Smart probes (SPs) are singly-labelled fluorescent probes used for the sequence-specific recognition of nucleic acid targets [1
]. They possess stem-loop, hairpin conformation, with a fluorescent dye on one end of the oligonucleotide sequence and guanine bases on the other. The guanine bases act as quenchers for the fluorophore when in close proximity. Due to its low oxidation potential, guanine effects fluorescence quenching via photoinduced intramolecular electron transfer when in contact with, or in close proximity to, the fluorophore [2
]. The loop sequence of a SP is designed to be complementary to the sequence of the nucleic acid of interest, while the stem consists of self-complementary strands of about six base pairs. In the absence of the target nucleic acid, the SP exists as a hairpin, whereby the fluorescent label and the guanine quenchers are close to one another and the fluorescence signal is effectively quenched. However, when the perfectly complementary target is present, the SP spontaneously hybridizes with this target, thereby separating the fluorophore and the quencher, and thus, turning on fluorescence signal. If a nucleic acid sequence has a single-base mutation in it, the SP can differentiate between this mismatch and the target sequence by providing a relatively lower fluorescence signal compared to that of the perfect target, which indicates a less stable hybrid between the SP and the mismatch sequence. This exquisite signaling property makes SPs effective analytical tools in various applications, such as monitoring polymerase chain reactions (PCR), DNA polymerase fidelity, T4 polynucleotide kinase activity, adenosine triphosphate (ATP) detection, detection of UV-induced nucleic acid photodamage, genetic testing, and biomedical diagnostics [8
]. SPs are structurally similar to molecular beacons (MBs), except that guanine bases are used in SPs to replace an extrinsic quencher label used in MBs. Due to certain limitations inherent to the use of MBs, it is more advantageous to use SPs [14
]. In any event, both SPs and MBs may be used for similar applications.
MicroRNAs (miRNA) are short, non-coding ribonucleic acid strands that regulate several processes in biology [16
]. They contribute to the initiation and progression of various diseases, such as viral infections, neurological diseases, and cancer, and miRNA levels have been shown to correlate well with such diseased states [17
]. As a result, miRNAs constitute an important class of therapeutic and diagnostic (theranostic) biomarkers [18
]. Specifically, the let-7 family of miRNA has been implicated in breast and lung cancers, and their levels may be used as a diagnostic tool for detection [18
]. Given this fact, accurate detection of members of the let-7a,b,c homologous miRNA family is crucial to the early detection and prognosis of these types of cancer. Several methods have been used in the detection of miRNAs. These methods include PCR-based methods, in situ hybridization, microarray methods, and northern blotting [18
]. Other methods include electrochemical, colorimetric, spectroscopic, and nucleic acid amplification techniques [28
]. Although they are selective, PCR-based techniques require a purified RNA sample and can be time- and labor-intensive [36
]. Northern blotting presents low sensitivity, requires a large amount of sample, and is cumbersome [18
]. Microarray methods have the advantage of high throughput, but they have low sensitivity and specificity [18
]. The electrochemical, colorimetric, and spectroscopic techniques reported so far for miRNA detection are complicated, with procedures that are rather involved, thereby making them less attractive for routine miRNA detection [27
]. Nucleic acid amplification methods are sensitive, but they come with complex reaction or hybridization mixtures, and unintended mismatch nucleic acids may also be amplified due to non-specific hybridization, therefore presenting high levels of background signals [32
]. Thus, mismatch nucleic acid sequences may present a challenge, even with nucleic acid amplification methods that are very sensitive. In addition to all of these limitations, all the methods reported so far for miRNA detection involve a single probe for a single target miRNA sequence; none of the methods so far reported has used a single probe for multiple miRNA sequences.
It may be desirable to use a single probe for the recognition of several homologous, mixed-base target sequences. This is relevant in situations where multiple targets are to be simultaneously detected, especially where several members of the same homologous family code for the same type of cancer. For example, let-7a, -7b, and -7c have been implicated in the same type of cancer [21
]. Non-specific interactions between the probe and unintended (mismatch) sequences can in fact be exploited for good use in miRNA detection. Thus, a probe can interact with multiple sequences via specific and non-specific hybridizations, making the probe universal for detecting multiple sequences. Such a universal probe system is desirable and would be a good addition to the repertoire of tools available to scientists for cancer biomarker detection. Furthermore, when only one member of the homologous family is to be detected to the exclusion of the other members [22
], the universal probe system can be conditioned such that the same probe will sequence-specifically recognize the target of interest, to the exclusion of other, unintended homologous members.
Homogeneous methods that based on SPs are suitable for miRNA detection [38
]. They are simple and fast, and present good sensitivity and specificity. In the presence of single-base mutations, non-specific interaction between mismatch sequences and the SP may be significant, especially if mixed-base sequences are involved [40
]. The specificity of SPs in the face of non-specific interactions and sequence homology can be significantly improved by making use of nucleic acid blockers (NABs) [40
]. These are unlabeled nucleic acids that are complementary and specific to the mismatch sequence of interest. They prevent non-specific interactions between the probe and unintended nucleic acid (mismatch) sequences by specifically hybridizing with such mismatch sequences, thereby isolating the SP to hybridize exclusively with the target sequence of interest. Oligonucleotide blockers have been previously used in the detection of single nucleotide mutation [41
], and our research group recently reported a simple protocol involving the use of hairpin-shaped NABs for mixed-base miRNA detection [40
]. Thus, unlike other methods that have so far been used for miRNA detection, homogeneous methods based on the SP/NABs system can offer good sensitivity, while also presenting excellent discrimination between the miRNA target of interest and similar mismatch sequences, including single nucleotide polymorphism (SNP).
In the work being presented here, we designed and characterized a universal SP detection system for the recognition of let-7a, -7b, and -7c, which are homologous members of the same miRNA family, and are biomarkers for breast and lung cancers. The SP was designed to be strictly complementary to let-7a, and therefore, would hybridize with this target sequence-specifically. However, due to the sequence similarity between let-7a, let-7b, and let-7c, non-specific interaction between the SP and let-7b and let-7c is also significant. Therefore, the SP is expected to hybridize with all three sequences, i.e., let-7a,b,c, with essentially equal propensity. This single SP thus offers a universal detection for all three homologous sequences. In addition, for sequence-specific recognition of let-7a alone, without interference from homologous let-7b and let-7c, we introduced linear nucleic acid blockers (LNABs) to the medium to screen out possible interference due to non-specific interactions between the SP and let-7b and let-7c sequences. LNABs are non-fluorescent, linear oligonucleotides that are perfectly complementary to unintended mismatch sequences, and thereby, specifically hybridize with such sequences. The SP/LNABs system thus constitutes a universal detection system that is capable of simultaneous recognition of all three Let-7a, -7b, and -7c homologous members; furthermore, in the presence of the appropriate LNABs, the system is also capable of discriminating between the three sequences. To our knowledge, this is the first report on the use of a single SP system for the universal detection of several homologous miRNA sequences, which also provides exquisite discrimination amongst the sequences when required. This detection system is simple and fast, it does not involve a complicated hybridization procedure, it operates at room temperature, and no washing steps are required. Furthermore, this universal detection system presents good sensitivity and sequence-specific discrimination in vitro that is better than previously-reported miRNA detection methods. We wish to state that this work involves the use of linear NABs (LNABs), whose performance and blocking characteristics may be quite different from hairpin-shaped NABs [40
We have designed and characterized a SP for in vitro detection of let-7a, let-7b, and let-7c homologous miRNAs, which are breast and lung cancer biomarkers. The fluorescence signal of the SP switches on in the presence of any of the three homologous sequences, showing that this SP represents a universal probe for the simultaneous detection of the homologous miRNA sequences, either individually or as a mixture. When only one intended target (let-7a) was to be detected, LNABs were used to screen out unintended homologous sequences, i.e., let-7b and let-7c. So, the SP/LNABS system is also capable of providing sufficient discrimination between let-7a and the other homologous sequences of let-7b and let-7c. We found no evidence of non-specific LNABs/L7a interaction, which we ascribe to the relatively low LNABs:SP molar ratio of 6:1 used in this work. The universal SP system provides a good detection limit and sensitivity for a let-7a target. The SP used here can also be used for the recognition of various let-7 miRNA hairpin precursors, in which case the SP interacts with the stem regions of such miRNAs. The approach presented in this work can be adapted for any other homologous miRNA family. This new detection system is sensitive, selective, simple, fast, usable at room temperature, and does not involve any washing or isolation steps. It provides relative fluorescence signal levels that rival nucleic acid amplification methods, and it may be a suitable mix-and-read homogeneous platform for in vitro detection of various homologous miRNAs.