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Tetraloop receptor motif investing
One of the key features that dictate non-coding RNA functions is their 3D structures 6—8. As a result, understanding the characteristics of RNA 3D structures becomes a critical element of biological research. One approach to identify the characteristics of RNA is finding recurring structural components across various types of RNAs.
Those recurring structural components are called RNA structural motifs and considered as building blocks of the RNA architectures 10 , The importance of RNA structural motifs is shown in many contexts, including how different molecules engage with RNA through interactions in the known motif regions 12— Moreover, it has been shown that RNA motifs can be used in building structural elements in nanotechnology Finding RNA structural motifs of similar characteristics and identifying variations of them can help in understanding the basics of RNA functions and aid in the rising new directions of RNA-inspired research.
These methods have discovered many instances of known motifs, such as kink-turn 12 , reverse kink-turn 19 , sarcin-ricin 13 , C-loop 20 and E-loop In addition to finding instances of known motif families, these computational methods also have identified new motif families. These methods attempt to put all the instances of a motif family into the same cluster. But they cannot manage to do so in many cases due to the inherent variations of structural features in the motif instances.
If these methods allow too much flexibility to encompass these variations, they run the risk of putting instances of different motif families together. As a result, they choose the option to be rigid to some extent. Consequently, the instances of one family get separated into multiple groups in these clustering results. The details of these separations into groups and the corresponding implications is an area worth investigating, but there is not much work focusing in this direction yet.
A few research were conducted to extensively analyze the variations in a couple of well-known motif families, such as kink-turn and sarcin-ricin. Leontis et al. The extent of acceptable sequence variations, especially the isosteric variants, to achieve desired structural features of known motif families is also shown in simulation-based experiments 27 , The potential application of an agonist RIG-I aptamer in cancer therapy is suggested by observations of immune response to tumor cells through RIG-I activation.
Radiation induces translocation of small nuclear RNAs U1 and U2 to the cytoplasm, where they form a complex with RIG-I and trigger downstream signaling, leading to activation of interferon genes. Transcription of IFN genes can sensitize cancer cells and mobilize immune cells to the tumor microenvironment. The selection of aptamer-discriminating pathogens or toxins may be less prone to isolating false positive sequences than cell SELEX. Currently, several bacterial, viral, and parasitic pathogens were subjected for aptamer selection to detect or inhibit infection.
Given the physicochemical and economic advantages of aptamers over antibodies, the development of treatment and diagnosis against infectious organisms based on aptamers may prove as viable in less developed countries. The conjugation of aptamers and other functional oligonucleotides into one chimeric construct is a way to specifically deliver TNAs without a need for a transfection reagent.
The majority if not all of chimeric constructs are designed to affect post-transcriptional regulation upon binding to the specific receptor and successful trafficking to the cytoplasm. Knocking down the expression of certain genes is an intensely researched approach in the context of nucleic-acid-based therapies. The first proof-of-concept for in vivo in a xenograft model targeted delivery of therapeutic miRNA relied on antagonistic aptamer GL T targeting Axl, a receptor tyrosine kinase an oncogene overexpressed in several human cancers and tumor suppressor let-7g miRNA.
Synergistic chimeras downregulated let-7g target genes in tumor cells, leading to apoptosis, decreased cell proliferation, and a reduction in tumor size. The versatility of this approach was confirmed by using alternative linking strategies and a combination of diverse aptamers and miRNAs. Interestingly, no increase in antagonizing potential was observed when two identical anti-miRs connected in tandem were co-delivered, but the tandem of two different anti-miRs anti-miR and anti-miRb reduced respective miRNAs and increased target protein levels.
Every phase of the viral life cycle depends on the host cell. Aptamer-mediated blocking of either a cellular receptor or a viral glycoprotein is a viable strategy to prevent the infection of cells. Together with aptamers targeting proteins and enzymes crucial for virus replication, we can combat virus infection throughout the whole viral replication cycle. The feasibility of this idea has been tested in the experimental model of humanized mice by targeting the expression of HIV-1 viral proteins tat and rev and host CD4 receptor that is required for HIV-1 entry as well as transportin 3 TNPO3 necessary for viral integration.
The long-term inhibition of HIV replication in a cell culture system relies on the cellular transcription of aptamers incorporated into the terminal loop of an shRNA targeting the tat—rev region. However, on one side, these sequences can abrogate functional conformations of an aptamer, but on the other side, proper design and experimental validation of aptamer adjacent sequences can stabilize transcripts and increase the resistance to degradation. If the integrity of the human body is not compromised by wounds, bites, etc.
Application of a formulation of aerosol containing therapeutic aptamers or a gel with aptamers or aptamer—siRNA chimeras might protect against viral infections. It seems that even a small percentage of repaired pre-mRNA becomes sufficient to generate phenotypic effects. Decoy oligonucleotides bind to a corresponding transcription factor in order to inhibit binding to its specific target genome sequences present in promoters and enhancers, thus attenuating transcription of corresponding genes.
However, for chimeric aptamers carrying TNA, rapid internalization and release to the cytoplasm are crucial processes. Although many aptamers that efficiently delivered TNA into the cytoplasm were described, the process of their passage through the membrane without help from external carriers remains elusive and is represented with a complex set of limitations.
At the end of the s and the beginning of this millennium, several pioneering studies introduced the notion of the programmable design of RNA nanoparticles assembled using long-range RNA tetraloop-receptor interacting motifs — or structural RNA motifs derived from the DNA-packaging motor of bacteriophage phi29 that can serve as a building block to engineer NANPs via a bottom-up assembly , and implement therapeutic aptamers embedded into their structures.
In the latter case, the independent folding of several domains is the main hallmark of packaging RNA pRNA monomers for their further controlled assembly. This spatial arrangement has been proven, in multiple in vitro or in vivo studies, as an effective vector of diverse functional RNAs. CD4-specific aptamer or folate on one subunit delivered to targeted cells can introduce an interlocked subunit carrying siRNA against the survivin or luciferase genes.
Ex vivo treatment of nasopharyngeal epidermal carcinoma cells with dimers containing folate and siRNA—survivin suppressed tumor formation in athymic nude mice after the axillary injection of targeted cells. The treatment has the potential to inhibit inflammation in ischemic strokes and other neuroinflammatory diseases. In vivo biodistribution of RNA nanoparticles was tumor specific with negligible or no accumulation in healthy organs and tissues. The doxorubicin intercalated into the pRNA-3WJ scaffold with high loading efficiency and then selectively entered annexin A2 positive cells through receptor-mediated endocytosis.
Doxorubicin associated with pRNA scaffolds is slowly released, which is advantageous upon systematic application. This means that the remaining positions can be used for displaying other functional moieties. Additionally, some recent data suggest that aptamer-mediated delivery of naked, large, functional RNA NANPs leads to the internalization of payloads in a nondisrupted state for more than 2 h.
Similarly, as for chimeric aptamers, the release of NANPs from endosomes currently represents one of the main obstacles in the application of naked NANPs in vivo. It would be interesting if we could engineer nucleic acid structures that would respond to acidic environment analogically as certain viral fusogenic proteins.
Ultimately, such features could lead to development of multifunctional NANPs synthesized purely from nucleic acids Figure 5. Figure 5. Cell-specific aptamers facilitate targeted delivery of NANPs. Fluorescent aptamers would provide tracking of NANPs from the cell surface, through the endosome compartment, to cytoplasm.
Go to: Use of Aptamers in Nanorobot Construction. Although this is true also for many reported NANPs delivered by aptamers, DNA origami-based structures promise a cell-specific delivery and logical release of therapeutic cargo without entering the diseased cell. The following works are illustrative of the potential of aptamers for the creation of nanorobots combining diagnostics with therapeutics that are based on the aptamer logic-gated checks minimalizing off-target effects.
The logical trigger is activated only in the presence of cellular receptors that are recognized by a pair of NANP-associated aptamers. The first DNA origami-based logic-gated nanorobot in the form of a hexagonal barrel consisting of two halves linked together by single-stranded scaffold hinges was locked by two DNA aptamers Figure 4D. In the inactive state, therapeutic cargo is hidden inside the barrel and the functional payload is exposed and activated only when both aptamers simultaneously recognize target proteins protein keys , which will cause dissociation of aptamer lock duplexes.
The process operates equivalently to a logic AND gate. The specificity of the aptamer-encoded logic gating strategy has been proven by six different robots displaying pairwise combinations of aptamer locks from a set of three aptamer sequences: 41t, TE17, and sgc8c. Several experiments tested the functionality and robustness of nanorobots. Nanorobots with specific aptamer pairs were able to distinguish matching antigens on target NKL cells in a mixture of receptor-negative cells.
The result showed an induced growth arrest in NKL cells in a dose-dependent fashion. Recently, another study tested the feasibility of logic-gated nanorobots targeting various tumors in vivo. In the open state DNA, the origami nanorobot adopts sheet conformation, whereas the closed particle has a cylindrical shape. The tube structure encapsulates four blood coagulation factor thrombin molecules, effectively hiding them from circulating platelets and plasma fibrinogen.
The closed inactive state is ensured by several fastener two-strand sequences containing duplex fibers and modified DNA aptamers AS targeting the solid tumor-specific molecular marker nucleolin. Four AS aptamers were located also at both ends of the tube. The robustness and functionality were tested in a mouse model of breast and lung cancer or melanoma as well as in Bama miniature pigs.
As hypothesized, the nanorobots accumulated at the tumor where binding of aptamer fasteners to nucleolin unlocked the nanotubes. Thus, exposed thrombin triggered the formation of intravascular blood clots specifically at the tumor site. Clogged vessels resulted finally in tumor infarction. This is partly due to the nonexistence of large-scale productions of RNA via synthetic or biological processes which we use for DNA.
One of the most promising strategies would be to employ RNA aptamers that emit a fluorescent signal upon binding a fluorophore. RNA aptamer—fluorophore pairs should feature five essential properties: i it should be as bright as possible to be sensitive up to one molecule resolution.
Various types of fluorogens are used for attachment to the RNA aptamers: environment-sensitive fluorogens, molecular rotor fluorogens, and quenched fluorogens. However, the nonspecific DNA binding capacity of these dyes can lead to significant unwanted background fluorescence when used in the cell-based assay. This can be suppressed by creating chemical derivatives from initial dyes no longer capable of nonspecific DNA binding, but preserving their fluorogenic capacity and becoming fluorescent only upon specific interaction with DNA and RNA aptamers.
They are poorly fluorescent in their unbound form in a fluid environment. However, fluorescence can be restored by restricting intramolecular movements either by strongly increasing medium viscosity or upon specific interaction with a nucleic acid. Examples of such molecular rotors are malachite green, patent blue, , thiazole orange, , and others.
Quenched fluorogens are dyes obtained by appending a quenching group to a fluorescent organic dye. Recently, another generation of RNA aptamers mimicking the green fluorescent protein has been generated and their potential tested in vitro and in vivo. The detection system consisted of small-molecule-binding aptamers linked to Spinach. The cellular interior presents several obstacles for efficient function of in vitro selected aptamers, including but not limited to RNA degradation and incorrect folding.
Reverse transcribed aptamers were then cloned into a plasmid fused with tRNALys. After induction of transcription in transformed E. The selected aptamer a 49 nt long Broccoli was less dependent on magnesium ions and exhibited higher thermostability than Spinach or Spinach2. Improved Spinach2 was subsequently used for virus tracking. Characterization of Broccoli stability in the context of various scaffolds showed that tRNA scaffold, and not Broccoli, triggers RNA cleavage both in bacterial and mammalian cells.
The deeper analysis found that the F29 scaffold contains a sequence similar to the transcription terminator. Despite the presence of intact V5-Broccoli transcript in-gel analysis, the fluorescent signal in mammalian cells was outcompeted by FBroccoli. Re-engineering of F29 resulted in an F30 scaffold that, together with Broccoli, is transcribed as a single strand adopting a conformation with robust fluorescence. Furthermore, both F30 arms can incorporate at least two additional Broccoli aptamers, which increases the fluorescence of the construct.
Corn selectively binds 3,5-difluorohydroxybenzylideneimidazolinoneoxime DFHO , a chromophore found in the red fluorescent protein, and induces its fluorescence in the yellow spectrum. In a solution, this aptamer unexpectedly functions as a homodimer with the quasi-symmetric structure of the ligand-binding site.
That means that even though the two units exhibit an identical primary structure, there are certain minor differences in conformations. In this case, the three adenosines that are at the DFHO-binding site adopt different conformations in each subunit. Interestingly, there is no intermolecular Watson—Crick base pairing at the interface of dimers. The decrease in size can potentially make shorter aptamers more favorable for fusion with transcripts of interest or incorporation into RNA nanoparticles as the smaller structure has a lower probability to interfere with the rest of the tagged molecule.
The conundrum of real-time visualization of RNA NANP co-transcriptional assembly in vivo may be resolved by incorporation of fluorogenic split aptamers. The strategy of using the split aptamer system is based on the physical separation of the aptamer strand into two separate parts. When both nonfunctional halves reassociate together, the functional aptamer is formed, which is detected by a fluorescent signal.
Therefore, a model system such as split Spinach or split Broccoli represents an important tool for monitoring the dynamics of RNA reassembly in vitro. Fluorescent aptamers as a tool in NANP assembly. B Assembly of 3D nanostructures is possible during the parallel transcription of all templates.
C Embedding split light-up aptamers into conditionally pairwise reshaping NANPs enables the monitoring of their interactions in real time. Cell-specific delivery of fully functional nucleic acid nanoparticles is ideal, but the need for a more controllable system is of immediate concern. Inspired by split protein systems, our research group and collaborators developed a technique based on a pair of RNA—DNA hybrids with embedded split functionalities.
Separated hybrids are inactive, and to become functional, hybrids require reassociation of cognate double strands. Versatility of the technique was confirmed in mammalian cells and in vivo with the triggering of various functionalities and silencing of several genes HIV-1 genes, eGFP, or glutathione S-transferaseP1 , fluorescence resonance energy transfer FRET , and aptamer binding the malachite green Figure 6C.
When functionally interdependent shape-switching NANPs interact together, the formation of Broc and Coli reassemble into an active fluorescent aptamer Figure 6C. In this single-stranded RNA origami construct, Spinach and Mango aptamers are positioned in close proximity to induce high FRET signals between the two fluorophores. Due to the versatility of nanoparticles to respond to conformational changes, two different nanomechanical constructs were designed.
Therefore, this system has potential to expand our knowledge of basic RNA biology in vivo and for broader applications regarding nanomedicine. Natural vehicles for the delivery of functional RNAs can open a perspective field of diverse aptamer technologies. Upon intracellular assembly, genetically encoded TNAs or NANPs can be trafficked into the natural vesicles or engineered virus-like particles. We envisage that NANP loading will be based on and specifically mediated by aptamers binding to proteins characteristic for vesicles.
In this section, we discuss described observations and experimental efforts loading the vesicles with RNA. However, the relevance and importance of intercellular delivery of EVs enriched with small ncRNAs remains to be elucidated. Even more puzzling is the inter- and trans-kingdom exchange of EVs containing exRNAs that may have global evolutional and ecological influence. Once in the cytosol, small ncRNAs could then perform a given function. Therefore, employment of EVs in biotechnology and therapy may revolutionize respective fields of biology.
Extracellular vesicles are a heterogeneous group of small phospholipid membrane-enclosed vesicles that are released into the extracellular environment by various cell types. In humans, EVs are secreted from healthy, malignant, or virus-infected cells and are involved in many physiological as well pathophysiological processes, such as cancer progression, the immune response, cell proliferation, cell migration, angio-genesis, and extracellular matrix remodeling.
Based on the size and biogenesis, EVs can be further organized into apoptotic bodies, microvesicles, and exosomes that are the subject of a growing area of research. However, it seems that some proteins and RNAs are specifically enriched in exosomes. Our current understanding proposes that exosomes constitute a sophisticated system of cell-to-cell communication.
Two approaches exist for incorporation of RNAs with therapeutically relevant properties into exosomes. Either vesicles isolated from cell lines are loaded with siRNAs connected to a lipid compound such as cholesterol Figure 7A or endogenous siRNAs are selectively taken up by exosomes from the transgenic construct Figure 7B.
Figure 7. Schematic description proposing NANP loading into exosomes or lentiviruses. C NANPs are loaded into viral particles via the aptamers binding to virus proteins. One concern regarding the former is the method of cholesterol conjugation to siRNA which may affect the loading of siRNA into vesicles and potentially interfere with the gene-silencing ability of siRNA. Therefore, the optimization of siRNA chemical modification patterns is necessary for efficient gene downregulation and reproducible and scalable exosome-mediated delivery.
Placing cholesterol on the arrowhead resulted in partial loading of RNA into the vesicles. The authors used the later system for linking arrowheads with aptamers and folate for targeting exosomes loaded with siRNA against survivin in three cancer models. Similarly, folate-displaying exosomes suppressed colorectal cancer xenografts. Therefore, it is necessary to understand mechanistically how genetically encoded RNAs are directed and enriched in exosomes in order to develop effective exosome-based therapeutics.
In this model, miRNAs in excess compared to their cellular target mRNAs are passively transported to exosomes for disposal. In fact, more processes may be involved. As discussed in previous sections, aptamers can function as a delivery agent.
Therefore, an aptamer could potentially target delivery of therapeutic RNA to exosomes. Of the 56 exosome-associated clones that were found, 29 shared a similar sequence.
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Fraser Find articles by Marie E. Wrote the paper: LW SZ. Received Feb 6; Accepted Oct 8. Associated Data Supplementary Materials Figure S1: Secondary structures and front and back stereo views of all interactions in this study. Gray indicates nucleotides that do not belong to the loop-helix interaction. Secondary structures are drawn in the Leontis-Westhof notation see Figure 1C.
B Two views of an overlay of eight 1. C Secondary structures of the four Subclass 1 Indiv structures and the reference 1. Gray bases are not shown in the three-dimensional depictions in panels D, E and F. Three stereoviews are shown, color-coded as indicated in Panel C. JPG pone. Superposition is based on the backbones of 4 nucleotides of the receptor and either one or two nucleotides from the loop T2, T3 in the case of tetraloops. Green and blue indicate the two loop nucleotides having different backbone geometries but analogous base positions as other structures in the subclass see Panel B.
Epub Aug 5. DOI: The most common example of the latter is the GAAA tetraloop—11 nt tetraloop receptor motif. Biophysical characterization of this motif provided evidence for the modularity of RNA structure, with applications spanning improved crystallization methods to RNA tectonics.
In this review, we identify and compare types of GNRA tetraloop—receptor interactions.