Eun-Kyung
Lim‡
ab,
Kyeonghye
Guk‡
ac,
Hyeran
Kim
b,
Bong-Hyun
Chung
*abc and
Juyeon
Jung
*abc
aBioNanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology, 305-806, Daejeon, Republic of Korea. E-mail: chungbh@kribb.re.kr; jjung@kribb.re.kr; Fax: +82-42-860-4209; Tel: +82-42-860-4192
bBioNano Health Guard Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 305-806, Daejeon, Republic of Korea
cNanobiotechnology Major, School of Engineering, University of Science and Technology (UST), 125 Gwahak-ro, Yuseong-gu, Daejeon, 305-806, Republic of Korea
First published on 28th October 2015
A peptide-based molecular beacon (PEP-MB) was prepared for the simple, rapid, and specific detection of H1N1 viruses using a fluorescence resonance energy transfer (FRET) system. The PEP-MB exhibited minimal fluorescence in its “closed” hairpin structure. However, in the presence of H1N1 viruses, the specific recognition of the hemagglutinin (HA) protein of H1 strains by the PEP-MB causes the beacon to assume an “open” structure that emits strong fluorescence. The PEP-MB could detect H1N1 viruses within 15 min or even 5 min and can exhibit strong fluorescence even at low viral concentrations, with a detection limit of 4 copies.
In this study, we have developed a peptide-based molecular beacon (PEP-MB) that acts as a smart probe for the rapid, simple and sensitive detection of H1N1 viruses. The PEP-MB included two complementary oligonucleotides in the stem region and a specific peptide sequence in the central loop region that targets a highly conserved region in the HA protein of H1 strains (HA1).24,27–33 The PEP-MB alone formed a hairpin structure that exhibited fluorescence quenching; however, the fluorescence of beacon molecules increased after these molecules specifically recognized the HA1 protein on the H1N1 viruses (Fig. 1).
Fig. 1 Schematic illustration of a peptide-based molecular beacon (PEP-MB) for the simple and rapid detection of influenza A subtype H1N1 viruses (H1N1 viruses). |
The PEP-MB was prepared for specific H1N1 virus detection using a fluorescence resonance energy transfer (FRET) system in which a peptide that enabled the specific recognition of the HA1 protein was chemically conjugated with two oligonucleotides, an organic fluorophore (Cy3)-modified oligonucleotide (Oligo-Cy3) and a black hole quencher 2 (BHQ2)-modified oligonucleotide (Oligo-BHQ2); one oligonucleotide was placed on each side of the peptide, using sulfo-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) as a cross-linker (Fig. S1 and ESI†). The amine groups of the peptide were activated by maleimide groups; subsequently, the thiol groups of the oligonucleotides were conjugated with these amine groups.34,35
In particular, because the two oligonucleotides of the PEP-MB consist of complementary sequences, the PEP-MB formed a hairpin structure due to the base pairing of these oligonucleotides. The distance between the fluorophore donor (Cy3, maximal emission at 570 nm) and the fluorophore acceptor (BHQ2, absorption from 560 to 670 nm) was thus extremely small. The interaction between these oligonucleotides, which involved the excited Oligo-Cy3 fluorophore transferring its excitation energy to the BHQ2 group of Oligo-BHQ2, resulted in the strong quenching of fluorescence after annealing relative to before annealing (Fig. S2, ESI†).36–47
In addition, the peptide sequence used in this study was known to have a high affinity for the HA proteins of the H1 and H3 strains. However, the PEP-MB, which was created by modifying both ends of this peptide with oligomers, was found to have higher affinity for H1N1 viruses (with a dissociation constant (Kd) of 4.9 nM) than for other strains (with Kd values of 326 nM, 289 nM, and 159 nM for the H3N2, H5N2, and H6N5 viruses, respectively) (Fig. 2a).24,48
PEP-MB molecules were annealed to form hairpin loop structures and thereby reduce non-specific fluorescence signals. The quenching efficiency of PEP-MB was determined by annealing a fixed concentration of Oligo-Cy3 with increasing concentrations of Oligo-BHQ2. Fluorescence intensities were little different overall before PEP-MB annealing. However, their intensities were slightly increased over 4.0 nmol of Oligo-BHQ2 despite not annealing due to a high concentration of the quencher (Fig. 2b(i)). The comparisons of the fluorescence intensities before and after PEP-MB annealing (ΔF = Fbefore − Fafter) revealed that the maximal quenching efficiency was observed with 4 nmol of Oligo-BHQ2 (Fig. 2b(ii)). This value was chosen as the optimal condition for use in further experiments examining the specific detection of influenza A subtype H1N1 viruses (H1N1 viruses) by the PEP-MB.
The PEP-MB was designed for the rapid and simple detection of the H1N1 virus; thus, minimal PEP-MB fluorescence emission was observed with the beacon in the “closed” hairpin structure in the absence of H1N1 viruses. However, strong fluorescence was emitted by beacons in the “open” structure, which was adopted in the presence of H1N1 viruses, because the distance between the fluorophore donor Cy3 and the quencher BHQ2 increased due to a spontaneous conformational change of the peptide in the PEP-MB. Based on this phenomenon, we evaluated the specific detection ability of the PEP-MB for H1N1 viruses relative to other viral subtypes (specifically, H3N2, H5N2 and H6N5 viruses). A control molecular beacon (Control MB) was designed with a 17-mer random peptide sequence that did not correspond to the target protein and with Oligo-Cy3 (donor) and Oligo-BHQ2 (acceptor) sequences identical to the sequences in the PEP-MB.30
The detection efficiency was measured by examining the fluorescence intensities before and after virus treatment. All viruses were obtained from the BioNano Health Guard Research Center (H-GUARD). The fluorescence of the PEP-MB was largely quenched before virus treatment (non-treatment (NT)); however, this fluorescence changed after 30 min of virus treatment (with relative fluorescence intensities calculated as ΔF/FNT (in %), where ΔF = F − FNT) (Fig. 3a). After the beacon was exposed to the H1N1 virus, the fluorescence intensity was markedly increased by approximately 23-fold relative to the NT fluorescence (H1N1: 2275.9%) due to the binding of the PEP-MB to the target HA1 protein. In comparison, the PEP-MB fluorescence intensity was only slightly enhanced by incubation with other viruses (H3N2: 379.3%; H5N2: 17.2%; and H6N5: 220.7%) (Fig. 3b). The specific binding of the PEP-MB to the target viral HA1 protein induced a spontaneous conformational change in the beacon that promoted the opening of the hairpin structure and increased the distance between the Cy3 and BHQ2 groups. The control MB exhibited negligible fluorescence intensity enhancements in response to all tested viruses (Fig. 3b).
We also examined the detection ability and sensitivity of the PEP-MB (35 pmol) with respect to the incubation time. Fluorescence was detected every 5 min for 15 min of incubation with viruses (103 copies per well). Minimal fluorescence was observed in the absence of viruses (the NT condition) (with relative fluorescence intensities ΔF/FNT (in %), where ΔF = F − FNT). As expected, stronger fluorescence was detected with H1N1 viruses than with other viruses (Fig. 4a). Within 5 min, H1N1 treatment produced a fluorescence intensity 5.7-fold higher than the NT intensity (571.9%). The fluorescence intensity gradually increased as the incubation time increased (with an increase of 97.81% at 10 min relative to 5 min). The PEP-MB was clearly able to detect the H1N1 virus after only 15 min of incubation, which produced a fluorescence intensity 16-fold higher than the NT intensity (1630%) (Fig. S3a, ESI†). No notable changes in PEP-MB fluorescence were observed after treatment with the other examined viruses (H3N2, H5N2 and H6N5 viruses), confirming that PEP-MB could be used for the rapid and specific detection of H1N1 viruses.
The sensitivity of PEP-MB was then investigated by measuring the fluorescence intensities obtained with different concentrations of H1N1 viruses (0, 10, 102 and 103 copies per well) and different incubation times (5, 10 and 15 min) at room temperature (Fig. 4b and ESI†). PEP-MB fluorescence was detected every 5 min after the addition of the aforementioned concentrations of the H1N1 virus. As indicated in Fig. 4b, the fluorescence intensity was markedly increased after the addition of the virus. After short incubations (5 or 10 min), the PEP-MB could be used to rapidly detect H1N1 viruses (at a concentration of 103 copies per well) after short incubations of 5 and 10 min, which produced high relative fluorescence intensities of 532.4% and 885.7%, respectively, compared with the NT intensity. Even at low H1N1 concentration (101 copies per well), the fluorescence intensities after treatment remained higher than the NT fluorescence intensities (by 111.8% at 5 min, 385.7% at 10 min and 200.0% at 15 min) (Fig. S3b, ESI†). These differences in fluorescence intensity are sufficient to effectively detect the target (H1N1 viruses). Based on these results, we confirmed that the PEP-MB could be used for the rapid, straightforward, and highly sensitive detection of H1N1 viruses.
In conclusion, we have described a simple, rapid and highly sensitive detection method for H1N1 viruses using a PEP-MB, a peptide-based molecular beacon with a peptide that specifically interacted with the HA1 protein. The PEP-MB could be used to directly detect H1N1 viruses by producing intense fluorescence; thus, this approach constituted an alternative to genetic techniques, such as PCR assays. This was also capable of detecting low concentrations (above 4 copies) of H1N1 viruses within just 15 min. Based on these results, it is likely that the PEP-MB has great potential for use in multiplex assays of influenza viruses in which several fluorophores and peptide sequences allow for the detection of interactions with specific HA proteins.
This study was supported by a National Research Foundation of Korea (NRF) grant (NRF-2014M3A6B2060507, NRF-2013M3C1A3064462), which was funded by the Ministry of Education, Science and Technology, and by the KRIBB Research Initiative Program of the Republic of Korea.
Footnotes |
† Electronic supplementary information (ESI) available: Experimental details; preparation and characterization of PEP-MB, viruses, measurements of PEP-MB binding affinity on viruses. See DOI: 10.1039/c5cc05684e |
‡ The authors contributed equally to this work. |
This journal is © The Royal Society of Chemistry 2016 |