A novel platform using homobifunctional hydrazide for enrichment and isolation of urinary circulating RNAs

Abstract Changes in specific circulating RNA (circRNA) expressions can serve as diagnostic noninvasive biomarkers for prostate cancer (PCa). However, there are still unmet needs, such as unclear types and roles of circRNAs, PCa detection in benign prostatic hyperplasia (BPH) by unstandardized methods, and limitations of sample volume capacity and low circRNA concentrations. This study reports a simple and rapid circRNA enrichment and isolation technique named “HAZIS‐CirR” for the analysis of urinary circRNAs. The method utilizes homobifunctional hydrazides with amine‐modified zeolite and polyvinylidene fluoride (PVDF) syringe filtration for combining electrostatic and covalent coupling and size‐based filtration, and it offers instrument‐free isolation of circRNAs in 20 min without volume limitation, thermoregulation, and lysis. HAZIS‐CirR has high capture efficiency (82.03%–92.38%) and a 10‐fold more sensitive detection limit (20 fM) than before enrichment (200 fM). The clinical utility of HAZIS‐CirR is confirmed by analyzing circulating mRNAs and circulating miRNAs in 89 urine samples. Furthermore, three miRNA panels that differentiate PCa from BPH and control, PCa from control, and BPH from control, respectively, are established by comparing miRNA levels. HAZIS‐CirR will be used as an optimal and established method for the enrichment and isolation of circRNAs as diagnostic, prognostic, and predictive biomarkers in human cancers.

invasiveness, high risk, high cost, and tumor location and size limitations. 8,9 In addition, because of the heterogeneity of circulating NAs by type and stage of cancer, it is possible to monitor cancer metastasis and cancer treatment prognosis through molecular characterization. 10,11 Due to these advantages, circulating NAs are used as biomarkers for early or accurate diagnosis of cancer, and the diagnostic platform used to detect them should be highly sensitive, such as end-point, real-time, and droplet-digital PCR. However, the low sensitivity due to low concentrations of circulating NAs in liquid specimens is one challenge to consider in the development of a powerful diagnostic platform. Currently, there is a lack of discussion about the enrichment of high-concentration circulating NAs using a large sample volume.
Prostate cancer (PCa) is one of the most common malignancies in men worldwide and the second leading cause of all cancer-related deaths. 12,13 Prostate-specific antigen (PSA) testing has been widely used for the early diagnosis of PCa, consequently increasing the number of treatable patients with PCa and dramatically reducing the death rate from PCa. 14 PSA is only present in prostate tissue, and PSA levels are elevated in the serum of patients with PCa. 15 However, PSA levels are variously increased by many factors. 16 Especially, benign prostatic hyperplasia (BPH) is the most common condition associated with elevated PSA values that can be mistaken for PCa. In the PSA gray zone of 4-10 ng ml À1 , some limitations were closely related to certain nonmalignant conditions, such as BPH and prostatitis. [17][18][19] The low sensitivity and specificity of the PSA test lead to erroneous diagnosis and treatment, because PCa patients sometimes have positive BPH findings with or without clinical symptoms. Erroneous diagnosis can cause a lot of stress for patients and put a lot of burden on the healthcare system. 20,21 Therefore, there is an unmet need for more effective biomarkers for PCa diagnosis. Recently, due to the various advantages of circulating NAs, PCa diagnostic platforms for circulating RNAs (cir-cRNAs), including circulating miRNA and circulating mRNA, are being intensively developed. [22][23][24] miRNA is a noncoding RNA molecule approximately 22 nucleotides long (17-27 nts) that preferentially binds to the 3 0 UTR of mRNA and acts as a post-transcriptional regulator, influencing important cellular processes, such as cell cycle, proliferation, and apoptosis. 25,26 In patients with PCa, circulating miRNA-141-3p and miRNA-375-3p are increased compared to control patients and are known as promising biomarkers. 22,27,28 However, the role of these miRNAs is still unclear, and there are limitations to the discovery of PCa-specific miRNA biomarkers among numerous miRNAs. Furthermore, previous research has mostly focused on circulating miRNA rather than circulating mRNA.
One of the important considerations for circRNA analysis is the sample extraction method to obtain high-concentration circRNAs. [29][30][31][32] To capture rare circRNAs, conventional isolation and purification technologies using phenol-chloroform-based, spin-column-based, or bead-based methods have been proposed. [33][34][35] These approaches are expensive, time-consuming (>30 min), labor-intensive, depend on hazardous chemicals, such as phenol, and require large or specialized equipment for centrifugation and temperature control. In addition, they use a lysis buffer containing a chaotropic reagent, such as guanidine thiocyanate, which increases the genetic background derived from noncancerous cells. It is also impossible to process a large volume of clinical specimens, so the reproducibility is poor due to the use of low concentrations of circRNAs. Therefore, developing a universal technology for the enrichment and isolation of circRNAs with high reproducibility remains a challenge.
Here, we developed a novel platform called "HAZIS-CirR" for the simple and rapid enrichment and isolation of circRNAs from the urine of patients with PCa, BPH, and control patients by combining adipic acid dihydrazide (ADH), amine-modified zeolite (AZ), and PVDF syringe filtration. ADH is low-cost, water-soluble, and odorless with low toxicity. As a nucleophilic molecule, ADH binds covalently to amine groups. It is mainly used as a crosslinking agent in the manufacture of mechanical latex films and injectable oxidized hyaluronic acid hydrogels due to its reactive hydrazide group at each end of the molecule. [36][37][38] Zeolites are natural mesoporous and microporous aluminosilicates widely used in various industrial processes, such as separation, catalysis, and sensors because of their low-cost, structural stability, and biocompatibility advantages. [39][40][41][42] The large surface area relative to the volume of zeolite increases its reactivity with various biomaterials, so zeolite is easily manufactured into functionalized materials for the binding of biomolecules through surface modification. We exploited these properties of ADH to couple AZ and cir-cRNAs through covalent and electrostatic interaction. Furthermore, we used PVDF syringe filters, which can handle large samples and large volumes easily and simply without relying on large equipment, such as centrifuge and vacuum pump. The PVDF syringe filter requires only a suitable syringe for the sample volume, can quickly separate substances larger than the filter pore size without external contamination, and allows washing debris smaller than the pore size of the filter.
We analyzed several circulating mRNAs and circulating miRNAs using a total of 89 urine samples in HAZIS-CirR. Our results demonstrated that circRNAs can be concentrated and extracted quickly, inexpensively, and effectively using urine from patients with PCa (55 samples), BPH (24 samples), and control patients (10 samples), and circulating mRNAs and circulating miRNAs that can be used as PCa biomarkers in patients with BPH. Furthermore, we identified three miRNA panels that distinguish PCa from BPH and control samples

| Enrichment and isolation of circRNAs using HAZIS-CirR
HAZIS-CirR is a novel platform for the enrichment and isolation of cir-cRNAs based on the molecular characteristics of AZ and ADH and the size-based filtering of PVDF sterilized filters. As a porous structure and amine group donor, AZ can effectively capture circRNAs due to the electrostatic coupling between the positively charged amino group and the negatively charged phosphate of the circRNAs and can also capture ADH and circRNAs due to the covalent coupling. ADH is a pH-sensitive linker and a nonchaotropic reagent, with a C4 backbone and two reactive hydrazide groups (C ONHNH 2 ). The hydrazide group of ADH can react with carbonyl groups of circRNAs to form a reactive hydrazone bond. In addition, because it has two nucleophilic primary amine groups and one aldehyde group per hydrazide group, it can react with amine-reactive and aldehyde-reactive groups of AZ and circRNAs. The PVDF syringe filter can process samples without limitation of sample volume and allows simple, quick, and user-friendly sample preparation. The combination of these three materials-AZ, ADH, and PVDF syringe filter-enables simple and rapid enrichment and isolation of circRNAs.

| Characterization of HAZIS-CirR
We first characterized HAZIS-CirR to identify the molecular properties and binding mechanisms between AZ, ADH, and NAs. The shape and size distribution of zeolite were confirmed from scanning electron microscope (SEM) images and dynamic light scattering (DLS) analysis ( Figure 2a,b). Zeolite is about 2-18 μm (mostly 6-14 μm) and maintains a stable structure after silanization and even after interaction with ADH and NAs. Fourier-transform infrared spectroscopy (FTIR) results ( Figure 2c) for pure zeolite showed absorption peaks at 464 cm À1 (symmetric bending of TO, T: tetrahedrally bonded Si or Al), 547 cm À1 (double six-membered rings of TOT symmetric stretching), 667 cm À1 (SiOSi symmetric stretching), 777 cm À1 (symmetric TOT stretching), 1028 cm À1 (SiOAl asymmetric stretching of TO), and 1654 cm À1 (OH bending) and broad absorption bands at 3467-3572 cm À1 (hydroxyl group). After silanization, AZ showed an increase in the intensity of the absorption peaks at 1654 cm À1 and the broad absorption bands at 3467-3572 cm À1 due to NH stretching and NH bending, respectively. These results verified that amine groups are formed on the AZ surface. In addition, the ADH-modified AZ (HAZ) showed additional absorption peaks at 1471 cm À1 (CN stretching), 1535 cm À1 (NH bending), 2864 cm À1 (CH stretching and bending), 2925 cm À1 (CH stretching), and 3317 cm À1 (NH stretching), increased intensity of the absorption peak at 1654 cm À1 (C O stretching) and increased intensity of the broad absorption bands at 3467-3572 cm À1 (NH stretching and bending) due to ADH ( Figure S2A). These FTIR results provided spectroscopic evidence that AZ and ADH can interact to form an imine bond ( Figure 1b) and that hydrazide is generated on the surface of AZ, which can be used as a functional group.
Furthermore, the FTIR data for HAZ with NAs (HAZ-NAs) showed that absorption peaks representing hydrazide (1471, 1535, 2864, 2925, and 3317 cm À1 ) are decreased by the imine bond, hydrazine bond, and electrostatic attraction, and the broad absorption peaks (3317-3572 cm À1 ) caused by NAs are increased. On the basis of the FTIR data, AZ and ADH can be used to capture circRNAs. Figure 2d shows the zeta-potential of pure zeolite, AZ, HAZ, and HAZ-NAs.
Pure zeolite showed a zeta-potential of À44.58 mV attributed to the OH and oxygen-containing groups on the zeolite surface. For AZ, the zeta-potential increased to À11.62 mV compared to pure zeolite due to the immobilized amine group on the zeolite surface. The zetapotential of À13.82 mV obtained for HAZ is similar to that of AZ. This result is attributed to the interaction between AZ and ADH and the amines and aldehydes of the hydrazide groups. The high zetapotential of HAZ-NAs (À33.92 mV) can be explained by the strong Our zeta-potential data confirmed that AZ and HAZ have high zetapotential values and that electric bonds are formed from the interaction of HAZ with the negative charge of circRNAs. These characterization results indicate that HAZIS-CirR can be used as a new platform for the enrichment and isolation of circRNAs.

| Optimization of HAZIS-CirR
We next determined the optimized concentrations of AZ and ADH in HAZIS-CirR. Optimization of the AZ and ADH concentrations is essential to avoid the low-binding affinity of circRNAs due to the low concentration and low capture efficiency of circRNAs because of the interference by molecules of high concentration. The optimization was set to 1 ml of the sample, and AZ and ADH were added at the same ratio according to the change in the sample volume. We varied the concentrations of AZ and ADH, which revealed that the best capture efficiency of RNAs was obtained when 5 mg of AZ and 50 mg of ADH were used per 1 ml of sample (Figure 3a ribosomal RNA (18S rRNA) target genes. It was calculated using the formula value of the standard curve obtained by serial dilution of hsa-mir-21-5p ss mimics ( Figure S3 and Table S1) and T7 in vitro transcribed RNAs ( Figure S4A-C and Table S2). As shown in Figure 3c and Table S3, the detection limit before and after HAZIS-CirR remained the same (1.204 Â 10 3 copies reaction À1 ), and the enrichment and isolation of circRNAs using hsa-mir-21-5p ss mimics could be carried out with high efficiency (82.03%-92.38%). Long RNA fragments (>100 bp) can be concentrated and isolated with high efficiency at both 150 bp (82.14%-95.48%) and 112 bp (80.37%-94.53%) size ( Figure S4D,E and Table S4). These results showed that HAZIS-CirR has a similar capture efficiency at both long RNA fragments (150 bp and 112 bp) and short RNA fragments (hsa-mir-21-5p ss mimic, 20 bp) and can isolate circRNAs of various lengths. Furthermore, we compared the capture efficiency of hsa-mir-21-5p ss mimics before and after HAZIS-CirR using a large volume of samples and human serum. When the hsa-mir-21-5p ss mimics were concentrated using a large volume (from 1 ml to 100 μl) of sample in HAZIS-CirR, the sensitivity of the detection limit increased 10-fold to 20 fM (1.204 Â 10 2 copies reaction À1 ) compared to not proceeding with enrichment ( Figure 3d). As shown in Figure S5A and Table S5, it was confirmed that the hsa-mir-21-5p ss mimic can be concentrated and isolated with high efficiency in both 5 ml (78.65%-94.51%) and 10 ml (82.49%-98.25%). These results showed that HAZIS-CirR is useful for various clinical sample volumes (1-10 ml). In addition, the hsa-mir-21-5p ss mimic can be concentrated and isolated with high efficiency in the human serum sample (80.62%-96.36%) ( Figure S5B and Table S6). These results showed that HAZIS-CirR is useful for a variety of liquid biopsies including urine, blood plasma. Our optimization data indicate that HAZIS-CirR can increase the circRNA capture

| Circulating mRNAs in clinical specimens using HAZIS-CirR
We analyzed the circulating mRNAs in 84 urine samples to confirm the clinical utility of HAZIS-CirR for circulating mRNA-based PCa diagnosis ( Figure S6) using the PCA3 and the TMPRSS2-ERG gene fusion as the biomarkers and the relative quantification (RQ) values were calculated (Figure 4a,b). The PCA3 gene is a long noncoding RNA overexpressed in PCa cells, and it has recently been used in clinical applications as a PCa biomarker. 43,44 The ERG oncogene is known to be overexpressed in more than 50% of PCa cases, and ERG overexpression is induced by fusion with TMPRSS2, a prostate-specific and androgen-regulating gene, and TMPRSS2-ERG gene fusion is a mutation found in 40%-70% of PCa cases. [45][46][47] Recent studies have highlighted the value of this biomarker combination. 48

| Circulating miRNAs in clinical specimens using HAZIS-CirR
In addition, we also analyzed the circulating miRNAs in 62 urine samples to confirm the clinical utility of HAZIS-CirR for circulating miRNA-based PCa diagnosis (Figures 5 and S6). To this end, we used three known PCa biomarkers (miR-21-5p, miR-141-3p, and miR-375-3p) as circulating miRNA biomarkers, and the RQ values were calculated. MiR-21-5p is an oncogenic microRNA and is known to be overexpressed in several cancers, including PCa. 50 p < 0.05), and miR-375-3p (mean RQ = 7.57 ± 8.21; p < 0.01) were overexpressed compared to control patients. This suggests that there must be a significant difference in these biomarkers between patients with PCa and BPH. Additionally, miR-141-3p (p < 0.05) and miR-375-3p (p < 0.05) were overexpressed in PCa compared to BPH, and there was no significant difference in miR-21-5p (p < 0.075). As a result, we were able to verify that miR-141-3p and miR-375-3p can be used as biomarkers to discriminate patients with PCa from those with BPH.

| DISCUSSION
High-efficiency NAs sampling is required for noninvasive liquid biopsy applications such as early cancer detection and treatment prognosis monitoring. However, the conventional methods have disadvantages, such as time-consuming, expensive, small sample processing capacity, and contamination by genomic NAs due to chaotropic and harmful reagents, besides the complexity of using equipment, such as large centrifuge, vacuum pump, and thermoregulator (Table 1). Our new platform, HAZIS-CirR, allows simple and rapid enrichment and isolation of circRNAs using HHs to complement the existing methods.
HHs, also known as carbonyl-reactive crosslinkers, react with aldehydes of oxidized sugars to form stable conjugates through the hydrazone bond. 41,42,55 HHs are commonly used for the preparation of specific glycoprotein conjugates and glycoprotein labeling and immobilization, and many studies have focused on protein interactions with HHs with regards to antibody labeling, metabolic labeling, circulating protein, and hydrazide-modified nanomaterials, in addition to cancer-related proteins for cancer diagnosis and therapy. [56][57][58][59][60] However, despite several advantages of HHs, the applications for molecular diagnostic platforms have been insufficiently explored. We reported a sample preparation that combines AZ and syringe filtration for the first time to develop a novel molecular diagnostic platform utilizing the advantages of HHs. HAZIS-CirR is inexpensive and does not use chaotropic reagents and lysis buffers that can damage NAs or increase the genomic background, and the entire process is completed within 20 min. In addition, it is low-cost, simple, user-friendly, and does not require electric equipment, such as a large centrifuge, vacuum pump, and thermoregulator (Table 1)

| Analysis of circRNAs
The RQ value of circRNAs was calculated using the comparative C t method with 18S rRNA for mRNA and U6 snRNA for miRNA as the respective reference gene. The RQ value of gene expression was computed using Equations (1)- (3): The p values were determined by the unpaired t-test, and graphs were obtained using GraphPad Prism 8 statistical software (GraphPad Software, San Diego, CA, USA).

| Clinical specimens
For clinical analysis, urine samples were collected from 55 patients with PCa, 24 patients with BPH, and 10 control patients at the Asan Medical Center (AMC), Seoul, South Korea ( Figure S6 and Table S8).
All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of AMC. The use of patient information in this study was approved by the AMC (IRB no. 2019-1312). Urine samples were selected from patients of similar age. The PSA levels of patients with PCa were relatively higher than those of the other groups (Table S8).

| CONCLUSION
HAZIS-CirR platform enables to concentrate rare circRNAs using large sample volumes, allowing extraction of high-concentration circRNAs within 20 min in prostate cancers. HAZIS-CirR does not use chaotropic and hazardous reagents because there is no cell lysis process, and it does not require electricity-consuming equipment, such as centrifuge and thermoregulator. We identified three PCa miRNAs panels and one circulating mRNA using HAZIS-CirR but only focused on diagnosis. This is a limited study and does not reflect the various functions of circRNAs. In addition, further studies are needed to fundamentally investigate the role of circRNAs and explore potential biological functional mechanisms, such as intercellular communication.
However, the potential of circRNAs as cancer biomarkers to overcome human health problems is attractive and cannot be underestimated. In this respect, the development of molecular diagnostic platforms utilizing nanotechnology, such as HAZIS-CirR, will help create more accurate and stable diagnostic and treatment approaches.
We envision that HAZIS-CirR will provide a new dimension for overcoming cancer by concentrating and extracting circulating NAs in various clinical fields not only for early and accurate diagnosis of cancer but also for metastasis confirmation and treatment prognosis.

CONFLICT OF INTERESTS
The authors declare no competing interests.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.