WO2023286073A1 - Réseau nanohybride basé sur le polystyrène fluorescent pour l'estimation des mir circulants acellulaires - Google Patents

Réseau nanohybride basé sur le polystyrène fluorescent pour l'estimation des mir circulants acellulaires Download PDF

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WO2023286073A1
WO2023286073A1 PCT/IN2022/050239 IN2022050239W WO2023286073A1 WO 2023286073 A1 WO2023286073 A1 WO 2023286073A1 IN 2022050239 W IN2022050239 W IN 2022050239W WO 2023286073 A1 WO2023286073 A1 WO 2023286073A1
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ccf
mirs
poly
mir
nano
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Pradyumna Kumar Mishra
Arpit Bhargava
Ruchita SHANDILYA
Irina Yu GORYACHEVA
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Indian Council Of Medical Research
Saratov State University, Russian Federation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays

Definitions

  • the present invention relates to analytical field that describes a method for quantitative as well as qualitative assessment of circulating cell-free miRNAs (ccf-miRs) of interest using a nano- polystyrene based fluorescent array.
  • the current invention also provides a oligonucleotide - nano-polystyrene-fluorophore labelled locked nucleic acid based flow cytometry method for the precise quantitative estimation of ccf-miRs of interest and applications thereof in subsequent analysis of the altered biological processes during certain infections.
  • MicroRNAs are 18-25 nucleotides, short, non-coding sequences that contribute to abundant class of endogenously occurring RNA molecules (Herkenhoff et al., 2018; Gebert and MacRae, 2019). miRNAs play a pivotal role in the regulation of gene expression through binding to the 3' untranslated regions (3’UTR) of the target mRNAs, thereby influencing the translational repression and target gene modulation (Catalanotto et al., 2016; Rissland et al., 2017; O'Brien et al., 2018; Van Peer et al., 2018).
  • Non-coding sequences influence many fundamental cellular machineries such as cell differentiation, proliferation, growth, mobility, apoptosis, cellular stress response, immuno-regulation as well as disease progression (Roy et al., 2016; Yerukala and Ho 2017; Mahdiannasser and Karami, 2018). Dysregulation of the fundamental biological processes indicate a strong correlation with the altered expression of miRNAs in various pathological conditions highlighting their potential as significantly attractive biomarkers (Rinnerthaler et al., 2016; Ragusa et al., 2017; Liu et al., 2018; Han et al., 2019).
  • miRNAs are mostly detected in the cellular micro-environment, however a small fraction of miRNAs are found in the extracellular milieu that are known as circulating cell-free miRNAs (ccf-miRs). These miRNA species can be detected from various biological fluids such as serum, plasma, urine, saliva, tears, breast milk, colostrum, peritoneal fluid, cerebrospinal fluid, bronchial lavage, seminal fluid and follicular fluid (Singh et al., 2018).
  • biological fluids such as serum, plasma, urine, saliva, tears, breast milk, colostrum, peritoneal fluid, cerebrospinal fluid, bronchial lavage, seminal fluid and follicular fluid (Singh et al., 2018).
  • miRNAs may differ from other miRNAs of the same family by a single nucleotide, stressing on the need for development of an array with good mismatch discrimination potential (Tiberio et al., 2015; Filipow and Laczmahski, 2019).
  • Sritabutra and Soonwera, 2013 discloses electrochemical detection of miRNA in case of the clinical samples of breast cancer.
  • the article describes an electrochemical method for detection of miR-21 as a model for detection of breast cancer using a complicated assembly of a glassy carbon electrode coated with carboxylated multi-walled carbon nanotubes and conjugated with a complementary capture probe.
  • Boonyuan et al, 2014 discusses the detection of biomarkers with biosensors for early diagnosis of cancer. It is a review article describing the types of electrochemical, optical, mechanical and other biosensors that have been developed till date for the detection of the nucleic acid and protein based cancer biomarkers.
  • Hakan et al, 2013 demonstrates a biosensing platform for naked eye detection of miRNA.
  • the article describes a paper based method for the visual detection of the presence or absence of target miRNA using a poly(vinylidene fluoride) thin paper impregnated with positively charged poly(3-alkoxy-4-methylthiophene) as luminescent reporters.
  • Li et al, 2013 suggests a method for isothermally sensitive detection of serum miRNAs using hairpin probe-based rolling circle amplification (HP-RCA). It explains a method which involves PCR based amplification and subsequent detection of the miRNAs using by SYBR Green II dye.
  • Yarding Hu et al, 2015 discloses a label-free biosensor with nanometer scale for the detection of trace miRNAs/oligonucleotides using localized surface plasmon resonance (LSPR).
  • LSPR localized surface plasmon resonance
  • Zhang et al, 2010 reports and defines a fluorescent metal nano-shells synthesized as a molecular imaging agent to detect single microRNA (miRNA) molecules in the cells positive to lung cancer. It describes a fluorescence based in-situ hybridization method which uses spectrophotometer for the detection of signals to identify cellular miRNAs in lung cancer settings.
  • miRNA microRNA
  • CN104155447A discloses a preparation method of a sandwich type lung cancer tumor marker biosensor.
  • the patent application provides a method for fabrication of an immunesensor using gold @ silver core-shell nanorod and reducing graphene for the electrochemical based detection of lung cancerassociated antigen markers.
  • CN109722481A discloses the methods of microRNA in the electrocatalysis amplification biosensor detection of lung carcinoma cells. It describes a method for the electrocatalytic based detection of cellular miRNAs using a capture probe conjugated to AuNP/FeCN/GCE electrode.
  • CN 105463077 A relates to a biosensor for visually detecting microRNA combining a nanogold probe with a gene chip.
  • the patent application provides a method for fabrication of chip based sensor for the visual detection of lung cancer associated antigen markers using a microarray based approach.
  • US7825229B2 describes the various polynucleotides such as miRNAs, miRNA precursors, and associated nucleic acids and their use for diagnosis, prognosis, and treatment of lung cancer. It describes in detail about the methods and compositions for linear amplification and labeling of a targeted nucleic acid which may be used in hybridization techniques like Microarray analysis.
  • US7709616B2 discloses miRNAs and the method of detection in case of prostrate and lung cancer. It describes the types of nucleic acids and their compositions that can be used for diagnosis, prognosis, and treatment of lung cancer. It also describes the amplification based methods for identification of these disease-associated biomolecules.
  • US7709616B2 provides a method of detecting a variety of miRNA concentrations simultaneously by fluorescence method in case of lung cancer.
  • the present invention overcomes the complications of the prior art and provides rapid, reproducible and selective fluorescence based quantitative as well as qualitative method for estimation of ccf-miRs of interest with high specificity and sensitivity.
  • the novel ONPS (poly-T oligonucleotide sequences on the nano-polystyrene)-fLNA based nano-hybrid assembly discussed herein provides a sensitive and direct method for the quantification of ccf-miRs of interest through flow cytometry without the need of any amplification steps.
  • the method involves four distinct steps of conjugation and hybridization: (i) attachment of a 3* -end amine-modified poly-T tailed oligonucleotide sequence with the carboxyl groups on the surface of the nano-polystyrene using carbodiimide conjugation chemistry; (ii) hybridization of the poly- A tailed ccf-miRs of interest with the complementary poly-T sequence on nano- polystyrene; (iii) second hybridization of a fluorescent molecule (6FAM)- labelled, target ccf- miR complementary locked nucleic acid (fLNA) probe with the ccf-miRs of interest; and (iv) evaluation of the sensing capability of the methodology through fluorescence based detection using flow cytometry.
  • 6FAM fluorescent molecule
  • fLNA target ccf- miR complementary locked nucleic acid
  • the novelty of the method is the detection of the fluorescence response generated by the composite nano-hybrid assembly formed in the presence of the target ccf-miR in a given isolated sample.
  • the target ccf-miR will be sandwiched between the two frameworks that will be appropriately gated and determined in flow cytometry. Contrastingly, the presence of non- complementary ccf-miRs would not result into formation of the composite assembly which will not induce any signal generation due to highly specific nature of the fLNA indicating the high selectivity of the method.
  • Figure 1 Schematic representation of the processes involved in the array methodology for determination of the ccf-miRs in a given sample using the flow cytometry.
  • Figure 2 Schematic illustrating the hybridization reactions involved in the preparation of sandwiched nano-hybrid structure with respective facets for the determination of specific ccf- miRs.
  • Figure 3 Schematic illustration of the hybridization and detection of the target poly-A tailed ccf-miRs by the developed nano-polystyrene based method following complementary base pair matching.
  • nano-hybrid array refers to an appropriate flow cytometry method that is based on detection of the fluorescence generated by the sandwiched assembly on recognition of the ccf-miR of interest in a given isolated sample.
  • nano-polystyrene comprise of a highly charged, relatively hydrophilic, a 'fluffy* surface copolymerized with carboxylic acid containing polymers that makes them electrosterically stable structures. This forms the base for the development of the capture facet of the developed nano-hybrid array method.
  • carboxylicde conjugation herein represents the coupling chemistry between the carboxylic groups on the surface of nano-polystyrene and the amine-groups of the poly-T oligonucleotide sequences.
  • Carbodiimide chemistry involves EDC-NHS based activation of the carboxyl groups followed by hybridization with the amine groups present at 3* -position of the poly-T oligonucleotide sequences which yields stable conjugates in aqueous solutions.
  • oligonucleotide sequence is a customised oligonucleotide sequence with poly-T nucleotides at the 5* -end and amine-modification at the 3* -end. This sequence would serve as a binding site for the effective hybridization of the ccf-miRs available in a given isolated samples. This hybridization depends upon the cooperative binding of the poly-A tailed ccf-miRs with the poly-T sequences of the capture hybrid resulting in the formation of complementary A-T base pairing via hydrogen bonds and attachment of the available ccf-miRs in the given isolated sample.
  • ccf-miRs refers to specific cell-free circulating miRNAs which are found to be significantly altered in several pathologies.
  • the altered expression of these ccf- miRs may provide the idea of the altered biological processes contributing to the occurrence, development and progression of a disease.
  • poly-T poly-A hybrid
  • poly-T : poly-A hybrid refers to an assembled framework formed as a result of hybridization of the poly-adenylated ccf-miR sequences with the poly-T oligonucleotide attached to the nano-polystyrene on the basis of complementary base pairing. This hybrid acts as the capture element of the developed array.
  • ccf-miR : fLNA hybrid stated herein refers to the hybridized structure formed after the complementary attachment of the miR-specific fLNA probe with the seed sequence of the ccf- miR of interest.
  • the “sandwiched nano-hybrid assembly” mentioned herein denote the complex sandwich structure formed after the capture of the poly-adenylated ccf-miRs with the poly(T) sequence of the ONPS and hybridization of the miR-specific fLNA probe with the complementary sequence of the target ccf-miRs.
  • This nano-hybrid assembly will generate a detectable fluorescent signal that will be examined using flow cytometry.
  • the shift in the fluorescence intensity observed as a result of precise detection of the target ccf-miRs can significantly indicate the selectivity and sensitivity of the nano-hybrid sandwiched assembly.
  • the present disclosure describes a method for the real-time, accurate, and rapid recognition of the circulating cell-free miRNAs (ccf-miRs) in a sandwiched assembly by using the developed nano-hybrid array method.
  • the detection process involves two hybridization reactions. Firstly, the formation of a “poly-T : poly-A” hybrid that comprise of an assembly of the poly- adenylated ccf-miRs previously isolated from the plasma samples with a 30 bp of poly-T oligonucleotide sequence conjugated on the nano-polystyrene surface.
  • the detection limits of developed array offer a proof-of-concept of sensitivity of the present invention providing a significant relevance in the real-time analysis of ccf-miRs in isolated samples.
  • the present invention selectively determines ccf-miRs of interest in the samples irrespective of the presence of other ccf-miR species, a crucial feature for development of any characterization array protocol.
  • the developed nano-hybrid assembly based methodology allows rapid, reproducible and real- time quantification of disease specific ccf-miRs with high specificity without the requirement of any complex & time-consuming amplification steps.
  • the developed methodology might assist in high sensitive and selective characterization of the aberrantly expressed ccf-miRs in isolated samples that would assist in prediction of the altered biological processes for further analysis of different pathologies.
  • the proposed array methodology is based on the competent hybridization of the miR-specific fLNA (6-FAM labelled LNA) probe with the ccf-miRs of interest captured by the poly-T oligonucleotide sequence conjugated to the surface of nano-polystyrene. This sandwiched complex exhibits a strong fluorescence-based signal which is easily detected by flow cytometry.
  • the methodology requires appropriate positioning of the poly-T oligonucleotide sequences on the nano-poly styrene (ONPS) for the effective capture of poly-adenylated ccf-miRs.
  • the formed complex is then allowed to bind with customized fLNA probes to identify the specific ccf-miR.
  • the fLNA probes are conformationally restricted nucleotide analogs that comprise of a ribose molecule in which 2'-oxygen and the 4 '-carbon are associated with a methylene group to mimick RNA sugar conformation.
  • the fLNA probes bind with the target miRNA molecules in a sequence-specific manner abiding by the Watson-Crick base pairing rule and exhibit high affinity towards the complementary target miRNAs sequences.
  • Such probes offer significant melting temperature (Tm) difference between a perfectly matched ccf-miR of interest and a mismatched ccf-miR, thereby offer good mismatch discrimination, provided with optimal hybridization conditions (Vester B, Wengel, 2004; Mishra and Mukhopadhyay, 2013).
  • the generalized workflow of the array include (i) effective attachment of the poly- A tailed ccf- miRs of interest with the poly-T tailed oligonucleotide sequence conjugated to the surface of nano-polystyrene that result in the formation of the poly-A ccf-miR: poly-T hybrid; (ii) formation of a second ccf-miR : fLNA hybrid structure on specific complementary attachment of the miR-specific fLNA probe with the ccf-miRs of interest that exhibit a detectable fluorescent response; and (iii) assessment of the fluorescent signal generated by the sandwiched structure of poly-T : poly-A ccf-miR : miR-specific fLNA using flow cytometry.
  • the magnitude of fluorescence intensity relative to the detection of ccf-miRs of interest would help in the quantitative determination of target ccf-miRs in a given isolated sample.
  • the development of array method comprise of four major steps of conjugation and hybridization. Fabrication of the ONPS capture hybrid involved the initial washing and activation of carboxyl- functionalized nano-polystyrene by EDC-NHS coupling reaction. This is subsequently followed by conjugation to the amine-terminated poly-T oligonucleotide sequence. The capture hybrid serves as a template for the binding of the entire poly-A tailed ccf-miRs available in a given sample.
  • the ccf-miRs from the plasma samples are isolated using the recommended kit protocol which is then subjected to poly-adenylation reaction. Incubation of the poly-A tailed ccf-miRs with the poly-T oligonucleotide sequence conjugated nano-polystyrene that result in generation of poly-A : poly-T hybrid between the capture facet and available ccf-miRs.
  • the ccf-miR of interest is detected by hybridization of the miR-specific fLNA probe with the captured target ccf-miRs on the basis of complementary base pairing. Such hybridization will induce generation of a fluorescent signal which is detected by flow cytometry using proper voltage and gate settings.
  • the current invention provides a real-time and reproducible approach for the quantitative estimation of ccf-miRs from isolated samples that withholds great promise for early disease risk assessment.
  • the authors initially processed the nano-poly styrene for washing and activation prior to conjugation with the poly-T sequences.
  • the preliminary process involved pre-activation of the carboxyl-groups on the surface of nano- polystyrene that was further coupled with amine-modified customised oligonucleotide probe via the formation of amide linkage. This coupling was promoted by EDC.HC1 (N-Ethyl-N'-(3- dimethylaminopropyl) carbodimide, Hydrochloride) and N-hydroxysuccinimide (NHS).
  • EDC.HC1 N-Ethyl-N'-(3- dimethylaminopropyl) carbodimide, Hydrochloride
  • NHS N-hydroxysuccinimide
  • the nano-polystyrene were initially washed using wash buffer followed by addition of the 50 ⁇ L each of EDC and NHS solutions (10 mg/mL solution prepared in the activation buffer i.e. 50 nm of MES buffer, pH 5.5) and incubation at room temperature.
  • EDC and NHS solutions 10 mg/mL solution prepared in the activation buffer i.e. 50 nm of MES buffer, pH 5.5
  • a 20 ⁇ L of 3* end amine-modified poly-T oligonucleotide solution (10 pM) was added. This mixture was allowed to incubate for 3 hours at room temperature with intermittent vortexing.
  • 0.05% w/v solution of Tween-20 was added. The mixture was finally vortexed and centrifuged followed by the removal of supernatant.
  • the methodology illustrated in the present invention provides a pragmatic, highly reliable array that is capable of providing results within 65 minutes.
  • This promptness makes the method highly attractive for the use as an analytical array for characterization particularly for the quantitative determination of ccf-miRs in the given samples wherein reliability, sensitivity and promptness are prerequisite features.
  • the methodology exhibited high selectivity which was evaluated using flow cytometry. The results showed no significant change in the fluorescence intensity of the developed complex nano-polystyrene based array in the absence of any ccf-miRs (i.e. in the blank solution free of nucleotides).
  • the probability of finding the desired ccf-miR sequence in the real-time samples is extremely low, unless the sample is enriched in the target ccf-miRs species.
  • the selectivity results demonstrate that the array provides a highly precise methodology for the specific detection of target ccf-miR even in low concentrations.
  • the present methodology could provide a remarkable approach for the detection of ccf-miRs in intricate samples. Remarkable results of the selective and sensitive nature of the developed array were obtained when assessed in samples enriched in miR-U6 and miR-16-5p isolated from the plasma samples.
  • miR-U6 In comparison to the ubiquitously expressed miR-U6, a significant difference was observed in the magnitude of the fluorescence intensity of miR-16-5p which is found to be altered in various pathologies.
  • the miR-U6 was used as control for comparison as these species are highly stable and conserved among the miRNA species (Lou et al., 2015; Duan et al., 2018).
  • the prime aspect of the present invention involves quantitative determination of ccf- miR of interest (miR-U6 and miR-16-5p) using the developed array methodology by following the below mentioned sequential steps: a. Obtaining specific ccf-miRs of interest from the plasma followed by poly-A tailing. This is subsequently followed by a PCR amplification of the target ccf-miRs, which serve as positive controls and sample devoid of any nucleic acids i.e., only the conjugate nano-polystyrene which is considered as a negative control. The ccf-miRs were isolated from the plasma samples using the recommended manufacturer’s protocol (Macherey- Nagel, Germany).
  • the isolated ccf-miRs were then subjected to poly-adenylation reaction using the standardized laboratory protocol of Invitrogen poly-A tailing kit (ThermoFisher, USA).
  • the poly-A tailed ccf-miRs sample comprising of a complex of ccf-miRs belonging to different families was denoted as the test sample.
  • each nano-polystyrene can be coated with a number of oligonucleotides.
  • the carboxyl groups on the surface of the nano- polystyrene were activated that result in the formation of a reactive intermediate which was stabilized by addition of NHS.
  • the activated carboxyl groups were conjugated with the terminal amine group of the oligonucleotide sequence through amide bond formation.
  • the poly-A tailed ccf-miRs of interest was allowed to bind to the poly-T tailed oligonucleotide sequence adhered to the nano-polystyrene resulting in the formation of poly-T : poly- A hybrid structures. This mixture was allowed to incubate for 5 minutes followed by immediate cooling in ice.
  • the identification of ccf-miRs of interest utilizes a highly specific miR-sequence complementary fLNA probe based on complementary base pairing.
  • the “fLNA probe” herein described refers to a locked nucleic acid probe that is labelled with a fluorescent molecule namely, 6-carboxyfluorescein (6FAM) at the 3' terminus of the probe sequence. This forms the detecting facet of the proposed array methodology.
  • the fLNA probe consists of 17 nucleobases with fifteen DNA bases and two 2* O-methyl RNA bases. This probe is customised to possess sequence complementary to the seed sequence of target ccf-miR.
  • the fLNA probe possessing complementarity with the target ccf-miR generate a fluorescent response upon detection of ccf-miRs of interest in the given sample. This mixture was incubated at 50°C for 60 minutes and immediately cooled on ice.
  • the fluorescent signals were analysed using flow cytometry.
  • the customised fLNA probe upon hybridization with the target ccf-miR exhibits an increase in the fluorescence intensity with excitation and emission maxima of 485 and 520 nm respectively. Owing to the high specificity, fLNA probe will bind only to those target ccf-miR sequences which are captured by the poly- T oligonucleotide bound nano-polystyrene disregarding other ccf-miRs, thereby displaying the high selectivity of the developed array method.
  • Signal assessment through flow cytometry The assessment of signals through flow cytometry involves blank, negative control, positive control and test samples.
  • a blank comprising of unconjugated nano-polystyrene was assessed to optimize voltage and gate settings as well as nullify the auto fluorescence.
  • the positive controls were tested to assess the detection capability of the developed array to accurately determine the ccf-miRs of interest in the enriched samples that demonstrated a positive correlation of the fluorescence intensity with the detected ccf-miRs.
  • the results obtained after the analysis of the positive controls may be used as a reference for the quantitative determination of the target ccf- miRs in the intricate poly-adenylated test samples. It is important to note that the sequence of the steps of the present invention as mentioned herein must be strictly followed in order attain the efficient determination of the ccf-miRs of interest in real-time settings.
  • the current invention can be further implemented as a revolutionary, simplified, routine analytical procedure capable of quantitative estimation of ccf-miRs and accomplishing fluorescence-based detection within 65 minutes.
  • This developed invention amalgamates the specific affinity of the poly-T tailed oligonucleotide sequence towards the poly-A tails of the ccf-miR sequences and the high complementarity of the fLNA probe. This results in the formation of sandwiched assembly between which the target ccf-miRs is captured.
  • Such hybridization allows the precise detection of only the ccf-miRs of interest, disregarding other available ccf-miRs in the intricate sample containing different families of ccf-miRs.
  • the present innovation henceforth, combines the liquid biopsy and fluorescence sensing methods as a pragmatic analytical characterization approach.
  • the present invention provides a method for estimation of ccf-miRs of interest in an isolated sample by an array, said method comprising the following sequential steps: a. Obtaining specific ccf-miRs of interest from the isolated biological samples b. attaching poly-adenine molecules with ccf-miRs of interest to generate poly-A tailed ccf-miRs; c. Capture of the poly-A tailed ccf-miRs by a poly T oligonucleotide sequence adhered to the surface of nano-polystyrene spherical structures making the capture facet resulting in the formation of poly-A:poly-T hybrid structures; d.
  • fLNA locked nucleic acid
  • step (c) in the capture facet comprising the poly-A:poly-T hybrid structures obtained in step (c) is allowed to hybridize with the poly-adenylated ccf-miRs sequences of step (b) for 5 min at 60-65°C and allowed to cool in ice to arrest the process of linearization.
  • step (d) the highly specific complementary fLNA probe in the range of 50- 100 pl was added and allowed to incubate for 60 minutes at 50-55°C.
  • the detection of the fluorescent signal generated using flow cytometry comprises a. analyzing a blank sample comprising of only nano-polystyrene that is completely devoid of any nucleic acids; b. analyzing the negative control comprising of the poly-T oligonucleotide sequences conjugated nano-polystyrene to analyze and nullify any auto fluorescence generated if any; c. testing the positive controls comprising of poly-A tailed ccf-miRs of interest that demonstrate a positive correlation of the fluorescence intensity with the ccf-miRs detected; d. using the positive controls of step (c) as a reference standard for the quantitative determination of the ccf-miRs in the samples under test.
  • the molecule of interest is a defined family of ccf-miRs.
  • the defined family of ccf-miRs of interest is selected from the group comprising U6 and miR-16-5p, miR-29c, miR-126, miR-146-b, miR-182, miR-210, miR-375 and miR-486-5p.
  • nano-polystyrene spherical structures are 1500-2500 nm in size range and are surface functionalized with carboxyl groups, resulting in formation of the rigid structure for the binding of the amine-modified poly-T oligonucleotide sequence for the fabrication of the capture facet of the nano-hybrid.
  • the oligonucleotide sequence comprises a poly-T sequence with amine-modification at the 3* -end that binds with the activated carboxyl groups on the surface of nano-polystyrene for the preparation of the capture facet, wherein this sequence acts as a template for hybridization of the ccf-miRs of interest based on the base-pair matching.
  • the poly-T oligonucleotide is capable of capturing the poly-A tailed ccf-miRs in a given sample resulting in the formation of poly-T : poly-A ccf-miR hybrid.
  • fLNA probe indicates the 6FAM labelled miR-specific LNA probes that is capable of binding with the ccf-miRs of interest captured by the nano-polystyrene for detection.
  • the fLNA probe is capable of generating a fluorescent signal on hybridization with the target ccf-miRs.
  • flow cytometry is used for the detection of the fluorescent signal generated after the formation of sandwiched structure in which ccf- miRs are stacked in between the poly-T and fLNA probe sequences.
  • the poly-T tailed oligonucleotide sequences used are appropriately designed to capture poly-adenylated ccf-miRs species in a given isolated samples.
  • the nano-polystyrene with surface carboxyl groups are suitably washed and activated for the attachment of the poly-T tailed oligonucleotide sequences.
  • the poly-T: poly-A hybrid structure is formed on incubation with the poly-adenylated ccf-miRs.
  • fLNA probes are customised for the specific hybridisation with the ccf-miR molecule of interest, in particular miR-U6 and miR- 16-5p.
  • the fluorescence is generated by the fluorescence generating molecule 6-carboxyfluorscein (6FAM).
  • the sample is a solution containing poly- adenylated ccf-miRs extracted from a biological fluid preferably plasma, serum, saliva, urine, bronchial-alveolar lavage and pleural effusions.
  • Yet another embodiment of the present invention provides an array, wherein said array is comprised of the following facets: a) a capture facet which comprise of poly-T sequence coated nano-polystyrene spherical structures; b) a fluorescent miR-specific detector facet, complementary 6FAM labelled LNA probes.
  • a detector probe solution comprising of a miR-specific appropriately customised LNA capable of hybridizing with the captured ccf-miRs of interest;
  • an optional suitably prepared wash buffer comprising of 0.1X PBST for the washing and re-suspension of the sandwich nano-hybrid required in the preparation of samples for flow cytometry analysis;
  • Yet another embodiment of the present invention provides in vitro use of the developed array as explained above for the qualitative and quantitative estimation of the ccf-miRs of interest in at least one isolated sample using fluorescence detection based flow cytometry method that comprise of: a) a capture hybrid solution comprising of the nano-polystyrene spheres conjugated to the customised poly-T oligonucleotide sequences; b) a detector probe solution comprising of a miR-specific appropriately customised LNA capable of hybridizing with the captured ccf-miRs of interest; c) an optional suitably prepared wash buffer comprising of 0.1X PBST for the washing and re-suspension of the sandwich nano-hybrid required in the preparation of samples for flow cytometry analysis; for implementation of the method as elucidated in the above embodiments.
  • Yet another embodiment of the present invention provides use of the method as explained above in early diagnosis and prognosis of severe diseases including non-small cell lung cancer.
  • Nucleospin® miRNA plasma kit for ccf-miRs isolation was procured from Macherey-Nagel, Germany.
  • Poly-A tailing kit was procured from Invitogen (Thermofisher Scientific, US).
  • PrimeScript 1st strand cDNA synthesis kit was obtained from Takara (Japan).
  • Taq 2X mastermix was obtained from NEB (USA).
  • CML latex coated beads, EDC (l-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) were procured from Thermofisher Scientific (USA).
  • MES 2-(N-morpholino)ethanesulfonic acid
  • Tween 20 was procured from Bio-Rad Laboratories (USA).
  • oligonucleotides whose sequences are mentioned in the table below were appropriately customized in accordance to the specificity and requirement.
  • Activation buffer A 50 mM MBS buffer, pH 5.5 was prepared by dissolving 976.2 mg of MBS (2-(N-morpholino) ethanesulfonic acid) in 100 mL of distilled water.
  • Washing buffer 0. IX phosphate-buffered saline with Tween® detergent (0. IX PBST) was used washing buffer. The buffer was prepared using 500 ⁇ L of stock solution of 20X PBS into a 99.5 mL of distilled water. To this solution, 5 mL of tween 20 was added to prepare a working solution of 0.1X PBST.
  • EDC solution 10 mg/mL solution was prepared in activation buffer (Note: Always use freshly prepared solution)
  • Amine-modified oligonucleotide & LNA probe The amine-modified poly-T oligonucleotide sequences and fLNA probes (U6 and miR-16-5p specific) were reconstituted in nuclease-free water.
  • the plasma samples were subjected to ccf-miRs isolation as per the prescribed instructions of a commercialized kit (Nucleospin® miRNA plasma kit, Macherey-Nagel). Briefly, plasma was added to the silica column to allow circulating miRNAs to bind on the membrane. The membrane was subjected to a series of washing for the removal of impurities. The bound ccf- miRs fraction was then eluted and quantified using Nanodrop 2000 spectrophotometer. The isolated ccf-miRs were then subjected to poly-adenylation reaction using the recommended laboratory protocol of Invitrogen poly-A tailing kit (ThermoFisher Scientific).
  • the isolated ccf-miRs were mixed with the appropriate volumes of 5X EPAP buffer, MnCh, ATP solution, EPAP enzyme and nuclease free water. The mixture was subjected to the previously standardised thermocycler program for poly-adenylation reaction at 37°C for 60 minutes and 4°C.
  • Thermocycler program for poly-adenylation reaction For the preparation of target ccf-miRs enriched samples, the poly-adenylated ccf-miR sequence (lOOOng) was subjected to reverse transcription reaction. The cDNA was synthesised as per the directions of the PrimeScript 1st strand cDNA synthesis kit (Takara, Japan) using the below mentioned thermocycler program. For the preparation of ccf-miRs enriched samples, the obtained cDNAs were ligated with miR-specific primer sets and amplified using conventional PCR method in GeneAmp® PCR system 9700 (Applied Biosystems) using Taq 2X Master Mix (NEB, US). The PCR products so obtained were further subjected to poly-adenylation reaction as per the preceding protocol for the preparation of ccf-miRs-enriched samples which will serve as the positive control.
  • the nano-polystyrene comprise of spherical structures with carboxylic groups on the surface making them electrosterically stable.
  • the carboxyl groups were initially pre-activated using EDC-NHS protocol.
  • a suspension (4% w/v) comprising of 2.204 x 10 9 billion nano-polystyrene spheres was subjected to washing with 1 mL washing buffer followed by centrifugation at 10,000 x g for 10 minutes.
  • EDC solution 50 ⁇ L of EDC solution and 50 ⁇ L of NHS solution freshly prepared in activation buffer (50mM MES buffer) were added and vortexed properly. This mixture was allowed to incubate for 20 minutes at room temperature. To this solution, 20 ⁇ L of 3 ’end amine-modified poly(T) tailed oligonucleotide solution (10 pM) was added. Subsequently, a second aliquot of 50 ⁇ L of EDC solution and 50 ⁇ L of NHS solution was added to the above mixture for efficient binding. The final mixture was vortexed thoroughly and incubated for 3 hours at room temperature with intermittent vortexing.
  • activation buffer 50mM MES buffer
  • the performance ability of the developed array was assessed through flow cytometry which involved blank, negative control and positive control.
  • the samples devoid of any nucleic acids served as negative controls while the samples enriched in miR-16-5p and U6 acted as positive controls. All these samples were tested against a blank solution which does not incorporate any capture and detector facet (i.e blank nano-polystyrene). This was necessary to analyze and nullify the auto-fluorescence (if any) generated by the conjugated nano-polystyrene to prevent the interference with the fluorescence intensity of the developed array.
  • a blank sample comprising of only nano-polystyrene that is completely devoid of any nucleic acids was analyzed.
  • the blank was analyzed in the Attune NXT flow cytometer to adjust gating and voltage settings.
  • BL1-H channel using 488 nm excitation and the 530/30 nm bandpass filter were set.
  • negative control and positive control were analysed.
  • the negative control comprised of the poly-T oligonucleotide sequences conjugated nano-polystyrene to analyze and nullify any auto fluorescence generated if any.
  • the negative controls demonstrated no shift in the fluorescent intensity due to absence of ccf-miRs available for binding.
  • the positive controls comprising of poly-A tailed ccf-miRs of interest was tested that demonstrated a positive correlation of the fluorescence intensity with the ccf-miRs detected.
  • a significantly observable shift in the fluorescence intensity was observed in the samples enriched in target ccf-miR species.
  • Example 2 Detailed protocol of the developed array and implementation of the protocol for the detection of miR-16-5p and miR-U6
  • the array method described herein is based on the detection of the sandwiched structure generated by the formation of a poly-T : poly-A ccf-miR : fLNA probe assembly.
  • the sandwiched hybrid is formed as a result of binding of the target ccf-miR with the capture facet on nano-polystyrene and miR-specific fLNA probe.
  • the capture facet comprising of poly(T) tailed oligonucleotide sequence (10 ⁇ M) conjugated nano-polystyrene was allowed to hybridize with the poly-adenylated ccf-miRs sequences available in the samples (20 ⁇ L) for 5 min at 65°C. This was allowed to cool in ice to arrest the process of linearization. Subsequently, the highly specific 6-FAM labelled U6 and miR-16-5p complementary fLNA probe (10 pM) capable of identifying only U6 and miR-16-5p species was added and allowed to incubate for 60 minutes at 50°C. This sandwiched framework was analyzed using flow cytometry with respect to negative controls.
  • the array protocol followed in the present invention and illustrated in the figure 1 involves the following major steps: (i) capture of the available poly(A) tailed ccf-miRs in the samples by the poly(T) tailed oligonucleotide conjugated to the nano-polystyrene; (ii) hybridization of the target ccf-miR with the miR-specific fLNA probe; and (iii) fluorescent detection using flow cytometry.
  • the present methodology offers simplified, quantitative, selective identification of the ccf-miRs of interest present in the given isolated samples.
  • the present invention comprise of a methodology that involves efficient conjugation of the target poly(A) tailed U6 and miR-16-5p to the poly(T) tailed oligonucleotide sequence conjugated to the surface of the nano-polystyrene followed by the proficient and selective hybridization to the miR-specific fLNA probe on the basis of complementary base pairing. This forms a sandwiched structure and generates signals that are detected through flow cytometry.
  • the table 2 mentioned below summarizes optimized experimental variables for the detection of miR-16-5p and miR-U6 according to the standardized protocol.
  • the efficiency of methodology in detection of ccf-miRs was assessed in low sample volumes.
  • the volume of conjugated nano-polystyrene and fLNA probe solution used were kept constant while varying volumes of ccf-miRs were used.
  • the results demonstrated that the developed nano-hybrid could effectively determine ccf-miRs of interest in low sample volumes.
  • the fluorescence intensity decreased with the increase in the sample volume. This may be probably due to increase in the competition for binding with the poly-T oligonucleotide sequences and LNA probe hybridization.
  • the ideal sample volume selected for analysis was 1-20 ⁇ l.
  • the selectivity tests involved the analysis of the fluorescent signal generated upon detection of the ccf-miRs of interest with respective miR-specific fLNA probes using the detailed protocol described in example 2.
  • the fluorescence response generated in the presence and absence of target ccf-miRs captured on the nano-polystyrene was compared.
  • the capability of the array to identify only the target sequence precisely demonstrated the selectivity of the methodology.
  • the developed array was evaluated for real time detection capability of the endogenous level of a target ccf-miRs in the samples obtained after isolation of the ccf-miRs from plasma followed by poly-adenylation reaction.
  • the analysis was performed in accordance with the previously mentioned protocol as detailed in example 2.
  • test samples containing a known concentration of ccf-miRs (1 ng) isolated from the plasma was used for determination of miR-U6 and miR-16-5p.
  • the results demonstrated an evident shift in the fluorescence spectra clearly indicating the selective nature of the developed array methodology in detection of the target ccf-miRs in the presence of different ccf-miRs.
  • the present invention could be able to detect minute concentrations of ccf-miRs of interest present in an isolated sample due to high affinity and complementarity of the fLNA probes with the respective ccf- miRs. The results are illustrated in table below.
  • the developed methodology was evaluated for the feasibility of the array as a routine analytical approach for the sensitive estimation of minuscule quantum of a target ccf-miRs in the intricate samples obtained after isolation from plasma. These analyses were performed in accordance with the previously mentioned protocol mentioned in example 2 but using a different set of samples in order to check the high sensitivity of the developed array directly in the isolated samples without any reverse transcription and amplification processes.
  • the interesting results obtained by the array methodology confer the precise detection of miR-U6 and miR-16-5p in the isolated samples at low concentrations.
  • the reproducibility of the array methodology was determined by comparative analysis of the fluorescence responses generated by the sandwiched nano-hybrid framework comprising of capture and detector hybrids fabricated using the standardised protocol (as mentioned in the example 2) for the detection of target U6 and miR-16-5p in the ccf-miRs enriched samples.
  • Table 8 Reproducibility of the array in detection of miR-U6 in isolated samples followed by poly- A tailing
  • the method discussed herein involves a combination of oligonucleotide conjugated nano- polystyrene as capture facet and customised fLNA as the detector sequence of the composite nano-hybrid framework that provide a precise and rapid ccf-miR quantitative estimation array.
  • the array method offers generation of reliable data while avoiding time-consuming and intricate processing steps. Additionally, the developed array opens the opportunity for customization of the protocol for focussed and multiplexed arrays targeting a definite subset of ccf-miR species.
  • PK Beloglazova NV
  • Goryacheva IY Water-dispersed luminescent quantum dots for miRNA detection. TrAC. Trends Anal. Chem. 2019; 111:197-205. https://doi.Org/10.1016/i.trac.2018.12.022
  • MicroRNA-106b serves as a prognostic biomarker and is associated with cell proliferation, migration, and invasion in osteosarcoma. Oncol Lett. 2019 Sep;18(3):3342-3348. https://doi.org/10.3892/ol.2019.10666 30. Yang YN, Bian LQ, Ling XD, Fang CY, Jiang SL. MicroRNA-421 promotes proliferation and invasion of non-small cell lung cancer cells through targeting PDCD4. Pathol Res Pract. 2019 Jul 23:152555. httns://doi.ora/10.1016/i.prp.2019.152555

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Abstract

La présente invention concerne un procédé basé sur une cytométrie en flux pour la reconnaissance précise et l'estimation quantitative des miARN circulants acellulaires (ccf-miR). Le réseau est basé sur la capture des ccf-miR par la formation d'un nouveau composite nanohybride comprenant un hybride oligonucléotide-latex de nano-polystyrène (ONPS) et un acide nucléique verrouillé marqué par un fluorophore hautement spécifique (fLNA) assemblés ensemble. Le procédé comprend les deux étapes distinctes suivantes : a) capture des ccf-miR par hybridation des queues poly-A des ccf-miR avec les séquences poly-T conjuguées au nano-polystyrène ; et b) identification des ccf-miR cibles avec des sondes fLNA spécifiques générant un signal détectable indiquant la présence de la cible dans un échantillon donné. La présente invention concerne également l'application dudit procédé dans l'analyse ultérieure des processus biologiques altérés pour le diagnostic précoce et le pronostic de maladies graves.
PCT/IN2022/050239 2021-07-13 2022-03-15 Réseau nanohybride basé sur le polystyrène fluorescent pour l'estimation des mir circulants acellulaires WO2023286073A1 (fr)

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