid substrates. But, if the β-carboxyl group in the side chain of aspartate or the γ-carboxyl group in the side chain of glutamate is used for covalent linkage with the surface of the nanoparticles, the negative charge is lost, thus leaving the remainder of the molecule as a recognizable substrate by the transporter  are responsible for the transport of glucose from the circulation into target cells. As these are facilitative, they also function in the release of glucose from the cells into circulation in certain tissues . GLUT1, a representative member in GLUT family, is expressed ubiquitously; it is the transporter responsible for glucose uptake in erythrocytes and also for glucose transfer across the blood–brain barrier; it is also the transporter that supports glucose delivery into tumor cells as it is overexpressed in tumors . Glioma D-glucose (DGlu-NP) have been used successfully for glioma therapy by targeting the blood–brain barrier as well as the tumor -co-poly(trimethylene carbonate) nanoparticles functionalized with 2-deoxy-he tumor . Compare in vivo . To opti in vivo . Unlike 18F-fluoro-2-deoxy-D-glucose in positron emission tomography in in vivo tumor diagnosis. Thus, this transporter can be used not only for tumor-specific drug delivery but also for tumor-specific delivery of imaging probes. Shan et al. prepared γ-Fe2O3 nanoparticles coated with dimercaptosuccinic acid and modified with 2-deoxy-D-glucose (γ-Fe2O3@DMSA-DG NPs) to target to GLUT1-overexpressed breast cancer for tumor imaging , a hallmark of all cancers. This is the basis of the effective use of  imaging . It was er cells .The size of the linkers used to link glucose onto the surface of the nanoparticles impact on the interaction of the ligand with GLUT1. D-glucose to suppress the uptake of Glc-QDs.GLUT4 is also a facilitative GLUTs, but unlike GLUT1, this transporter is expressed only in cardiac muscle, skeletal muscle and adipocytes; it is also the only facilitative GLUTss that responds to insulin . InsulinSodium-dependent multivitamin transporter (SMVT/SLC5A6) is an important transporter obligatory for the uptake of the vitamins biotin and pantothenate; it is highly expressed in placenta, intestine, brain, liver, lung, kidney and heart . Biotin in vitro was enhanced more than twofold compared to unmodified erythrocytes. In vivo studies showed that the therapeutic index of liver targeting was enhanced threefold with biotinylation. As many other vitamin transporters, SMVT is also expressed at higher levels in tumors than in normal tissues. SMVT expressed in non-intestinal tissues has also been targeted for site-specific drug delivery. The overexpression of SMVT in cancer cells is also of use for simultaneous delivery of not only anticancer drugs but also imaging probes. +-dependence and the affinity for choline  are responsible for cellular uptake of this compound. There are two distinct classes of ChTs, primarily based on Na choline . The Na+The structural components in choline necessary for interacting with the transporter include the positively charged quaternary ammonium group and the hydroxyl group ; therefoGliomas are the most common type of primary central nervous system cancer in adults . The gliin vitro and then in mouse xenografts in vivo. Choline derivative-modified micelles were also effective in improving glioma therapy via ChT dual-targeted strategy , which exhibits functional features that are uniquely suited to satisfy the increased demands for amino acids in tumor cells . The upregulation of several amino acid transporters in tumors has been documented; one of these transporters is ATBor cells . This tror cells , cervicaor cells , estrogeor cells , and panor cells . The upr0,+ as the target, we developed amino acid-conjugated liposomes for enhanced anticancer efficacy and decreased off-target side effects , which show abundant expression of ATB0,+; the uptake was inhibited by free glycine and lysine  mediates cellular uptake of almost all neutral amino acids, but unlike ATBependent . LAT1 prependent . Therefoependent . In the ependent . Comparedelivery . Glutama+, following which the conformation shifts to an inward-facing configuration to facilitate the release of the substrate and the co-transported ion inside the cell  is a highly polar zwitterionic molecule obligatory for the transfer of long-chain fatty acids across the inner mitochondrial membrane for subsequent β-oxidation. L-Carnitine can be synthesized from lysine endogenously, but a majority of L-carnitine in the body is from intestinal absorption of dietary L-carnitine  targeting to OCTN2 enhanced oral bioavailability. There was an optimal value for the surface density of L-carnitine; we found 10% L-carnitine to provide the maxima enhancement of nanoparticle uptake. This phenomenon was also observed in other studies  is a water-soluble vitamin, and transporters for vitamin C belong to SLC23 family . SVCT1 (P-glycoprotein (P-gp/ABCB1/MDR1). As the primary function of MDR1 is to efflux drugs out of the cells, its utility in facilitating drug entry into cells remains doubtful. Nonetheless, there was a report that linking the substrate of P-gp to the surface of nanoparticles significantly increased the uptake of nanoparticles (P-gp inhibitors (cyclosporine A and verapamil) in P-gp overexpressed cells.ATP-binding cassette (ABC) transporters is a major superfamily of membrane transporters, which bind and hydrolyze ATP to drive the efflux of various compounds out of cells. Several of the drugs that are used routinely in clinical practice are substrates for this transporter. This includes paclitaxel, docetaxel, and doxorubicin. The most-investigated member of the ABC transporter family is articles . The sub+-coupled if the zwitterionic L-carnitine is the substrate, but is Na+-independent if cationic molecules are substrates; in this case, L-carnitine transport is mostly unidirectional preferring movement of the substrate from outside to inside whereas organic cation transport could be in either direction . If the cargo consists of proteins, peptides or plasmid DNA, this pathway might lead to inactivation of the cargo because of the involvement of lysosomal degrading enzymes. The caveolin-dependent endocytosis avoids the lysosomal compartment and hence the cargo remains intact and unaffected. This pathway is used by some pathogens such as viruses and bacteria : (i) the transporters follow the recycling route in which once the substrate-conjugated nanoparticles along with their cargo are dissociated from the transporter, the transporter recycles back to the plasma membrane; (ii) the transporters follow the transcytosis route in which they stay bound to the nanoparticles in the pinched-off vesicles that originate on one side of the cell, travel across the cell, and then fuse with the membrane on the other side of the cell to exit; (iii) the transporters follow the fate of the nanoparticles and their cargo where the transporters get degraded in endosome/lysosomes. Our studies show that all three routes play a role in different proportions depending not only on the transporter but also on the cell type. In one of our studies with LAT1-targeted nanoparticles with cultured cell lines, the amount of LAT1 protein was decreased at the plasma membrane but increased in cytoplasm at the beginning of the uptake process , but none of the targeted nanoparticles, either cell-surface receptor-assisted or cell-surface transporter-assisted, has actually reached the market. This does not necessarily mean that the idea of transporter-assisted nanoparticles has not potential. The field itself is at its beginning stage and additional research is needed to validate the eventual success or failure of this approach.VG and JS conceived the review, decided on the general outline, and finalized the manuscript. LK wrote the first draft of the manuscript. YB, QY, and ZH critiqued and revised the manuscript. All authors read the final version of the manuscript and approved it for submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.