The modes of binding and fluorescence property of molecular probes that recognise RNA are significantly dependent on their secondary structure. RNA GQs play a crucial role in a number of cellular processes such as pre-mRNA slicing and polyadenylation, mRNA targeting and translation.12, 209 As a number of putative RNA GQ motifs have been identified within the human transcriptome,210-212 there arises a critical need for the development of specific and reliable detection methods for these structures. Tang and coworkers recently reported a cyanine-based dye CyT as a promising sensor for selective detection of RNA GQ among other RNA secondary structures (Fig. 11).213 So far, CyT is the only red emission-based molecular probe reported for the detection of RNA GQ in live cells of human origin. The sensing capability of the probe was studied in the presence of various forms of RNA, including single-stranded, double-stranded, hairpin structure, and three- and four-way junctions, in addition to the quadruplex structure. CyT exhibited negligible fluorescence in the unbound state and fluorescence enhancement (?em = 595 nm and??ex = 532 nm) of over 1000-fold was observed in the presence of RNA GQ compared to a mere 25-fold increase with non-GQ RNAs. Selective recognition of RNA GQ results from the disassembly of CyT J-aggregates and binding of the monomer to RNA GQ, which result in restricting the molecular rotation and prevention of non-radiating transition. In agreement with the binding preference, CyT displayed a relatively high binding constant (order of 105 M-1) for RNA GQ compared to non-GQ RNA structures (order of 104 M-1). Remarkably, the probe exhibited high quantum yields upon binding to two equivalents of RNA GQs (?F = 0.95 for Tel22, 0.92 for VEGF, 0.79 for TRF2, 0.74 for BCL-2, and 0.58 for NRAS oligonucleotides). The selective staining of non-canonical RNA GQs in the presence of various non-GQ RNA forms (like ssRNA, duplex RNA, short hairpin RNA and RNA junction structures) was demonstrated in a polyacrylamide gel electrophoresis experiment. The probe was successfully implemented to detect RNA GQ in fixed and live cells of human origin. Fluorescence microscopy study revealed that incubation of live A549 cells with CyT (5 ?M) for 24 h illuminated the cytoplasm in red, reiterating the potential of the probe for the detection of endogenous RNA GQs in real time. However, there remains a dearth of conformation-specific fluorescence probes, especially far-red probes for efficient detection and imaging of different RNA GQs under in cellulo and invivo conditions.
Human telomeric repeat-containing RNA (TERRA) plays an important role,along with its DNA counterpart, in the process of telomere shortening and elongation.214 TERRA of various lengths solely adopts a parallel GQ structure (TERRA GQ) under in vitro and in vivo conditions and the major challenge is to selectively distinguish it from DNA GQs that possess polymorphic structures including parallel (3 + 1) hybrid, basket, and chair topologies.215 Yong Shao and co-workers identified 5,10,15,20-tetrakis(3,5-dihydroxyphenyl)porphyrin (TOHdPP) from the pool of porphyrin derivatives as a selective fluorescence probe for TERRA GQ (Fig. 17 & Table 4).216 TOHdPP interacts with TERRA GQ tetrad through ?-stacking and triggers an efficient electron communication between the tetraphenyl substituents and porphyrin macrocycle, as required by the hyperporphyrin effect. This was further validated by the observed enhanced and red-shifted fluorescence emission band at 732 nm. TOHdPP showed 60-fold brighter fluorescence intensity (?ex = 467 nm) with RNA GQ (rUAG4), compared to its DNA counterpart (TAG4). It is interesting to note that TERRA parallel GQ structures selectively convert TOHdPP to the hyperporphyrin state,ref and similar transformation is unfavourable for varieties of DNA GQs with parallel topology. The hydrogen bonding interactions of hydroxyl groups of TOHdPP with the phosphate backbone and/or the ribose/U residues have been suggested as being responsible for the observed exclusive RNA GQ selectivity.These results are interesting attributes, making TOHdPP a promising probe for RNA GQ, especially TERRA.
In contrast to known classical turn-on probes, David Monochaud and co-workers devised an alternative strategy, referred as “twice-as-smart” GQ ligands.217-219 These ligands act as smart probes with high affinity and selectivity for GQs, and exhibit fluorescence turn-on property-based on fluorogenic electron distribution. This unprecedented design strategy that employs the red-edge effect (REE: acharacteristic property defined as the dependence of the wavelength of the emission maximum on the excitation wavelength ?emmax = f ?ex)has immensely widened the scope of the probe’s emission wavelength despite the lack of emission in the red region, exclusively. The probeoperates through a quadruplex-promoted conformational switch that assembles its four guanines into an intramolecular G-quartet (smart ligand). The first smart ligand PyroTASQ for DNA/RNA GQexhibited turn-on fluorescence through a quadruplex-promoted conformational switch and was exploited to light-up the quadruplex in polyacrylamide gel electrophoresis. However, it was only partially successful inin cellulo imaging due to its propensity for aggregation.217 To overcome this limitation of aggregation, naphtho-TASQ (N-TASQ) was designed and studied exclusively for cellular imaging. N-TASQ exists in its open conformation when free in solution, and the fluorescence of its naphthalene template is quenched by the four surrounding guanines via intramolecular-photoinduced electron transfer. However, upon interaction with GQ, the intramolecular G-quartet formation leads to redistribution of the guanine electrons onto the four guanines and relieves the template from its electronic restraint, restoring the probe fluorescence (Fig. 18a). Although N-TASQ absorbs below 320 nm, hampering its application in confocal imaging (commonly equipped with lasers adjusted at 408, 488 and 555 nm), its multiphoton absorption (> 600 nm) property has allowed unbiased detection of RNA GQs in live cells (no fixation or forced permeabilization is necessary) by two-photon microscopy.218The fluorescence response of the probe is associated with better accessibility of the external G-quartet created by loop-free environment of any quadruplex, thus, offering selectivity for particular GQs over other NA conformations. However, given the authors’ judicious hypothesis for N-TASQ with the unique spectroscopic property of REE, compatibility of N-TASQ for confocal imaging was discovered.219 Hence, they could explore the options of convenient visualisation of RNA GQs in live cells using all the standard emission filters of a confocal microscope (DAPI: 495 nm; FITC: 495-590 nm; Alexa: 585 nm and above), observing better quantum gain through the FITC filter (Fig. 18b). This unparalleled strategy elicited small molecule-based fluorescent probes for GQ imaging in live cells, contrary to antibody-based approaches that are limited to fixed and permeabilised cells.
Miscellaneous
In the past few years, stimuli-responsive fluorescence probes have gained more attention, owing to on-demand or analyte-specific activation to target, and space-time specificity.220-222 These probes provide an additional opportunity to detect the generation, accumulation, and transportation of specific analyte flux across subcellular locales or organelles such as cell nucleus, cell membrane, and mitochondria. Especially, fluorescence combination probes with signalling assisted by the additional recognition event (DNA or protein binding), which are provided with built-in correction for false positives, is highly beneficial for probing specific biological activity in a cellular environment and has the potential to be used in diagnostic and therapeutic applications.223-227 These probes also help in avoiding unwanted background fluorescence from the other biomolecules and cellular compartments, apart from increased sensitivity and selectivity towards the stimuli (pH, ROS or chemical substrates) or analyte (biomolecules) of interest. The majority of DNA binding probes are almost non-fluorescent in the unbound state and exhibit turn-on fluorescence response in the presence of DNA. Such turn-on fluorescence response, specifically in the presence of DNA, inspired us to design a stimuli-responsive fluorescence probe in combination with DNA, particularly making use of our turn-on NIR fluorescence probe QCy-DT (Fig. 19a).94 In probe QCy-DT, free phenolic-OH group is available for functionalisation with a large variety of chemical or enzymatically cleavable groups to develop promising stimuli-responsive probes for various biological applications. We developed a phenyl boronic acid-functionalised quinone-cyanine probe QCy-BA,in combination with DNA (i.e., QCy-BA?DNA), which serves as a superior NIR fluorescence combination probe for cellular hydrogen peroxide (H2O2) over other reactive oxygen species (ROS) (Fig. 19a).228In the presence of H2O2, probe QCy-BA converts to DNA minor groove binding QCy-DT,which shows turn-on NIR-fluorescence in the presence of exogenous orendogenous (cellular) DNA. In the absence of exogenous or cellular DNA, the probe QCy-BAdoes not show any fluorescence response in the NIR-region, and this helps in achieving double check (built-in-correction) on the observed signal output. Selective turn-on NIR-fluorescence response of combination sensor QCy-BA?DNA, in the presence of H2O2, encouraged us to study its cellular uptake properties and detection of the exogenous and endogenous level of H2O2 in normal and primary cells. Further, cell viability assay in HeLa cells showed that more than 80% cells were viable at 25 ?M concentration. These results revealed that probe QCy-BA is cell-permeable and non-toxic at working concentration (5 ?M). To evaluate the detection of endogenously elevated levels of H2O2, live HeLa cells were treated with epidermal growth factor (EGF, 50 ng/mL). EGF binds to epidermal growth factor receptor (EGFR), which activates the NOx/PI3K signalling pathways and stimulates the production of H2O2 endogenously. In the presence ofQCy-BA (5 ?M), FACS analysis showed an increase in fluorescence on the PerCP channel, and the presence of H2O2 scavenger N-acetyl-L-cysteine (NAC) cells resulted in decreased fluorescence in the PerCP channel (Fig. 19c). This confirms that probe QCy-BA is capable of detecting endogenous levels of H2O2 in live cells. Further, probe QCy-BA (5 ?M) detected endogenously elevated concentrations of H2O2by DNA damaging agents such as BrdU and doxorubicin in HeLa as well as primary MRC5 cells (human primary lung fibroblasts) (Fig. 19b). Further, glucose oxidase assay confirmed the use of the combination probe QCy-BA?DNA for probing H2O2 generated in situ by the oxidation of glucose to gluconic acid. In the presence of catalase enzyme, which converts H2O2 to water and oxygen, QCy-BA?Drew-AT showed reduced fluorescence emission at 650 nm, and hence, the combination probe is a promising tool for monitoring in situ turnover of H2O2. Interestingly, QCy-BA showed 10-fold higher fluorescence in cells compared to the commercially available ROS probe 2′,7′-dichlorofluorescein diacetate (DCFDA), which generally exhibits 3 to 4-fold fluorescence response. Confocal fluorescence images of QCy-BA (5 ?M) in the cancer cell-line HeLa and primary MRC5 cells showed promising red fluorescence enhancement in cell nucleus in the presence of H2O2 (100 ?M), whereas cells incubated with QCy-BA alone did not show any fluorescence in the red channel (Fig. 19d). This suggests that probe QCy-BA is a superior probe for detection of exogenous and endogenously elevated levels of H2O2 in normal and primary cells. These versatile properties make our probe QCy-BA a potential NIR-fluorescence probe for the detection of H2O2 flux in the cell and open new avenues to ascertain a relationship between the oxidative stress in cancer and aging-related diseases.Therefore, stimuli-responsive fluorescence combination probe (QCy-BA) with signalling assisted by an additional recognition event (DNA binding) is highly beneficial for probing specific biological molecules, events or activities in a cellular environment and has the potential to be used in numerous diagnostic and therapeutic applications. Thus, our fluorescence combination probe provides an additional advantage to detect the generation, accumulation, and transportation of H2O2 in cells. Also, unwanted background fluorescence from the other parts of cellular organisms is avoided and improves selectivity towards the stimuli of interest.To the best of our knowledge, combination probe QCy-BA?DNA is the first of its kind used for the detection of normal and elevated levels of H2O2 in live cells and in primary cells with NIR-fluorescence. We believe the design concept of this combination probe would inspire the future development range of effective far-red fluorescence probes for the detection, quantification and detailed analysis of several biomolecules for in cellulo and in vivo imaging, diagnostic and theranostic applications.
Simultaneous detection of different RNA targets can provide a bigger picture and deeper insight into their subcellular dynamics. However, imaging endogenous RNA accurately in live cells in real time is not an easy task. Some of the powerful methods for imaging specific mRNAs enlist the use of fluorescent proteins, labelled antisense probes, aptamers and recently reported bioorthogonal conjugation that enable real-time mRNA visualisation.229-231 However, it requires careful optimisation of absorption and emission profiles to avoid cross-excitation among different probes and simultaneous detection of various targets.
There have been reports describing the techniques involved in imaging RNA,106, 229 and herein we chose to describe few labelled antisense probes and bioorthogonal conjugation strategy with far-red fluorescence labels to achieve high signal-to-background ratio for RNA imaging.230, 232-233 Seitz and co-workers exploited, for the first time, single dye forced intercalation (FIT) probes, emitting in the red region (?590 nm), to address this challenge (Fig. 20a).232 This technique involved hybridisation of a DNA FIT-probe containing quinoline blue (QB) with a complementary RNA target, resulting in the intercalation of the chromophore between the nucleotide base pair. Restricted rotation around the methine bridge leads to increased lifetimeof the excited state, leading to fluorescence enhancement. The red emission (?em = 610 nm and??ex = 560 nm, Fig. 20b) and enhanced fluorescence (190-fold) of QB upon hybridisation with complementary RNA enabled its use for multiplexing, in combination with the orthogonal BO (cyan emission) and TO (green emission) probes, without any cross-excitation. The use of locked NA (LNA) monomer on the backbone, adjacent to the dye nucleotide, enhanced its quantum yield (?F(DNA) = 0.25; ?F (LNA) = 0.46) as compared to when the DNA backbone was present. TO- and QB-FIT probes were used for dual colour imaging of polyadenylated and oskar mRNA in oocytes of Drosphila melanogaster by means of wash-free fluorescent in situ hybridisation (FISH) and super resolution microscopy (STED).
In a similar study, Yavin et al. reported red emitting quinoline blue (BisQ) as an FIT probe attached to peptide NA (PNA) to detect single-point mutation of mRNA in malignant cell lines using KRAS as the model gene (Fig. 16c).233 One of the probes PNA1 ((D-Lys)4-CCTCGABisQTACCGCATCC-NH2) had the dye located opposite to Uracil/Thymine, and the other probe PNA2 ((D-Lys)4-CCTCGACTBisQTACCGCATCC-NH2) had the dye positioned opposite to Guanine. Both probes were designed to be complementary to mRNA of the Panc1 cancer cells with a single mismatch adjacent to the dye. The KRAS oncogene has frequent single-base mutations associated with tumorigenesis. To detect mRNA from KRAS in living cells, the probes PNA1 and PNA2 were incubated with three cancer cell lines (Panc1, BxPC-3, and HT-29); among these, only Panc1 contained the mutated form of mRNA (G to A point mutation) in the KRAS gene. Red fluorescence signal was observed solely for the hybridisation of mRNA of Panc1 with PNA1, while the other two cell lines produced a negligible response. However, in spite of the promising fluorescence response of PNA2 with DNA and RNA in test tubes, it gave a poor signal with live cells. The unique property of PNA1 for selective imaging of the live cells with a point mutation in KRAS makes it a promising approach for detection of cancer and other disease conditions.
Bioorthogonal conjugation is a robust strategy to accomplish requisite sensitivity and selectivity for NA detection and imaging in live cells.234 For instance, antisense probes are designed such that the reactive groups are brought into proximity upon hybridisation with RNA template for fluorogenic reaction, which produces a detectable fluorescence signal. Neal K. Devaraj and coworkers used tetrazine ligation-based bioorthogonal conjugation for the detection of mRNA in live cells (Fig. 21).230 NIR emitting quinone cyanine dye functionalised with phenyl vinyl ether group was used as a dienophile for tetrazine ligation reaction, which cages the phenoxide moiety, and fluorescence of the dye was quenched through internal charge transfer (ICT) (Fig. 21a).228 In particular, RNA binding sequence with vinyl ether (rBP-VE-Cy) and tetrazine group (rBP-Tz) were subjected to tetrazine ligation and turn-on fluorescence in the presence of in vitro transcribed mRNA transcript sfGFP-3′ BT (Fig. 21b), whichencodes for fluorescence protein GFP bearing two probe RNA recognition sequences within the 3′-UTR region. Utility of RNA template-assisted tetrazine ligation reaction and in situ generation of the probewas tested in CHO cells transfected with sfGFP-3′ BT and appropriately functionalised oligonucleotides rBP-VE-Cy (25 nM) and rBP-Tz (25 nM). As expected,significant fluorescence emission was observed with predominant localisation of the in situ generated probe in the cytoplasm (Fig. 21c). A similar bioorthogonal conjugation strategy that employs red fluorescence trimethine cyanine probe and sequence-specific short PNAs has been attempted for the detection of parallel GQ DNA.234 In general, development of sensitive and specific far-red fluorogenic template-assisted and bioorthogonal reaction-derived probes offer effective methods to detect and monitor RNA/DNA in cells and tissues, provided limitations of in cellulo and in vivo probe delivery are addressed.
Summary and Future Perspectives
The selected examples of far-red fluorescence probes discussed in the review article illustrate the incredible growth of fluorescence turn-on probes for canonical and non-canonical nucleic acid (NA) structures in the recent years. In view of rapid development in the area of ultrasensitive imaging techniques, it is essential to note that brightness, lifetime, ability to avoid auto-fluorescence from the surrounding environment and photostability of the far-red fluorescence probe would enhance the limit of spatial and temporal resolution, a sought-after feature for single molecule imaging-based techniques and applications. Therefore, it is imperative to appreciate the bright future of far-red fluorescence probes owing to their design, bioimaging properties, diagnostic and theranostic applications. The structure and function of canonical and non-canonical DNA and RNA directly or indirectly play a fundamental role in almost all the biological processes. In this context, structure or conformation-selective far-red fluorescence probes become indispensable molecular tools to understand the dynamics, localisation, transport and expression of canonical and non-canonical DNA and RNA in real time. In particular, various non-canonical NA structures (hairpin loops, internal loops, junctions, triplexes, G-quadruplexes and higher-ordered structures, homoduplexes like A-motif and poly-(GA)n duplex, three-way and four-way branches) need special attention in order to understand structure-function and biological implications. This is indeed a largely unexplored area waiting to be conquered by designing very selective, sensitive and biologically benign probes. In particular, designing highly target-specific fluorescent probes can create a revolution in addressing the drawbacks of immunofluorescence technology as it has concerns of imaging only in fixed cells due to impermeability of the antibodies used.
The intracellular dynamics of mRNA synthesis and localisation can be analysed at higher temporal resolution using small molecule-based selective fluorescence probes. In addition to bioimaging and structure-function study, these molecular probes can be utilised in conventional diagnostic and theranostic studies under several situations, through physical encapsulation or chemical conjugation. In addition, NIR probes find application in photodynamic therapy for selective destruction of cancer cells in tumour tissues.235 Moreover, far-red turn-on probes of NAs, which are specific to cellular organelles with high resolution, would substantially contribute to the advancements in the field of life sciences and medicine.19 The selective targeting of NAs (DNA/RNA/G-quadruplex and other non-canonical structures) in particular organelles like nucleus and mitochondria using highly conformation-specific and stimuli-responsive turn-on probes is an area that still needs to be explored. Contribution in this direction aids in understanding the interdependence of organelles and their cellular dynamics. For instance, mitochondria are the only organelle within the cell with extra-chromosomal DNA and are under the control of both nuclear and mitochondrial genome. Currently, mitochondria is a favourite topic of research, with recent discoveries of mitochondrial DNA (mtDNA) mutations and reactive oxygen/nitrogen species (ROS/RNS) leading to mitochondrial dysfunction that contributes massively to disease conditions such as cancer, hypertension,236 age-related disorders and neurodegenerative diseases including Alzheimer’s, Parkinson and Amyotrophic lateral sclerosis (ALS).237-239 Thus, developing selective far-red fluorescence probes for the detection of DNA mutations and damage specific to mitochondria240 might provide greater insights into the role of mtDNA in various disease conditions. This enhances our knowledge on disease pathogenesis and aids the development of effective diagnostic and theranostics probes. (Fig. 22).
It is essential to design conformation-specific probes to understand the intervention of quadruplex structural dynamics in a cellular process. Research findings over the last decade and ongoing research efforts reveal the ubiquitous presence of DNA and RNA G-quadruplexes and their possible involvement in the modulation of innumerable biological processes.129 Therefore, employing far-red fluorescence probes is an ideal strategy for studying structural dynamics of DNA and RNA G-quadruplexes, for their structure-function relation and role in various cellular processes. For example, a recentsurveyshows that the DNA sequence of the insulin-linked polymorphic region (ILPR) contains G-rich repeats (5?-ACAG4TGTG4-3?), which form non-canonical G-quadruplex structures and can easily be targeted to bridge the gap of disease mechanism and therapeuticsapproach.241-242 However, many reported probes for DNA G-quadruplex and RNA G-quadruplex detection are not selective or conformation-specific, and only a few of them have been used for cellular visualisation. These limitations need to be considered while designing new molecular probes. One must be cautious when a new probe is developed for DNA or RNA G-quadruplexes and greater care and attention must be devoted in determining whether the signal is coming solely from the specific DNA/RNA G-quadruplex or from other non-canonical and higher order DNA or RNA conformations or their complexes with biomolecules such as proteins. Such careful studies and observations not only help in designing conformation-specific turn-on probes but may also lead to the serendipitous discovery of selective probes for other non-canonical NA structures or their interactions with proteins, an area untouched so far. Developing probes for non-canonical DNA/RNA structures (not considered for developing probes so far) such as triplexes, i-motifs, homoduplexes (A-motif and poly-(GA)n duplex), hairpin loops, internal loops, junctions, three-way and four-way branches and higher-ordered structures is a challenging task and needs to be addressed with greater prominence.

x

Hi!
I'm Joan!

Would you like to get a custom essay? How about receiving a customized one?

Check it out