MSSRCA Powered Sensitive Point-of-Care Detection of HPV mRNA for Cervical Cancer Screening

doi:10.21203/rs.3.rs-2729544/v1

Download PDF

Article

MSSRCA Powered Sensitive Point-of-Care Detection of HPV mRNA for Cervical Cancer Screening

https://doi.org/10.21203/rs.3.rs-2729544/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Developing sensitive point-of-care testing (POCT) of oncogenic nucleic acids from HPV infection is essential in preventing cervical cancer, especially in resource-limited settings. Rolling circle amplification (RCA) is attractive in achieving POCT; however, still exists a bottleneck requiring multiple amplification steps with multiple detection probes due to the limited sensitivity. Here we developed a minimum secondary structured RCA technique (MSS-RCA) by designing a unique circular template, demonstrating significantly enhanced detection sensitivity with only one amplification step and one primer. We demonstrated that RCA amplification efficiency could be kinetically manipulated by controlling the secondary structure of the circular template. Introducing the invertase probe to MSS-RCA, Human Papillomavirus 16 (HPV16) E6/E7 nucleic acid target was detected with a personal glucose meter (PGM) with a sensitivity of 5 fM (50 zmol in 10 µL). This integrated MSS-RCA-PGM detection system was successfully applied to detect HPV16 E6/E7 mRNA extracted from 54 cervical swab samples reaching a positive predictive value of 100.00% and negative predictive values of 96.00% (77.77% to 99.40%, 95% CI). MSS-RCA-PGM provides a sensitive POCT platform for the detection of biomarkers for screening of cervical cancer or other diseases.

Biological sciences/Biotechnology/Assay systems

Health sciences/Biomarkers/Diagnostic markers

The continuous infection of human papillomavirus (HPV) contributes to the development of cervical cancer, one of the leading causes of mortality in women ^1,2. To address this issue, developed countries have introduced HPV vaccination and screening programs, significantly reducing the occurrence of cervical cancer. However, Due to lack of available vaccine or the resources for large screening programs, 90% of cervical cancer associated mortality happens in low and middle-income countries. Therefore, accurate and affordable point of care testing (POCT) technologies for sensitive detection of oncogenic nucleic acids from HPV infection are critical essential to achieve health equality^3-5. Comparing HPV DNA, HPV mRNA (especially E6/E7 mRNA) in high risk types is evidenced to be a more specific nucleic acid biomarker for the identification of women at risk of developing cervical carcinoma^6-9. Current commercial methods for HPV mRNA testing for high-risk types mainly include in-situ hybridization HPV tests and nucleic acid amplification tests (NAAT)¹⁰. Often, the in-situ hybridization tests require complicated experiment steps and skilled technicians with a high-equipped laboratory, which is not suitable for POCT purpose ¹¹. On the other hand, although it utilizes Polymerase Chain Reaction (PCR) as the tool to amplify the signal of nucleic acids with high sensitivity and specificity, NAAT is subject to precise temperature control, specified instrument, and long assay time, limiting its capacity in POCT. More importantly, the inclusion of heat-sensitive molecules could impact on the accuracy^12,13. As a result, isothermal NAAT such as Recombinase Polymerase Amplification (RPA), RCA, Nucleic Acid Sequence-based Amplification (NASBA), Loop-mediated Isothermal Amplification (LAMP), Strand Displacement Amplification (SDA), clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins (CRISPR/Cas) systems, et al, have been developed to resolve those issues associated with PCR^14,15. Particularly, Orakan et al reported an electrochemical biosensor combined with an isothermal target recycling amplification to detect HPV mRNA, which could detect HPV mRNA with portable measuring equipment in low-resource areas. However, the sensitivity of the method is 2.6 pM, which is not sensitive enough in accurate risk identification of cervical cancer in POCT¹⁶.

Simple and sensitive isothermal techniques for HPV mRNA detection remain elusive despite their significance in cervical cancer identification. As one of the most important and widely applied isothermal NTTA, RCA has become an attractive tool for biosensing diagnostics due to its efficiency, simplicity, and versatility^13,17. The linear amplification mode of RCA is fundamental for every RCA-based technique, although its sensitivity needs to be further improved by integrating with other detection platforms. Hybridization of RCA amplicons with different functionalized probes, combining RCA reaction with more than one enzymatic amplification steps, or designing more than one oligonucleotide are popular strategies applied in RCA to improve amplification efficiency and detection sensitivity^17-20. However, despite the enhancement of sensitivity, the integration strategies introduce additional background signals, contributing to the introduction of nonspecific signals and prolonged measurement time.

Active research has been carried out on improving the amplification efficiency and detection sensitivity of RCA directly without coupling with other techniques, including the selection of more efficient DNA polymerase^21,22，the addition of single-stranded DNA (ssDNA) binding proteins binding non-specifically to ssDNA that improve the amplification efficiency of DNA templates²³, the inclusion of an anti-sense oligonucleotide probe to hybridize with RCA products with a reduced secondary structure and accelerated amplification speed^24,25, and the utilisation of a more efficient circular template with higher amplification efficiency selected in vitro without a generic rule²⁶. That being said, these techniques are complex and lack genericity that is ideal for POCT purpose. Often, the size, sequence context, secondary structure, and topology of the circular template have been reported to affect the amplification efficiency of RCA^24-28. Although the secondary structure has been reported to be critical for high amplification efficiency in RCA, limited research has investigated the standardisation of the strategical design of a circular template from the amplification mechanism aspect.

Recently, a DNA sequence with minimum secondary structure was designed to acheive sensitive imaging with high resolution in situ²⁹. Inspired by this, we designed a circular template with a minimum secondary structure for the first time to improve amplification efficiency and detection sensitivity of RCA, named minimum DNA secondary structured-rolling circle amplification (MSS-RCA). The amplification kinetic between MSS-RCA and conventional secondary structured (SS-RCA) was compared by staining reaction product with DNA binding dye (SYBR Gold and SYBR Green Ⅰ). In a different amplification kinetic pattern from SS-RCA, MSS-RCA demonstrated higher amplification efficiency than SS-RCA at a lower concentration of primer and consequently high sensitivity (Fig. 1a). To power the MSS-RCA with the capability in POCT, this study took a step further, and uniquely realised the MSS-RCA on magnetic beads (MBs) with electrochemical signal by preparing a single-stranded DNA (ssDNA) modified invertase as a catalytic signal transduction probe.³⁰ As shown in Fig. 1b, the invertase-based signal amplification could be supremely qualified to combine with the MSS-RCA technique, creating a robust glucose electrochemical signal readable by a personal glucose meter (PGM). In the presence of the target sequence, a set of probes that are complementary to the target sequence were immobilized onto the MBs, which then initiated MSS-RCA to generate long ss-DNA repeats for invertase probe immobilization. Subsequently, invertase probes on the RCA amplicons could subsequently catalyze the hydrolysis of sucrose to glucose, producing a detectable signal by a PGM. MSS-RCA-PGM detection system was applied to detect the synthetic E6/E7 cDNA in about 2 h with a sensitivity of 5 fM (50 zmol in 10 µL), and high specificity for distinguishing single nucleotide polymorphism. The reliability of the MSS-RCA-PGM system was further validated by detecting E6/E7 cDNA of 54 clinic cervical swab samples; high concordance was observed between our system and the RT-qPCR. This system makes full use of MSS-RCA and the invertase to increase sensitivity and to directly quantify nucleic acids with a portable PGM readout, therefore permitting an alternative approach towards low-cost (below $30) on-site identifying of high-risk HPV towards the occurrence of cervical cancer, especially in resource-limited settings. This MSS-RCA-PGM detection platform is simple, generic, and programmable, which is adaptable to detect various biomarkers beyond nucleic acids with attractive characteristics.

Verification of MSS-RCA and SS-RCA. All of the circular templates used in different RCA for comparison are chemically 5’-phosphorylated. The length of the circular template is 50 nt, containing 5 tandem repeats. Minimum secondary structured circular template 1 (C1) was successfully designed and applied in MSS-RCA; a randomly generated circular template 2 (C2) with the same length while a different secondary structure was used in SS-RCA. Both MSS-RCA and SS-RCA were performed with circularization and amplification steps. Firstly, the chemically 5’-phosphorylated circular templated was circularized by Circligase. After purification, RCA mixture containing Phi29 DNA polymerase, primer and dNTPs was added into the solution to trigger the reaction (Fig. 2a). The ligation products were verified by denaturing polyacrylamide gel electrophoresis (PAGE). The circular template was then purified from the PAGE gel. Fig. 2b displays the electrophoretic identification result on 15% PAGE gel. After running electrophoresis, the gel was stained in SYBR GreenⅠsolution. As shown in Fig. 2b, the ligation products moved slower than that unligated circular template, and the band appeared at around 50 nt according to Marker, which indicates that some linear circular templates were formed into a covalent circular form by CircLigase. It is interesting to note that in ligated products and exonuclease treated by C2, two larger bands appear at higher molecule weight positions in C2, while there was no larger ligation product in C1. This phenomenon could be explained by the fact that in C2, the dimer and multimer were formed easier than C1 due to more secondary structure in C2 template (Table S2). The circularized template around 60 nt was collected from PAGE gel and purified and quantified by nanodrop precisely. The agarose gel result showed that RCA reactions were successfully conducted for both MSS-RCA and SS-RCA. Lane 2 represents the MSS-RCA product from Primer 1 hybridized with a C1 and extended by Phi29 polymerase. In lane 4, Primer 2 was hybridized with C2 and triggered the SS-RCA reaction.

In order to verify the structure of RCA products, the MSS-RCA and SS-RCA products were stained by SYBR Gold and then immobilized on a poly-lysine coated glass cover slide for imaging (Fig. 2c). The left figure shows images taken for the MSS-RCA product and the right one shows TIRF image from an SS-RCA product after 1 h with RCA reaction. Linear geometry product (more than 10 μm, in a single state) can be found in MSS-RCA, while much irregular geometry DNA was found in SS-RCA product. There was different conformation such as triangle, twist, hollow ellipse, and star-like plectonemic superhelical geometry. This linear geometry was verified by a longer RCA reaction time (6 h) with more linear DNA product with the size of more than 10 μm (Fig. 2d, the left panel). The structure of MSS-RCA products by AFM (Fig. 2d, the right panel) shows wires in a linear geometry of length in micrometre-sized were observed. The morphology of the observed DNA products was consistent with our hypothesis (minimum secondary structured-DNA circular template can generate long linear-shaped DNA by RCA). The difference in DNA size between TIRF and AFM images might be due to the fragility of single-strand DNA under the AFM tip. Additionally, an interesting phenomenon was observed that several long linear DNA was branched (Fig. 2d, the right panel). This can be explained by the fact that linear structured RCA products produced by MSS-RCA exposed more freely nucleobases in solution. When the products were added to the solution containing Ni²⁺for AFM imaging, the natural nucleobases adenine and cytidine of different long linear ssDNA molecules generated by MSS-RCA were bonded to each other by means of a transition-metal-catalyzed cross-coupling reaction³¹. These images prove that MSS-RCA can generate long linear DNA with minimum secondary structure, while there were lots of secondary structures contained in SS-RCA amplicons.

Kinetic study of MSS-RCA and SS-RCA. In order to investigate the amplification kinetics of different RCA reactions, different concentrations of primers were used to trigger MSS-RCA and SS-RCA reactions with the same concentration of circular template (Fig. 3). Circular template C1 consists of 5 repeats of minimum secondary structured DNA sequence and each repeat refer to a part of a DNA sequence in literature²⁹. As shown in Fig. 3a, the shape of C1 was predicted to be linear structure without any secondary structure, and the free energy of secondary structure of C1 was calculated to be 0.00 kcal/mol by NUPACK (http://www.nupack.org/). C2 consists of 5 repeats and each repeat unit was generated randomly by Sequence Manipulation Suite in GenScript (https://www.genscript.com/sms2/random_dna.html). Free energy of the secondary structure of C2 was calculated to be -9.96 kcal/mol, and the structure was predicted to contain two regions of secondary structure in C2. In order to prove the universal performance of our hypothesis in MSS-RCA design, another circular template with minimum secondary structure (C1.1) whose sequence was totally different from C1 was designed and applied for RCA kinetic study. The structure of C1.1 was predicted to be linear, and the free energy of the secondary structure was calculated to be 0.00 kcal/mol. As a comparison, circular template C3 used for SS-RCA contains three fifths of the same sequence as C2 and two-fifths of that the same as C1. The Free energy of the secondary structure of C3 was calculated to be -4.98 kcal/mol, and the structure was predicted to contain one region of secondary structure. The amplification DNA products were stained with SYBR Gold for detection of synthetic DNA. SYBR Gold binds to DNA by insertion of one dye molecule between two planar bases/base pairs of DNA and emits a strong fluorescence signal³². Fig. 3b shows the time profile of MSS-RCA reactions using C1 as the circular template and SS-RCA using C2 as the circular template with different concentrations of primers. As we can see that when the concentration of primers was at 200 nM, the signal-to-background ratio (S/B ratio) of the SS-RCA sample increased rapidly and reached the plateau at around 10 min. This phenomenon might be due to a giant DNA molecule with a complicated secondary structure being formed nearby Phi29 polymerase, interacting strongly with the circular template, resulting in a wane of amplification. This assumption could be indirectly verified by the research about generating a long linear-shaped RCA product that could accelerate amplification efficiency³³. While in the MSS-RCA reaction, the S/B ratio was unchanged in the first 20 min, then increased when the primer concentration was at 200 nM. This phenomenon might be attributed to the unwinding tasks, which were overloaded for Phi29 DNA polymerase due to excess primers wrapped around one circular template, hindering amplification³⁴. Additionally, it is interesting to note that when the concentration of primer decreased, the amplification efficiency of MSS-RCA was almost unchanged, but fluorescence intensity for SS-RCA decreased quickly, and almost no amplification was observed within 60 min when primer concentration was decreased to 20 nM. SYBR Green Ⅰ, another cyanine dye that binds to dsDNA in minor grooves and the fluorescence intensity is at least 11-fold higher than ssDNA, was also used to investigate the amplification kinetics of MSS-RCA and SS-RCA reaction ³⁵. As shown in Fig. S2, the result also showed that the amplification efficiency of MSS-RCA was higher than SS-RCA when the concentration of primer is at a lower range.

The kinetic study result showed that circular template C1.1 for MSS-RCA has a similar kinetic characteristic as C1. The SS-RCA reaction with C3 as a circular template also showed the same trend as that of SS-RCA with C2 as a circular template, and the amplification efficiency was lower than MSS-RCA at lower concentration of primer (Fig. 3c). Based on the comparison of SS-RCA kinetic from both Fig. 3b and Fig. 3c, we can see that the amplification efficiency of C3-SS-RCA was higher than C2-SS-RCA. When the concentration of primer was 40 nM, the signal increased and reached the plateau after 5 min for C3-SS-RCA, indicating the RCA amplification reaction was successfully conducted, while there was no obvious increment of signal in C2-SS-RCA. The difference in amplification efficiency is owing to the less extent of secondary structure in C3 compared to C2. We also noticed that the signal and amplification efficiency from the C1.1-MSS-RCA was lower than that in C1-MSS-RCA, although the kinetic trend was similar. This phenomenon can be attributed to two reasons:1) the staining efficiency of SYBR Gold for DNA is strongly affected by the sequence context^36,37. We stained different circular templates with SYBR Gold and compared their fluorescence intensity to verify this assumption. We found that at the same concentration, different circular template stained with SYBR Gold has different fluorescence intensity. The fluorescence intensity from C1 circular template was higher than C1.1 at different concentrations (Fig. S1). 2, The number of nucleic acid base pairs hybridized between primer and circular template in C1.1-Primer 1.1 was more than that in C1-Primer 1 hybrid complex (Table S3). A longer dsDNA takes more energy of Phi29 to unwind, thus leading to lower amplification efficiency. Thus, when the concentration of primer 1.1 was at 40 nM, it took more time than C1 to begin the amplification.

A more comprehensive study of the relationship between primer and circular template concentration using SYBR Gold was conducted to compare the difference of amplification kinetic between MSS-RCA and SS-RCA. As shown in Fig. 3d, the S/B ratio raised linearly with time in MSS-RCA. It is worth noting that the amplification kinetics of different primer to circular template ratios are different, the S/B ratio was peaked when the ratio of primer to circular template at 2:1 when amplification time at 60 min. Higher ratio of primer to circular template more than 2:1 resulted in lower S/B ratio signal (Fig. 3d). The phenomenon implied that a relatively higher concentration of primer in solution facilitated more primer binding to one circular template, and thus increased the energy required for Phi29 DNA polymerase to unwind the double strand DNA and thus decrease the efficiency of strand displacement in RCA reaction. For SS-RCA reaction, the peak of signal to background ratio was found at 40:1 of primer to circular template ratio, which is higher than the ratio found in MSS-RCA; this phenomenon might attribute to the secondary structure in the primer of SS-RCA formed dimer or multimer thus reduce its binding efficiency to circular template. The amplification kinetic of SS-RCA was also different from MSS- RCA, which has a clear plateau when reaction time at around 10 to 20 min. Similarly, SS-RCA efficiency decreased when the ratio of primer to the circular template was higher than 40:1 (Fig. 3e). The reason behind this phenomenon was the same as that in MSS-RCA: too many primers were wrapped around the same circular template, so there is more dsDNA ahead of Phi29 DNA polymerase to unwind, leading to lower reaction efficiency of RCA.

Magnetic beads-based RCA detection of nucleic acids. The performance of MSS-RCA was evaluated by applying it to detect the specific synthetic DNA of MSS-RCA was evaluated by applying it to detect the specific synthetic DNA target HPV E6/E7 gene. A set of probes for conducting SS-RCA on MBs was synthesized for comparison (Fig. 4a). Firstly, a biotin-labelled probe and a phosphate-labelled primer were designed to target a specific sequence in the E6/E7 gene specific to HPV and then ligated by T4 DNA ligase. Primer 1 was designed to hybridize with circular template C1 to trigger MSS-RCA, and primer 2 was used for hybridization with circular template C2 to conduct SS-RCA on MBs. Streptavidin-coated MBs then captured the ligation products. Adding the RCA mixture into the MBs complex generated the RCA products, which were then hybridized and detected by TexRed labelled DNA probe. As shown in Fig. 4b, after hybridization of the TexRed DNA probe on RCA amplicons, the fluorescence signal from all the fraction parts was collected and measured. Fig. 4c and Fig. 4d show the distribution of fraction signals for MSS-RCA and SS-RCA, respectively. Fig. 4e shows the result for fluorescence detection in elution fraction for MSS-RCA and SS-RCA on MBs, and the response signal of MSS-RCA with C1 circular template was much higher (about 18-fold) than SS-RCA with C2 circular template. The results suggested that the TexRed labelled DNA probe linked to MBs via specifically binding to MSS-RCA products. While the result of the fraction distribution in SS-RCA shows that although SS-RCA can be triggered on MBs, the elution signal in MSS-RCA with C1 circular template was much higher than SS-RCA with C2 circular template. It further confirmed that the amplification efficiency of SS-RCA and the binding efficiency of the signal probe were much lower than that of MSS-RCA. Finally, we determined the linear range of MBs-based MSS-RCA reaction for the detection of the synthetic E6/E7 DNA (4-4096 pM). The detection limit is defined as 4 pM (Fig. 4f and Fig. 4g), which is 1000 times more sensitive than the non-amplification method in the same detection platform (Fig. 4e). From this result; we also can deduce that MSS-RCA can produce a linear DNA scaffold with tens of thousands of nucleotides, which is consistent with what researchers usually found the size of RCA product before^38,39.The sensitivity of MSS-RCA is comparable to other RCA-based nucleic acid detection methods which require multiple enzymatic reaction step or coupling with more than one primer for amplification (Table S5). To our knowledge, it is the first time to achieve such a high sensitivity for an RCA-based fluorescence assay without additional amplification step, suggesting that MSS-RCA demonstrated huge potential in achieving high sensitivity and high efficiency in biomarker detection.

Detection of HPV E6/E7 mRNA by MSS-RCA-PGM system. In order to evaluate the feasibility of MSS-RCA in gene detection, an invertase probe was introduced to MSS-RCA to achieve the MSS-RCA-PGM system for detecting HPV E6/E7 mRNA in clinic samples. A synthetic HPV 16 E6/E7 gene sequence was first used to examine the performance of this system. As depicted in Fig. 5a, the detection process of the E6/E7 gene target includes hybridization and ligation step, MBs capture, conducting MSS-RCA on MBs, hybridization with invertase probe (ITP), and detection by PGM. Fig. 5 b-f is the characterization of different components in the MSS-RCA-PGM system^40,41. The ligation product is one of the components of the MSS-RCA-PGM system. First, we designed a biotin probe and a phosphate probe that target the cDNA of E6/E7 mRNA (Table S1). The former consists of an upstream hybridization region, a spacer, and 5’ biotin modification. The latter comprises a 5’ phosphate modification, a downstream hybridization region, and the primer for MSS-RCA. The correct size of the ligation product (109 nt) was only obtained in the presence of the two probes and the target in the ligation mixture, indicating successful probe design as well as the ligation reaction (Fig. 5b). The invertase probe (ITP) is another component in the system. The ITP is the invertase-ssDNA conjugate using copper-free click chemistry. The crosslinker SM(PEG)₂ first reacts with the amine of the lysine residue on the invertase to form amide bonds. The invertase with the maleimide group then reacts with the reduced thiol group to generate stable linkage. We performed SDS-PAGE to characterize the DNA-invertase conjugates. We further characterize the purified ITP using Uv-vis absorbance spectra. The invertase and ssDNA alone displayed their absorption peak at 280 nM or 260 nM, whereas the invertase-ssDNA conjugates displayed an absorption peak in between, indicating a successful conjugation (Fig. 5c). To investigate the functionalization of the ITP further, we performed scanning electron microscopy (SEM) to visualize MBs-MSS-RCA-ITP complex. The image illustrates that the invertase probe hybridizes with MSS-RCA amplicons with the help of modified

DNA strands. In addition, to monitor the ITP activity, a commercially available personal PGM (Bayer, China) was obtained; though engineered for whole blood glucose measurement, the instrument showed stable quantification of glucose concentration in our sucrose conversion buffer (Fig. S5). We further optimized and compared the sucrose conversion efficiency between invertase and ITP (Fig. S4 and Fig. S6), and our results indicated that the conjugation of ssDNA to the amine group of the invertase did not show a significant effect on its catalytic activity. Therefore, we confirmed that the target DNA could be detected quantitatively by performing each component and presented by a PGM. To verify the synthesis of long DNA repeats and invertase hybridization on MBs, we analyzed the size of the products using dynamic light scattering (DLS). In a total of 90 minutes, the average size of the product increased gradually from 1205 nM to 1901 nM, indicating the continuous assembly of long DNA repeats on MBs (Fig. 5e). We compared the size difference between bare MBs, MBs+MSS-RCA, and MBs+MSS-RCA+ITP complex. The particle size of different stats increased from 1205 nM to 1989 nM, confirming the successful generation of the MBs-RCA-ITP complex (Fig. 5e). Fig. 5f showed the results of PGM readout from non-amplification and after MSS-RCA. The system yields much more glucose from sucrose after the MSS-RCA reaction, generation of binding sites for ITP hybridization allows high glucose production.

Optimization and examination of the MSS-RCA-PGM system. After establishing the components in the MSS-RCA-PGM system. We measured the PGM signal containing the 500 nM of both biotin and phosphate probes and synthesized cDNA from HPV16 E6/E7 mRNA in the concentration range of 100 aM to 1 nM to determine the sensitivity. Notably, the detection limit of the MSS-RCA-PGM system was as low as 5 fM in 10 µL (equivalent to 50 zmol of molecules per reaction), demonstrating its excellent sensitivity. In addition, the linearity of the PGM signal obtained across a broad range of concentrations (5 fM to 10 pM, R² =9929) implies that the MSS-RCA-PGM system can be utilized for quantitative target analysis (Fig. S7).

The shortest turnaround time for the two major steps (the MSS-RCA and the T4 ligation) required to validate the existence of 5 fM of the target in a sample was then determined (Fig. 6a, 6b). We first examined the time effect of the MSS-RCA on the
system and noticed that the PGM signal with 5 fM was discernible with the control group with an MSS-RCA time of 40 min. An MSS-RCA time of 20 min resulted in a lower background signal but also lower sensitivity. We further decrease the time for T4 ligation. Our result suggested that in a ligation reaction of 10 min, the PGM signal with 5 fM was distinguishable against the negative control (Fig. 6b). Additional reaction incubation made the target cDNA-containing reaction more distinguishable from the negative control reaction. Notably, the PGM signal rose marginally in the absence of the target cDNA. A possible source of this background signal might be the unspecific ligation of the biotin probe and the phosphate probe⁴². Collectively, the MSS-RCA-PGM system was able to detect the target cDNA in a ligation time of 10 min and an MSS-RCA time of 40 min. The corresponding calibration curve revealed a linear range of 5 fM to 10 pM (R² =9907), retaining its good linearity (Fig. 6c). In three repeated

experiments with 1 nM, 1 pM, and 5 fM E6/E7 cDNA, the relative standard deviations of PGM glucose signal measurements were 2.9%, 0.138%, and 0.17%, respectively. These findings suggested that the proposed method offered a platform for nucleic acid detection that was both sensitive and robust. In order to examine the applicability of MSS-RCA-PGM for discriminating the nucleic acid targets, the specificity of the system was tested with designed synthetic DNA sequences. The target E6/E7 cDNA, single-base mismatched DNA (S1), 10% mismatched DNA (S2), and 20% mismatched DNA (S3) were tested. As shown in Fig. 6d, in the presence of 100 pM of the target analytes, the S/B ratio of E6/E7 cDNA (1.79) was much higher than that from S1 (1.27), S2 (1.14) and S3 (1.00). In the presence of the target analyte at concentrations lower than 100 pM, the glucose signal was similar to that of the control. These results proved the excellent specificity of the proposed nucleic acid detection platform. We interpret the high specificity as follows: more ligation of the biotin and the phosphate probe with the perfectly matched target. Mismatched targets, though hybridized with the biotin probe or the phosphate, were not able to trigger MSS-RCA for ITP hybridization. The unligated phosphate probe will be removed from the MBs by the strand displacement activity of the Phi29 polymerase.

Performance of the MSS-RCA-PGM system for E6/E7 mRNA detection in clinical samples. Finally, the performance of the MSS-RCA-PGM (Fig. 7) was evaluated by the detection of HPV E6/E7 mRNA extracted in clinical samples. As depicted in Fig 7b, the pair of single-stranded biotin probes and single-stranded phosphate probes were designed to specifically hybridize the target at room temperature (Fig. 7b). Following lysis and gene extraction, the mRNA was firstly reverse transcribed to cDNA. RNase H was then applied to remove excess RNA molecules in the solution. Fifty-four anonymous clinical cervical swab samples were obtained from Zhongshan Hospital, and 29 of which were identified as HPV16 E6/E7 mRNA positive and 24 as HPV16 E6/E7 mRNA negative by RT-qPCR (Table S4). We analyzed the Receiver Operator Characteristics (ROC) of the data collected from clinical samples using the MSS-RCA-PGM system (Fig S8)^43,44. An area under the curve (AUC) of 0.9950 (CI, 0.8620 to 1.000), a sensitivity of 1 and a specificity of 0.9655 were achieved at a threshold of 1.85. Our result suggested that the MSS-RCA-PGM system reached the required sensitivity and specificity for cervical cancer screening, in high agreement with the results provided by the conventional RT-qPCR.

We successfully developed a sensitive and efficient MSS-RCA by designing a circular template with MSS to achieve maximum amplification efficiency at a low concentration of primers. It demonstrated that the secondary structure in the circular template is one of the most critical factors affecting the amplification kinetics of RCA. Being consistent with the AFM observation, TIRF images showed that linear geometry of RCA product could be found in MSS-RCA, while versatile shapes of DNA product were observed in conventional SS-RCA. The detection signal of MBs-based MSS-RCA was also much higher than SS-RCA for detection with a detection limit of 4 pM in 10 µL (40 amol molecules) on a microplate reader with TexRed as the signal probe. It is the first time to develop a simple, sensitive, and highly efficient linear RCA method to detect nucleic acids in one amplification step with one primer. To realize its potential in POCT, MSS-RCA, with the assistance of an invertase-labelled DNA probe was integrated with PGM to achieve MSS-RCA-PGM for sensitive detection of cDNA sequence from the HPV16 E6/E7 mRNA in clinical samples based on a personal PGM signal readout. With the simple sequence design of both the biotin probe and the phosphate probe for high-risk mRNA sequences, this platform has the potential to enable on-site, low-cost, and fast identifying the high risk of developing cervical cancer, especially for women in low and middle-income countries. The MSS-RCA-PGM detection platform provides a generic and cost-effective strategy for PGM-based isothermal POCT for the detection of nucleic acids and beyond. We anticipate improvements in turnaround time with optimization in sample pretreatment⁴⁵ and integration of microfluidic devices⁴⁶ with the goal of achieving an automatic and intelligent detection. Moreover, because of the simple incorporation of many receptors into the MSS-RCA-PGM system, the suggested technology has tremendous potential for providing patients with a single-device solution for quantitatively monitoring several biomarkers simultaneously on site in the future.

Reagents and Materials. The ssDNA/RNA CircLigase (D2602), Phi29 DNA polymerase (A3703) and the corresponding buffer were purchased from HaiGene Biotech Co., Ltd (Harbin, China). Water (Z18.2 MO) used throughout the experiments was generated by the Milli-Q water purification system. SYBR Gold (10000x in DMSO) and streptavidin-coated magnetic beads (65001) were purchased from Thermo Fisher Scientific Inc. (Shanghai, China). SYBR Green I (S171397) was purchased from Aladdin (Shanghai, China). The deoxynucleotide solution mixture (dNTPs) was ordered from Solarbio (Beijing, China). All oligonucleotides used in our research are synthesized and purified by HPLC at Shanghai Sangon Biological Engineering Technology & Services Co. Ltd. (Guangzhou, China) (Table S1). Other chemicals employed are of analytical reagent grade and are used as received. Streptavidin-modified magnetic beads (1 µm average diameter), T4 DNA ligase, and Protein Ladder (10 to 180 kDa) were purchased from Thermofisher Fisher scientific (Shanghai, China), and 10 mM deoxynucleotide solution mixture (dNTPs) were purchased from Solarbio (Beijing, China). Amicon Ultra-0.5 Centrifugal Filter Devices (Amicon Ultra 3 K device and 30 K devices) were purchased from Merck Millipore Ltd. (Billerica, MA). PEGylated SMCC crosslinker SM(PEG)₂ was purchased from TCI (Tokyo, Japan). Phi29 DNA polymerase was purchased from HaiGene Biotech (Harbin, China). Grade VII invertase from baker's yeast (S.cerevisiae) was purchased from Sigma-Aldrich (Shanghai, China). Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and Sucrose were purchased from Aladdin (Shanghai, China). SYBR™ Gold Nucleic Acid Gel Stain (10,000´ Concentrate in DMSO) was purchased from Thermo Fisher Scientific. All of the synthetic DNA sequences used in this study were obtained from Sangon Biotech (Shanghai, China). The sequences of the DNA are listed in Table 1. All reagents were purchased and used without further purification. Deionized water (18.2 MΩ) obtained from a Milli-Q water purification system was utilized in all the experiments. Room temperature (RT) denotes 22°C. Buffers used in this work: 1x PBS: 136.89 mM NaCl; 2.67 mM KCl; 8.1 mM Na₂HPO₄; 1.76 mM KH₂PO₄, pH 7.4. 1x PBST: 136.89 mM NaCl; 2.67 mM KCl; 8.1 mM Na₂HPO₄; 1.76 mM KH₂PO₄, pH 7.4, 0.05% Tween-20. Washing buffer: 10mM Tris-HCl, 5mM EDTA, 0.1M NaCl, 0.1% tween 20, pH 7.5. Invertase conversion buffer: 100 mM Sodium Acetate, pH 5.5. Diffusion buffer: 0.5 M ammonium acetate; 10 mM magnesium acetate; 1 mM EDTA, pH 8.0; 0.1% SDS.

Apparatus. DNA and protein concentration was measured by Thermo Scientific™ NanoDrop™ One Microvolume UV-Vis Spectrophotometer. The glucose signal was recorded on a Bayer contour TS personal glucose meter (Shanghai, China). Gel electrophoresis was performed using a Bio-Rad Electrophoresis system. SEM images were obtained using a HITACHI SEM (Regulus8100) system. Single-molecule TIRF assay was performed using a Nikon Ti2 microscope.

Probe design. The MSS-RCA-PGM system relies on a set of oligonucleotide probes that link the target with the primer for MSS-RCA. Briefly, the target sequence was found at GenBank (GenBank: MK343362.1; https://www.ncbi.nlm.nih.gov/nuccore/MK343362.1?report=fasta), and the cDNA sequence was generated accordingly. We analyzed the sequence of the target to identify candidate hybridization sites using standard parameters for Mfold (http://www.unafold.org/). For the biotin probe, we added a 5’ biotin label, a poly T spacer, and a hybridization sequence to complete the probe. The phosphate probe consists of a 5’ phosphorylated label, a hybridization region that hybridizes to 3’ half of the DNA target, and a primer for MSS-RCA.

After the design of the two probes, we used Mfold to inspect the melting temperature of the biotin probe and the phosphate probe. A melting temperature of greater than 40 is preferred for successful ligation.

Synthesis and purification of the circular template. The circular template was synthesized using a phosphorylated DNA template and the ssDNA ligase. Briefly, 10 µL of 100 µM of the phosphorylated circular template was mixed with 5 µL of 10x Circligase buffer, 0.5 µL of the circ ligase (500 U/µL), 2.5 µL of 50 mM MnCl₂ and ddH₂O to the final volume of 50 µL. The mixture was incubated at 60°C for 2 h and 95°C for 10 min to inactivate the enzyme. After that, the ligation product was run by 15% UREA PAGE. The ligated product was then peeled off from the gel and immersed in the diffusion buffer at 55℃ for 2 h. The mixture was then centrifuged at 15000 rpm for 20 min to collect the supernatant. After that, 3 volume of 99.8% ethanol was added to the supernatant to precipitate the DNA. The mixture was then kept at -80℃ overnight and centrifuged at 15 000 rpm at 4℃ for 30 min to collect the DNA pellet. Finally, the collected DNA pellet was dissolved in water and purified using the Sangon UNIQ-10 DNA purification kit. The concentration of the purified circular template was then quantified by NanoDrop Microvolume Spectrophotometers (ThermoFisher) and then diluted to a proper concentration (i.e.,100 nM).

DNA gel electrophoresis. For UREA PAGE analysis, 5 µL of DNA samples obtained from each section were mixed with 5 µL of formamide and heated at 95℃ for 20 min. After that, 5 µL of the above DNA samples were mixed with 1 µL of 6x loading buffer. 4 µL of the mixture was then run on a 15% Urea PAGE gel at 100 V and then stained with 1x SYBR Gold at RT for 15 minutes before visualization. For agarose gel electrophoresis to visualize RCA products, a 20 µL mixture containing 40 nM of the primer, 10 nM of the circular template, 500 µM of dNTPs, and 5U of Phi29 polymerase in 1x Phi29 buffer was incubated at 37°C for an hour. After that, 5 µL of the DNA sample was mixed with 1 µL of 6x loading buffer. 4 µL of the mixture was mixed with 1 µL of 10x SYBR Gold and 1 µL of 6x loading buffer. The mixture was then run on a 1% agarose gel at 120V until the loading dye reach the bottom of the gel.

Different RCA reaction. For agarose gel electrophoresis, MSS-RCA and SS-RCA were performed in a 20 µL volume containing 2.5 U Phi29 DNA polymerase, 250 µM deoxynucleoside triphosphate mix (dNTPs), 200 nM concentration of primer, and 40 nM of the purified circular template. The reaction was performed under 37℃ for 1 h. The RCA product (5 µL) was analyzed by gel electrophoresis using a 1.0% agarose gel, with 2x SYBR Gold contained in RCA products. The running condition was constant voltage at 100 V for 40 min. The DL15,000 DNAmarker (Takara, #3582A) was used as the standard for the evaluation of the gels. Time-dependent changes of different RCA in fluorescence signals were conducted under 37℃ in an 80 µL volume system containing 2.5 U Phi29 DNA polymerase, 250 µM deoxynucleoside triphosphate mix (dNTPs), different concentrations of primer, and different purified circular template. The RCA product (5 µL) was taken out with a time interval of 5 min. RCA products from different time points were measured in solutions containing 2x SYBR Gold/SYBR Green I.

Detection of RCA products by Atomic Force Microscopy (AFM). A freshly peeled mica was immediately immersed in H2O for 10 min and dried by nitrogen gas. 1x deposition buffer was applied to wash the mica twice. Then, 5 µL 100-fold diluted RCA products in 1x deposition buffer were dropped onto the mica for 20 min and dried softly by nitrogen gas. The sample was subsequently analyzed on a Nanoscale AFM instrument (Bruker, Multimode 8) using ScanAsyst mode in ambient air.

Detection of RCA products by fluorescence imaging. Mixtures (40 µL) consisting of 1× Phi 29 polymerase buffer, 20 nM primer, 20 nM purified circular template, and 500 µM dNTP were reacted at 37°C for 1 h. After the reaction, 10 µL RCA products were added to a 90 µL mixture containing 2 µL 100x SYBR Gold and 10 µL 10X PBS buffer. The mixture was incubated at room temperature for 5 minutes. Before imaging the SYBR Gold-stained RCA product, glass slides were cleaned with plasma cleaner for 5 min. After that, the glass surface was immediately coated with 0.01% poly(L-lysine). Finally, 8 µL of the SYBR Gold-stained DNA was applied to the treated glass slides. Slides were imaged on Ti2 microscope (Nikon) equipped with an Apo TIRF 100x Oil DIC N2 objective. The sample was excited by the FITC channel (excitation wavelength: 488nm, emission wavelength: 530 nm). The emission signal was imaged onto Andor DU-897 X-13707 EM CCD camera with a 50 ms exposure time and conversion camera gain of 1. Data analysis was performed using NIS elements and ImageJ.

Magnetic bead-based RCA product detection by TexRed labelled DNA signal probe. Different concentration of target DNA was hybridized with the 1 µM biotin-labelled probe and 1 µM phosphate-labelled probe in a 10 µL ligation system containing 1x T4 ligase buffer and 400U T4 ligase. The probe mixture was incubated at 95°C for 5 min, then add T4 -related mixtures (1x T4 ligase buffer and 400U T4 ligase) incubating at 37°C for 1.5 h, and 95°C inactivated for 10 min.

The 10 µL ligation product was incubated with a 40 µL mixture containing 4 µL 10x PBS, 3 µL 10 mg/mL streptavidin-coated magnetic beads (Dynabeads™ MyOne™ Streptavidin C1) on a rotator at room temperature for 30 min. Then the magnetic bead was washed once with 1x PBST once and 1x PBS twice. After that, an RCA mixture containing 1 nM purified circular template, 500 µM dNTP, 4 µL 10x Phi29 buffer, and 10 U Phi29 polymerase was added into the magnetic bead with the ligated product. The magnetic bead-based RCA reaction was performed on the rotator for 1.5 h at room temperature (22 ℃). For comparison of SS-RCA and MSS-RCA, 100 nM of target and 100 nM of circular template were used. When the RCA reaction finished, the supernatant was taken out. TexRed labelled DNA detect probe (50 µL 1 µM) was added into the magnetic beads and incubated for 30 min. Different product of the detection process was collected and measured on a microplate reader (PerkinElmer EnVision multimode plate reader 2105-0010, excitation: 595 nM; emission: 615 nM).

Conjugation of DNA and invertase by SM(PEG) ₂. 2OD of thiol-DNA was first dissolved in MilliQ water to a final concentration of 500 µM. DNA-thiol reduction reaction system: 0.1 M phosphate buffer (pH 5.5), 30 mM TCEP, and 500 µM, 2 OD thiol-DNA were wrapped with aluminum foil and incubated at room temperature for 2 h to reduce disulfide bonds in thiol-modified DNA strands. Excess TCEP was removed by Amicon-3K using 1XPBS 3 times. Weigh SM(PEG)₂ (crosslinker) and add DMSO to prepare the 10 mg/ml SM(PEG)2 solution. Meanwhile, prepare 10 mg/mL invertase solution in PBS. For each 400 µL of invertase solution, 0.7 mg SM(PEG)₂ in DMSO was added and mixed by a vortex for 2 min. Wrap the above solution with aluminum foil and react on a rotator at 4°C for 1.5-2 h on a rotator. After vortexing, the solution was placed on a shaker for 2 h at room temperature. The mixture was purified by Amicon-30 K using 1XPBS 3 times. The above solution of reduced thiol-DNA and the purified solution of SM(PEG)₂-activated invertase were mixed and kept at 4℃ on a rotator overnight. To remove unreacted thiol-DNA, the solution was purified by Amicon-30 K using 1x PBS and kept at 4°C until further use. The concentration of the invertase probe was measured by the absorbance at 280 nM.

SDS-PAGE characterization of the DNA-invertase conjugates. 4 µL of the sample was mixed with 1 µL of sample loading buffer (10% SDS, 50 mM TCEP, 20% Glycerol, 0.2 M Tris-HCl, pH 6.8, and 0.05% Bromphenolblue) and heated to 95℃ for 5 minutes. The mixture was run on a 4–20% gradient gel at 110V until the blue line almost reach the bottom of the gel.

Optimization of the invertase incubation conditions for PGM reading. 1 µL of the invertase probe (ITP) (0.05 mg/mL) was used in all experiments. pH optimization was performed in 100 mM of sodium acetate. The effect of temperature was examined by incubation of the invertase probe at different temperatures for 30 minutes in 100 mM sodium acetate, pH = 5.5. For sucrose concentration optimization, Sucrose was first dissolved in 100 mM sodium acetate, pH = 5.5, to get a final concentration of 1 M, and diluted with the same buffer to receive experimental concentration.

Kinetic analysis of the invertase before and after conjugation. 2 µL of the invertase solution or ITP (0.05 mg/mL) was mixed with 38 µL of the acetate buffer. The mixture was then incubated at 65°C. For every 30 minutes, 5 µL of the mixture was taken out and cooled down to RT for PGM measurement.

Detection of synthetic E6/E7 cDNA using the MSS-RCA-PGM system. Magnetic beads were firstly prepared. Streptavidin-modified MBs (3 µL, 10 mg/mL) were washed 2 times with 200 µL Washing buffer to remove the surfactants and then resuspended in 30 µL of 1x PBS. To have the T4 ligation and immobilization of primer onto MBs, 1 µL of the target analyte with various concentrations, 1 µL of the biotin probe (5 µM), 1 µL of the phosphate probe (5 µM), 1µL of 5x T4 ligation buffer, and 1 µL of T4 DNA ligase (1 U/µL) was mixed in a final volume of 10 µL. The reaction mixture was heated at 95°C for 10 min before adding the T4 ligase and slowly cooled down to room temperature. Then, 1 µL of T4 ligase (1 U/µL) was added to the MBs in 30 µL of 1x PBS and incubated at RT for 1 h unless otherwise noticed. The MSS-RCA was initiated by adding a 40 µL mixture containing 1 nM of the circular template, 500 µM dNTPs, 1x Phi29 buffer, and 2.5 U Phi29 DNA polymerase. The MSS-RCA was carried out at RT for 90 minutes and then terminated, removing the MSS-RCA mixture. For the comparison between systems with/without MSS-RCA amplification, 10 nM of the circular template was used. Finally, The invertase probe (40 µL of 0.01 mg/mL) was added to the MBs and mixed on a rotator for 30 min to immobilize ITP. Then, the MBs were washed 4 times with PBST (0.1% Tween 20) and then once with PBS to remove the unbound ITP. 15 µL of 600 mM sucrose in acetate buffer was added to the MB and incubated at 65°C for 30 minutes. A portion of the final solution was measured by a commercially available PGM.

Zeta size measurements. The ligation reaction was performed using 1 µM of biotin probe, 1µM of phosphate probe, and 1µM of the synthetic DNA target in the volume of 10 µL. The MSS-RCA was performed under RT (22℃) for the desired amount of time with an 80 µL mixture containing 10 nM of the circular template, 500 µM of dNTPs, 2 µL of Phi29 polymerase (10 U/µL) and 8 µL of 10x buffer. The protocol for invertase probe hybridization was the same as described above. The content was dispersed in 1 mL of 1x PBS.

Ethic statement. All patients eligible for this study accepted surgery and were followed up regularly. Patients provided written informed consent, and the study was carried out according to the Declaration of Helsinki. Ethics approval (#B2023-112-01) was obtained from the Institutional Research Ethics Committee of Sun Yat-sen University Cancer Center to use the clinical specimens for research purposes.

Clinical sample validation. Firstly, the total mRNA was extracted from the cervical tumor tissue samples by using TRIzol (Life Technologies). After the mRNA pellet was resuspended in DEPC water, the first strand of cDNA was synthesized using the color Reverse Transcription kit (EZB, A0010CGQ) based on the manufacturer's instruction. The RT-qPCR assays were carried out with the Taq Pro Universal SYBR qPCR Master Mix (Vazyme) using CFX real-time PCR systems (Bio-Rad, Hercules, CA, which were used for thermocycling and fluorescence detection. Real-time PCR amplification for HPV E6/E7 mRNA was performed using a total volume of 20 µL that contained 10 µL of 2× qPCR mix, 2 µL of both forward and reverse primer (1 µM), and 2 µL of template cDNA, and distilled water to give a final volume of 20 µL for each sample. In the amplification procedure, the relative expression of the mRNA concentration was represented as the cycle threshold (Ct) value. We defined a CT value to be significant (positive) if it was at least four cycles lower than the control CT value ⁴⁷. In order to detect cDNA obtained in clinical samples using the MSS-RCA-PGM system, 4 µL of RNAse H digested sample was used for T4 ligation based on previously established protocol.

Characterization of RCA products with/without invertase probe (ITP) by SEM. Standard protocol for E6/E7 cDNA detection using the MSS-RCA-PGM system was used for the preparation of the sample, except that 1 µM of the target was used in the ligation step. The samples were let dry in a desiccator, then a platinum film was applied to the glass surfaces using a sputter coater for 60 s in a vacuum. SEM images were obtained at a 5 kV accelerating voltage.

Acknowledgements

The work was financially supported by the National Natural Science Foundation of China (Grant 22174121, 22211530067, T2250710180), 2022 Natural Science Foundation of Guangdong Provincial Basic and Applied Basic Research Fund (Guangdong Hybribio), Shenzhen Bay Open Laboratory Fund 2021 by Shenzhen Bay Laboratory, the University Development Fund (UDF01002012) of Chinese University of Hong Kong, Shenzhen. We thank T. Wu (CUHK-SZ) for RT-qPCR data acquisition and analysis. We thank B. Chen and other colleagues at the Ciechanover Institute of Precision and Regenerative Medicine for AFM figure acquisition and analysis. We thank Bioimaging Core of Shenzhen Bay Laboratory for providing imaging support. We also would like to acknowledge Bioimaging Core engineer M. Yu, and Dr Z. Sun for assistance with Apo TIRF on Ti2 microscope (Nikon).

Author contributions

Y. L. conceived of the presented idea, designed and performed experiments, wrote the manuscript with input from all authors. S. T. planned, designed and carried out experiments, analysed the data, prepared figures and wrote the manuscript. T. Y. performed experiments. Yx L. participated in sequence design. D. S. and X. J. collected and prepared clinical samples, performed experiments, and contributed to the manuscript under the guidance of L. S. G. L. provided the administration and supervising of this project, including the conceiving of the idea, experiment design, trouble shooting, data analysis, figure illustration, manuscript writing, reviewing and revising.

Competing interests

The authors declare no competing interests.

Sung, H., Ferlay, J., Siegel, R. L., Laversanne, M., Soerjomataram, I., Jemal, A. & Bray, F. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: a cancer journal for clinicians 71, 209–249 (2021).
Kiran, S., Dar, A., Singh, S. K., Lee, K. Y. & Dutta, A. The deubiquitinase USP46 is essential for proliferation and tumor growth of HPV-transformed cancers. Molecular Cell 72, 823–835. e825 (2018).
Shiraz, A., Crawford, R., Egawa, N., Griffin, H. & Doorbar, J. The early detection of cervical cancer. The current and changing landscape of cervical disease detection. Cytopathology 31, 258–270 (2020).
Iftner, T., Neis, K.-J., Castanon, A., Landy, R., Holz, B., Woll-Herrmann, A., Iftner, A., Staebler, A., Wallwiener, D. & Hann von Weyhern, C. Longitudinal clinical performance of the RNA-based Aptima human papillomavirus (AHPV) assay in comparison to the DNA-based hybrid capture 2 HPV test in two consecutive screening rounds with a 6-year interval in Germany. Journal of Clinical Microbiology 57, e01177-01118 (2019).
Derbie, A., Mekonnen, D., Woldeamanuel, Y., Van Ostade, X. & Abebe, T. HPV E6/E7 mRNA test for the detection of high grade cervical intraepithelial neoplasia (CIN2+): a systematic review. Infectious Agents and Cancer 15, 1–10 (2020).
Rossi, P. G. & Rebolj, M. HPV mRNA testing in cervical cancer screening. The Lancet Oncology 23, e436 (2022).
Iftner, T., Becker, S., Neis, K.-J., Castanon, A., Iftner, A., Holz, B., Staebler, A., Henes, M., Rall, K. & Haedicke, J. Head-to-head comparison of the RNA-based aptima human papillomavirus (HPV) assay and the DNA-based hybrid capture 2 HPV test in a routine screening population of women aged 30 to 60 years in Germany. Journal of Clinical Microbiology 53, 2509–2516 (2015).
Forslund, O., Miriam Elfström, K., Lamin, H. & Dillner, J. HPV-mRNA and HPV‐DNA detection in samples taken up to seven years before severe dysplasia of cervix uteri. International Journal of Cancer 144, 1073–1081 (2019).
Sharma, B., Lakhanpal, V., Singh, K., Oberoi, L., Bedi, P. K. & Devi, P. Evaluation of HPV E6/E7 mRNA Detection in Clinically Suspected Cases of Cervical Cancer with Abnormal Cytology: Time to Upgrade the Screening Protocols. Journal of Laboratory Physicians 14, 336–342 (2022).
Poljak, M., Valenčak, A. O., Domjanič, G. G., Xu, L. & Arbyn, M. Commercially available molecular tests for human papillomaviruses: a global overview. Clinical Microbiology and Infection 26, 1144–1150 (2020).
Montag, M., Blankenstein, T. J., Shabani, N., Brüning, A. & Mylonas, I. Evaluation of two commercialised in situ hybridisation assays for detecting HPV-DNA in formalin-fixed, paraffin-embedded tissue. Archives of Gynecology and Obstetrics 284, 999–1005 (2011).
Lee, S. H., Park, S.-m., Kim, B. N., Kwon, O. S., Rho, W.-Y. & Jun, B.-H. Emerging ultrafast nucleic acid amplification technologies for next-generation molecular diagnostics. Biosensors and Bioelectronics 141, 111448 (2019).
Xu, L., Duan, J., Chen, J., Ding, S. & Cheng, W. Recent advances in rolling circle amplification-based biosensing strategies-A review. Analytica Chimica Acta 1148, 238187 (2021).
Li, Y., Li, S., Wang, J. & Liu, G. CRISPR/Cas systems towards next-generation biosensing. Trends in Biotechnology 37, 730–743 (2019).
Kang, T., Lu, J., Yu, T., Long, Y. & Liu, G. Advances in nucleic acid amplification techniques (NAATs): COVID-19 point-of-care diagnostics as an example. Biosensors and Bioelectronics, 114109 (2022).
Hanpanich, O., Lomae, A., Maruyama, A., Palaga, T., Chailapakul, O. & Ngamrojanavanich, N. Label-free detection of HPV mRNA with an artificial chaperone-enhanced MNAzyme (ACEzyme)-based electrochemical sensor. Biosensors and Bioelectronics 221, 114352 (2023).
Gu, L., Yan, W., Liu, L., Wang, S., Zhang, X. & Lyu, M. Research progress on rolling circle amplification (RCA)-based biomedical sensing. Pharmaceuticals 11, 35 (2018).
Garafutdinov, R., Sakhabutdinova, A., Gilvanov, A. & Chemeris, A. Rolling Circle Amplification as a Universal Method for the Analysis of a Wide Range of Biological Targets. Russian Journal of Bioorganic Chemistry 47, 1172–1189 (2021).
Chaibun, T., Puenpa, J., Ngamdee, T., Boonapatcharoen, N., Athamanolap, P., O’Mullane, A. P., Vongpunsawad, S., Poovorawan, Y., Lee, S. Y. & Lertanantawong, B. Rapid electrochemical detection of coronavirus SARS-CoV-2. Nature Communications 12, 1–10 (2021).
Li, J., Mohammed-Elsabagh, M., Paczkowski, F. & Li, Y. Circular nucleic acids: Discovery, functions and applications. ChemBioChem 21, 1547–1566 (2020).
Povilaitis, T., Alzbutas, G., Sukackaite, R., Siurkus, J. & Skirgaila, R. In vitro evolution of phi29 DNA polymerase using isothermal compartmentalized self replication technique. Protein Engineering, Design and selection 29, 617–628 (2016).
Zibulski, D. L., Schlichting, N. & Kabisch, J. HyperXpress: Rapid Single Vessel DNA Assembly and Protein Production in Microliterscale. Frontiers in Bioengineering and Biotechnology 10 (2022).
Inoue, J., Shigemori, Y. & Mikawa, T. Improvements of rolling circle amplification (RCA) efficiency and accuracy using Thermus thermophilus SSB mutant protein. Nucleic Acids Research 34, e69-e69 (2006).
Lin, C., Xie, M., Chen, J. J., Liu, Y. & Yan, H. Rolling-Circle Amplification of a DNA Nanojunction. Angewandte Chemie International Edition 45, 7537–7539 (2006).
Lin, C., Wang, X., Liu, Y., Seeman, N. C. & Yan, H. Rolling circle enzymatic replication of a complex multi-crossover DNA nanostructure. Journal of the American Chemical Society 129, 14475–14481 (2007).
Mao, Y., Liu, M., Tram, K., Gu, J., Salena, B. J., Jiang, Y. & Li, Y. Optimal DNA templates for rolling circle amplification revealed by in vitro selection. Chemistry–A European Journal 21, 8069–8074 (2015).
Liu, M., Zhang, Q., Li, Z., Gu, J., Brennan, J. D. & Li, Y. Programming a topologically constrained DNA nanostructure into a sensor. Nature Communications 7, 1–7 (2016).
Joffroy, B., Uca, Y. O., Prešern, D., Doye, J. P. K. & Schmidt, T. L. Rolling circle amplification shows a sinusoidal template length-dependent amplification bias. Nucleic Acids Research 46, 538–545 (2018).
Kishi, J. Y., Lapan, S. W., Beliveau, B. J., West, E. R., Zhu, A., Sasaki, H. M., Saka, S. K., Wang, Y., Cepko, C. L. & Yin, P. SABER amplifies FISH: enhanced multiplexed imaging of RNA and DNA in cells and tissues. Nature Methods 16, 533–544 (2019).
Xiang, Y. & Lu, Y. Using personal glucose meters and functional DNA sensors to quantify a variety of analytical targets. Nature Chemistry 3, 697–703 (2011).
Clever, G. H., Kaul, C. & Carell, T. DNA–metal base pairs. Angewandte Chemie International Edition 46, 6226–6236 (2007).
Kolbeck, P. J., Vanderlinden, W., Gemmecker, G., Gebhardt, C., Lehmann, M., Lak, A., Nicolaus, T., Cordes, T. & Lipfert, J. Molecular structure, DNA binding mode, photophysical properties and recommendations for use of SYBR Gold. Nucleic Acids Research 49, 5143–5158 (2021).
Wang, L., Song, K., Qu, Y., Chang, Y., Li, Z., Dong, C., Liu, M., Brennan, J. D. & Li, Y. Engineering Micrometer-Sized DNA Tracks for High‐Speed DNA Synthesis and Biosensing. Angewandte Chemie International Edition 59, 22947–22951 (2020).
Morin, J. A., Cao, F. J., Lázaro, J. M., Arias-Gonzalez, J. R., Valpuesta, J. M., Carrascosa, J. L., Salas, M. & Ibarra, B. Active DNA unwinding dynamics during processive DNA replication. Proceedings of the National Academy of Sciences 109, 8115–8120 (2012).
Zipper, H., Brunner, H., Bernhagen, J. & Vitzthum, F. Investigations on DNA intercalation and surface binding by SYBR Green I, its structure determination and methodological implications. Nucleic Acids Research 32, e103-e103 (2004).
Han, X., Wang, E., Cui, Y., Lin, Y., Chen, H., An, R., Liang, X. & Komiyama, M. The staining efficiency of cyanine dyes for single-stranded DNA is enormously dependent on nucleotide composition. Electrophoresis 40, 1708–1714 (2019).
Tuma, R. S., Beaudet, M. P., Jin, X., Jones, L. J., Cheung, C.-Y., Yue, S. & Singer, V. L. Characterization of SYBR Gold nucleic acid gel stain: a dye optimized for use with 300-nm ultraviolet transilluminators. Analytical Biochemistry 268, 278–288 (1999).
Baner, J., Nilsson, M., Mendel-Hartvig, M. & Landegren, U. Signal amplification of padlock probes by rolling circle replication. Nucleic Acids Research 26, 5073–5078 (1998).
Dean, F. B., Nelson, J. R., Giesler, T. L. & Lasken, R. S. Rapid amplification of plasmid and phage DNA using phi29 DNA polymerase and multiply-primed rolling circle amplification. Genome Research 11, 1095–1099 (2001).
Sainz-Polo, M. A., Ramírez-Escudero, M., Lafraya, A., González, B., Marín-Navarro, J., Polaina, J. & Sanz-Aparicio, J. J. J. o. B. C. Three-dimensional structure of Saccharomyces invertase: role of a non-catalytic domain in oligomerization and substrate specificity. Journal of Biological Chemistry 288, 9755–9766 (2013).
Berman, H., Henrick, K., Nakamura, H. J. N. S. & Biology, M. Announcing the worldwide protein data bank. Nature Structural & Molecular Biology 10, 980–980 (2003).
Liu, S., Ming, J., Lin, Y., Wang, C., Cheng, C., Liu, T. & Wang, L. Highly sensitive detection of T4 polynucleotide kinase activity by coupling split DNAzyme and ligation-triggered DNAzyme cascade amplification. Biosensors and Bioelectronics 55, 225–230 (2014).
Victorious, A., Zhang, Z., Chang, D., Maclachlan, R., Pandey, R., Xia, J., Gu, J., Hoare, T., Soleymani, L. & Li, Y. A DNA Barcode-Based Aptasensor Enables Rapid Testing of Porcine Epidemic Diarrhea Viruses in Swine Saliva Using Electrochemical Readout. Angewandte Chemie 134, e202204252 (2022).
Zweig, M. H. & Campbell, G. Receiver-operating characteristic (ROC) plots: a fundamental evaluation tool in clinical medicine. Clinical Chemistry 39, 561–577 (1993).
Patchsung, M., Jantarug, K., Pattama, A., Aphicho, K., Suraritdechachai, S., Meesawat, P., Sappakhaw, K., Leelahakorn, N., Ruenkam, T. & Wongsatit, T. Clinical validation of a Cas13-based assay for the detection of SARS-CoV-2 RNA. Nature Biomedical Engineering 4, 1140–1149 (2020).
Gómez-de Pedro, S., Berenguel-Alonso, M., Couceiro, P., Alonso-Chamarro, J. & Puyol, M. Automatic microfluidic system to perform multi-step magneto-biochemical assays. Sensors and Actuators B: Chemical 245, 477–483 (2017).
Lamarcq, L., Deeds, J., Ginzinger, D., Perry, J., Padmanabha, S. & Smith-McCune, K. Measurements of human papillomavirus transcripts by real time quantitative reverse transcription-polymerase chain reaction in samples collected for cervical cancer screening. The Journal of Molecular Diagnostics 4, 97–102 (2002).

There is NO Competing Interest.

MSSRCAsupplementaryinformation230323.docx

Download PDF

Version 1

posted

You are reading this latest preprint version

MSSRCA Powered Sensitive Point-of-Care Detection of HPV mRNA for Cervical Cancer Screening

Status:

Version 1

Abstract

Figures

Introduction

Results and discussion

Discussion

Methods

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1