WO2025055169A1 - A fluorescent probe to measure rna binding and elongation - Google Patents

Abstract

Provided are methods to synthesize novel probes and methods of using said probes to measure RNA binding and elongation. The novel probe is an RNA probe with a five prime cap structure and a detectable modified nucleoside triphosphate. The novel probe can be used to measure the activities and the kinetics of capped RNA-dependent RNA polymerases and cap-binding proteins. The novel probe can also be used to screen the mutant proteins and inhibitor compounds of the capped RNA-dependent RNA polymerases and cap-binding proteins.

Description

A FLUORESCENT PROBE TO MEASURE RNA BINDING AND ELONGATION Cross-Reference to Related Application

This application claims the benefit of U.S. Patent Application Serial No. 63/582,077, filed September 12, 2023, which is hereby incorporated by reference in its entirety including any tables, figures, or drawings..

Background Art

In recent years, scientific interest in measuring the detailed process of RNA binding and elongation has increased.It is important to profile and measure the binding and elongation kinetics of RNAsynthesis to derive a general understanding of the relationship of RNA processing and gene expression.RNA viruses, such as Influenza virus, Dengue virus, SARS-CoV-2 virus, and Yellow Fever virus, have affected the world widely. It is important to have an efficient and sensitive tool to measure viral RNA transcription and replication in real-time. Measuring RNA binding and elongation can have a variety of beneficial uses in the medical industry, including the vaccine and anti-viral drug development.

The known method for measuring RNA binding and elongation mainly involves the radioactive isotopes ³²P labeling on the nucleotide, whichrelies on capturing high energy β emission. Experimentsinvolving isotopes can only be conducted in licensed facilities with extensive protection equipment by authorized personnel, which can be harmful to the researchers and the surrounding environment.Furthermore, radioactive isotope-labeled RNA is by nature difficult to obtain, and the price of it has increased. The RNA extension assay, using radioactive isotope-labeled RNA,can only be measured at the endpointof the reaction,once because the energy from the isotope needs to be converted.Radio isotope-labeled RNA is also unstable and cannot be preserved for a long time due to its extremely short half-life (around 15 days).

Thus, a novel, inexpensive, easy-to-obtain, and environmentally friendly method of measuring RNA binding and elongation is needed.

Brief Summary of the Invention

The subject invention provides novel probes and methods to measure RNA binding and elongation. In certain embodiments, the novel probe is a fluorescent labelled RNA primer. In certain embodiments, the fluorescent labelled RNA primer is a capped RNA primer. In certain embodiments, the novel probe can be used tomeasure the activities of RNA-dependent RNA polymerases(RdRP), such as, for example,RNA-dependent RNA polymerases of viruses including, for example, viruses from the Orthomyxoviridae family, such as, for example, aninfluenza virus;the Flaviviridae family, such as, for example,Dengue virus, Zika virus, West Nile virus, and yellow fever virus;the Bunyavirales order, such as, for example, aRift Valley fever virus; the Coronaviridae family, such as, for example, SARS-CoV and SARS-CoV-2. 

In certain embodiments, the subject probe is about 10 to about 15 nucleotides long or about 11 nucleotides long.In certain embodiments, the subject probes, for example,m7G(5')ppp(5')(2'OMeA)pGGC/fluorescein-U/ACCAAG (SEQ IDNO: 1),m7(3'OMeG)(5')ppp(5')(2'OMeA)pGGC/fluorescein-U/ACCAAG (SEQ ID NO: 2) m7G(5')pppAGGC/fluorescein-U/ACCAAG (SEQ IDNO: 3),m7RpppN…/modified-N/N…,m7RpppNmN…/modified-N/N… ,m7RpppNmNmN…/modified-N/N…, can be transcribed from a specific DNA sequence, such as, for example, TAATACGACTCACTATA-AGGCTACCAAG (SEQ ID NO: 4), TAATACGACTCACTATA-AGGNTNNNNNN (SEQ ID NO: 5), ATTTAGGTGACACTATA -AGGNTNNNNNN (SEQ ID NO: 6), or AATTAACCCTCACTAAA -AGGNTNNNNNN (SEQ ID NO: 7),which has a 17-bp initiator sequencethat contains, for example, a T7 RNA polymerase promotor sequence(e.g., TAATACGACTCACTATA (SEQ ID NO: 8)), an SP6 RNA polymerase promotor sequence (ATTTAGGTGACACTATA (SEQ ID NO: 9)), or a T3 RNA polymerase promotor sequence (AATTAACCCTCACTAAA (SEQ ID NO: 10)).The sequence to be transcribed into the RNA probe can contain one or more sites for the incorporation of modified NTP or dNTP or NTP analog, for example, the T at position 22 of the template can be transcribed into fluorescent-U.

In certain embodiments, the subject probe is easily accessible, easy to handle, and stable enough. In certain embodiments, the subject probe can be stored at about -80°C for at least for one year. In certain embodiments, the probe can dramatically reduce the experiment time course from 2 days to 2 hours by replacing the currently widely used radio-isotope probe. In certain embodiments, the probe can be used in a real-time assay to monitor the enzyme reaction. In certain embodiment, the subject probe can be used in a variety of applications due to its radiation-free advantage, such as, for example, a gel assay, a plate assay, an in vitro assay, an in vivo assay, with a scanner or a plate reader or a fluorescent imager, and any combination thereof. In certain embodiment, the subject probe is multifunctional due to the use of organic compounds (e.g., fluorescein). Fluorescein can directly test the changes in fluorescence intensity and in fluorescence polarization in real-time, such as, for example, the subject probe can be used to monitor the progress of the enzyme reaction and profile the enzyme kinetics. Furthermore, it is competent in screening mutations and inhibitors, and testing cap-binding virus kinetics.

Brief Description of the Drawings

  FIG. 1:The electrophoresis image of the transcribed products and PAGE-purified probe analyzed in the denatured 20% polyacrylamide gel supplemented with 8M urea, and scanned by the laser scanner at the wavelength of 480 nm.

FIG. 2:The electrophoresis image of viral RNA dependent polymerase activity. Reaction mixtures (10 μL) of Group A contained 1 μM RNA template, 1 μM probe, 0.2 μM viral RNA dependent polymerase, 1x RNase inhibitor, 1mM MgCl ₂ , 1mM dithiothreitol (DTT) and 1x Tris-HCl (pH 8), 1mM rNTP.  Reaction mixtures (10 μL) of Group B contained the same ingredients as Group A but without rNTP.  Reaction mixtures (10 μL) of Group C contained the same ingredients as Group A but without rNTP and viral RNA dependent polymerase.

FIG. 3The viral RNA dependent polymerase activity and kinetics test in plate assay.The probe is bound and extended by the polymerase, leading to changes in fluorescent polarization over time. Reaction mixtures (10 μL) of Group A contained 1 μM RNA template, 1 μM probe, 0.2 μM viral RNA dependent polymerase, 1x RNase inhibitor, 1mM MgCl ₂ , 1mM dithiothreitol (DTT) and 1x Tris-HCl (pH 8), 1mM rNTP.  Reaction mixtures (10 μL) of Group B contained the same ingredient as Group A but without rNTP.  Reaction mixtures (10 μL) of Group C contained the same ingredients as Group A but without rNTP and viral RNA dependent polymerase. The reactions are incubated at 30°C in the plate reader for 1 hour. The fluorescent polarization readouts are recorded every 60 seconds.

FIG. 4:The kinetics curve for different mutants.  Reaction mixtures contained 1 μM RNA template, 1 μM probe, 0.2 μM viral RNA dependent polymerase or mutants for different groups, 1x RNase inhibitor, 1mM MgCl ₂ , 1mM dithiothreitol (DTT) and 1x Tris-HCl (pH 8), 1mM rNTP. The blank group isbasically the same as the others. group but without any polymerase. Reactions are incubated at 30°C for one hour and the fluorescent polarization signals are recorded every 60 seconds.

FIG. 5:The electrophoresis image of viral RNA polymerase inhibitor screening with the probe in a gel assay.Reaction mixtures contained 1 μM RNA template, 1 μM probe, 0.2 μM viral RNA dependent polymerase, 1x RNase inhibitor, 1mM MgCl ₂, 1mMdithiothreitol (DTT) and 1x Tris-HCl (pH 8), 1mM ribonucleoside triphosphate (rNTP), and different concentrations of target drug. After developed and incubated at 30°C for one hour, the reaction mixtures were analyzed in 10% polyacrylamide gel supplemented with 8M urea and scanned by the laser scanner at the wavelength of 480 nm.

  FIG. 6: The cap-binding protein bound to the probe,causing changes in the fluorescent polarization. The cap-binding protein was titrated 2 times into 5 groups and loaded to the reaction mixture containing 1 μM probe, 25mM HEPES (pH 7.4), 150mM NaCl, and 1mM dithiothreitol (DTT). The reactions were loaded into 384 well plates and analyzed in the plate reader. The readouts were recorded every 60s for 15 min.

Brief Description of the Sequences

SEQ ID NO: 1: Exemplary 11-nucleotideprobe with cap-1structure

SEQ ID NO: 2: Exemplary 11-nucleotide probe with cap-1 structure

SEQ ID NO: 3: Exemplary 11-nucleotide probe with cap-0 structure

SEQ ID NO: 4: DNA sequence encoding promotor sequence and exemplary probe

SEQ ID NO: 5: DNA sequence encoding promotor sequence and exemplary probe

SEQ ID NO: 6: DNA sequence encoding promotor sequence and exemplary probe

SEQ ID NO: 7: DNA sequence encoding promotor sequence and exemplary probe

SEQ ID NO: 8: a T7 RNA polymerase promotor sequence

SEQ ID NO:9: a SP6 RNA polymerase promotor sequence 

SEQ ID NO:10:a T3 RNA polymerase promotor sequence.

Detailed Disclosure of the Invention

Selected Definitions 

As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising”.The transitional terms/phrases (and any grammatical variations thereof) “comprising”, “comprises”, “comprise”, “consisting essentially of”, “consists essentially of”, “consisting” and “consists” can be used interchangeably.

The phrases “consisting essentially of” or “consists essentially of” indicate that the claim encompasses embodiments containing the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claim. 

The term “about” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured, i.e., the limitations of the measurement system.In the context of compositions containing amounts of ingredients where the terms “about” are used, these compositions contain the stated amount of the ingredient with a variation (error range) of 0-10% around the value (X ± 10%).In other contexts, the term “about” provides a variation (error range) of 0-10% around a given value (X ± 10%).As is apparent, this variation represents a range that is up to 10% above or below a given value, for example, X ± 1%, X ± 2%, X ± 3%, X ± 4%, X ± 5%, X ± 6%, X ± 7%, X ± 8%, X ± 9%, or X ± 10%.

In the present disclosure, ranges are stated in shorthand to avoid having to set out at length and describe each and every value within the range. Any appropriate value within the range can be selected, where appropriate, as the upper value, lower value, or the terminus of the range. For example, a range of 0.1-1.0 represents the terminal values of 0.1 and 1.0, as well as the intermediate values of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and all intermediate ranges encompassed within 0.1-1.0, such as 0.2-0.5, 0.2-0.8, 0.7-1.0, etc.Values having at least two significant digits within a range are envisioned, for example, a range of 5-10 indicates all the values between 5.0 and 10.0 as well as between 5.00 and 10.00 including the terminal values. When ranges are used herein, combinations and subcombinations of ranges (e.g., subranges within the disclosed range) and specific embodiments therein are explicitly included.

As used herein, the term“biological sample” refers to a sample obtained from a virus. 

As used herein, the terms “determining,” “measuring,” and “assessing,” and “assaying” are used interchangeably and include both quantitative and qualitative determinations.

As used herein, the term “nucleic acid” or “polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms (SNPs), and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene. 

As used herein, an “isolated” or “purified” nucleic acid molecule or polynucleotide is substantially free of other compounds, such as cellular or viral material, with which it is associated in nature. A purified or isolated polynucleotide (ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)) can be free of the genes or sequences that flank it in its naturally-occurring state.Alternatively, an “isolated” or “purified” nucleic acid molecule or polynucleotide may be RNA or genomic DNA purified from its naturally occurring source, such as a prokaryotic or eukaryotic cell and/or cellular material or viral material with which it is associated in nature.

As used herein, “probe” is a single-stranded, capped RNA with modified bases, used as a binding agent to detect the cap-binding ability of a cap-binding protein; or used as a primer to initiate the RNA synthesis of a cap-dependent viral RNA-dependent RNA polymerase, whose bioactivity is thus detectable by, for example, an imager, scanner, or reader.

The term “label” refers to a molecule detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include fluorescent dyes (fluorophores), fluorescent quenchers,luminescent agents, biotin, digoxigenin,or other molecules that can be made detectable, e.g., by incorporating into an oligonucleotide.The term includes combinations of labeling agents.All references cited herein are hereby incorporated by reference in their entirety.

Probes

The subject invention provides novel probes that can measure RNA binding and elongation using a label. In certain embodiments, the probe can be usedto measure the activities of the cap-dependent RNApolymerases. In certain embodiments, the probe can be used to measure the RNA cap binding ability of protein with cap-binding domains, for example, eIF4E protein.

In certain embodiments, the cap comprises of a guanine nucleotide connected to the RNA sequence via an 5’ to 5’ triphosphate linkage.In certain embodiments,this guanosine is methylated on the 7 position. In certain embodiments, the first base of the RNA is methylated on the 2 position. In certain embodiments, the second base of the RNA is also methylated on the 2 position. In certain embodiments, the probe is a five-prime (5’) capped polynucleotide probe, including the cap-0 ( _m7G _ppp), cap-1( _m7G _pppN _m), and cap-2 ( _m7G _pppN _mN _m) structures. In certain embodiments, the capped polynucleotide probe is a labelled, capped RNA polynucleotide.In certain embodiments, the novel probe is an RNA primer.In certain embodiments, the polynucleotide probe is about 7 to about 3000 bases in length, including, for example, about 10, about 11, about 12, about 13, about 14, or about 15 bases in length.Useful labels of the probe include fluorescent dyes (fluorophores), fluorescent quenchers, luminescent agents, biotin, digoxigenin, or other molecules that can be made detectable, e.g., by incorporating into an oligonucleotide. The label can include combinations of labeling agents, e.g., a combination of fluorophores.

Exemplary fluorophores include, but are not limited to, Alexa dyes (e.g., Alexa 350, Alexa 430, Alexa 488, etc.), AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy2, Cy3, Cy5, Cy5.5, Cy7, Cy7.5, Dylight dyes (Dylight405, Dylight488, Dylight549, Dylight550, Dylight 649, Dylight680, Dylight750, Dylight800), 6-FAM, fluorescein, FITC, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, ROX, R-Phycoerythrin (R-PE), Starbright Blue Dyes (e.g., Starbright Blue 520, Starbright Blue 700), TAMRA, TET, Tetramethylrhodamine, Texas Red, and TRITC. 

In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides of the probe can be labelled. In preferred embodiments, nucleotides at positions 5, 6, and/or 7 of the probe can be labelled. In certain embodiments, uridine, adenosine, cytidine, guanosine,thymidine, xanthosines, and inosines, or a nucleoside analog, such as Ribavirin 5'-triphosphate, can be labelled. In preferred embodiments, uridine is labelled. 

In certain embodiments, the probe is in-vitro transcribedbyDNA-dependent RNA polymerase. In preferred embodiments, the probe is in-vitro transcribed by aT7 RNA polymerase.In certain embodiments, the probe is capped by known capping methods.In certain embodiments, the probe is enzymatically capped by a capping enzyme. In preferred embodiments, the probe is enzymatically capped by Vaccinia Capping Systemor Faustovirus Capping Enzyme. In certain embodiments,the probe is co-transcriptionally capped witha capping analog. In certain embodiments, in the in-vitro transcription, a fluorescent dye-labeled uridine triphosphate (UTP), adenosine triphosphate(ATP), cytidine triphosphate (CTP), guanosine triphosphate (GTP)or thymidine triphosphate (TTP) is incorporated into the 5 ^th, 6 ^th, and/or7 ^thpositions of the probe. In certain embodiments, the subject probes, with a generalsequence of _m7G _pppN…/modified-N/N…, _m7G _pppN _mN…/modified-N/N…, _m7G _pppN _mN _mN…/modified-N/N…, can be transcribed from a specific DNA sequence composed of a promoter region and a probe region, for example,TAATACGACTCACTATA-AGGC TACCAAG (SEQ ID NO: 4),and is about 10 to about 15 nucleotides long or about 11 nucleotides long. 

In certain embodiments, the transcribed capped primer can contain one or more fluorescein with the fluorescein labeled UTP incorporated into the primer. The capping analog can allow the co-transcriptional capping of mRNA to produce the mRNA primer with a naturally occurring cap structure. With this fluorescent-capped RNA primer, RNA-dependent RNA polymerase (RdRp) activities in the gel and plate assays can be easily detected. 

Methods of Using the Probes

In certain embodiments, the subject invention comprises methods of using the subject probes to detect and measure the activities of RNA-dependent RNA polymerases (RdRP), such as, for example, RNA-dependent RNA polymerases of viruses including, for example, viruses from the Orthomyxoviridae family, such as, for example, an influenza virus; the Flaviviridae family, such as, for example, Dengue virus, Zika virus, West Nile virus, and yellow fever virus; or the Bunyavirales order, such as, for example, a Rift Valley fever virus.

In certain embodiments,the subject methodsprovide a method for detecting real-time polymerase activity through fluorescent polarization changes on a modern plate reader equipped with a fluorescent polarization module. Polarization is intrinsic physical property of any fluorescent molecules. Thus, the polarization readouts are less dye-dependent and less susceptible to environmental interferences such as pH, salt, temperature than assays based on fluorescence intensity. The polarization is determined from two measurements of fluorescence intensities in parallel and perpendicular plane under the polarized excitation light. 

In certain embodiments, the subject methods can be used to measure the activity and the real-time kinetics of viral RNA polymerase. In certain embodiments, viral RNA polymerase can be subjected to the in vitro transcription reaction containing the subject probe. When the probe is bound or extended by the viral RNA polymerase, the fluorescent polarization readouts will change due to the alteration of the total molecular weight of the RNA-protein-bound complex, which represents the binding kinetic of the viral RNA polymerase. In certain embodiments, the measurement of the viral RNA polymerase can use the subject probe to detect activity and real-time kinetics in a high through-put format, such as, for example, 96-well or 384-well or 1536-well format. In certain embodiments, the subject methods can be used to analyze RNA extension from viral RNA-dependent RNA polymerase by single-nucleotide resolution in an in-vitro transcription reaction. This method can involve preparing the reaction mix by adding necessary components, subjecting the reaction mix containing the subject probe to denaturing gel electrophoresis, and imaging / scanning the gel under a fluorescent gel scanner or imager.The migration distance of the extended products from the probe is proportional to the size of the products. Thus,single nucleotide difference can be revealed in the denaturing gel.

In certain embodiments, the subject methods and probes can be used to monitor and identify theactivity of amutant viral polymerase.For example, a mutant viral polymerasecan be subjected to an in vitro transcription reaction containing the subjectprobe and the signal representing activity can be detected from a plate reader or imager. In certain embodiments, the subject probe can bind to an RNA template and be extended by the mutant viral polymerase. The increasing or decreasing of the fluorescent polarization readout signals(in mP) compared to the wildtype and baseline represents change in the polymerase activity, for example, a mutant viral polymerase has a fluorescent polarization readout of about 70 mP, while wildtype viral polymerase has about a180 mP readout and a baseline of about 0 mP;thus, one can conclude that the mutant viral polymerase has reduced activity.Likewise, the increasing or decreasing of the extended RNA products in the denaturing gel electrophoresis can also represent the increase or decreasing of the viral polymerase activity.In certain embodiments, a mutation screening platform can be used to test a large number of mutations.The viral RNA polymerases can use the subject probes to detect the mutant activityin a high through-put format, for example, 96-well, 384-well, or 1536-well format.

In certain embodiments, the subject invention can be used in an inhibitor screening platform to identify compounds that inhibit the activity of capped-RNA dependent polymerases. For example, atest compoundcan be subjected to an in vitro transcription reaction containing the subject probe, and the differences in the fluorescent polarization readouts, for example, a test reaction with compound A has a fluorescent polarization readout of about 100 mP, while a reaction without compound Ahas a 180 mP readout and baseline of about 0 mP; thus, one can conclude that compound Acan inhibit the viral polymerase. Likewise, the decreasing of the extended RNA products in the denaturing gel electrophoresis in reaction with compound can also represent the ability to inhibit viral polymerase. In certain embodiments, aninhibitor screening platform can be used to test a large amount of compounds using the subject probe to detect the anti-viralactivity in high through-put format, for example, 96-well or 384-well or 1536-well format. 

In certain embodiments, the subject methods can be used to measure the real-time kinetics of a cap-binding protein.For example, a cap-binding protein can be subjected to an in vitro transcription reaction containing the subject probe. As the subject probe is bound by the cap-binding protein in the reaction, leading to an increase to the molecular weight of the RNA-protein-bound complex,the continuous, increasingfluorescent polarization readouts from a binding periodrepresents the binding kinetic of the cap-binding polymerase.In certain embodiments, the measurement of the cap-binding protein kineticscan use the subject probe to detectcap-binding in a high through-put format, for example, 96-well, 384-well, or 1536-well format. 

In certain embodiments, the subject invention can be used in an isotope-free assay for analyzing cap-binding protein and cap-dependent viral RNA-dependent RNA polymerases. Therefore, the subject methods can replace traditional isotope-labeled probes to measure RNA elongation and cap binding. 

Advantageously, the methods to produce the subject invention are more environmentally friendly, cost-effective, and easily obtainable than the traditional, isotope-labeled NTP methods.

Materials and Methods

Preparation and use of the fluorescein-labeled capped RNA probe

There are two methods to synthesizethe fluorescein-labeledcapped RNA probe. 

Method 1is in vitro transcription RNA capping, in whicha cap analogue was added to the reaction to initiate the RNA capping together with the probe synthesis. 

Method 2is enzymatic RNA capping, where the probe is first synthesized without the cap, and enzymatically capped in a separate reaction.

Method 1: the fluorescein-labeled capped RNA probe was in-vitro transcribed by DNA-dependent RNA polymerase supplemented withcap analogue and other necessary components.The DNA template sequence is designed to have one or more target sites in the transcription product region,which allows the DNA-dependent RNA polymerase to incorporate modifiedNTP, or fluorescent-NTP,or other made-detectable NTP, into the capped RNA probe. The synthesis reaction can be set up by mixing the following components:DNA template within the range ofabout 1µg to about 10µg, preferably about 0.5µg to about 2µg, Tris-HCl with a concentration within about 1mM to about  100mM, preferably about 10mM to about 50mM, pH value ofabout 6 to about 10, preferably about 7 to about 9, MgCl ₂, or other divalent metal ions in water-soluble salt form, including but not limited to Ca ²⁺, Cu ²⁺, Fe ²⁺, Co ²⁺, Ni ²⁺, Mn ²⁺, Zn ²⁺, Sn ²⁺, Cd ²⁺, Ba ²⁺, Hg ²⁺, Sr ²⁺, with the concentration ofabout 1mM to about 50mM, preferably in the range of about 2mM to about 10mM, NaCl within the range of about 5mM to about500mM, preferably about20mM to about100mM,  spermidine concentration with range ofabout 0.5mM to about10mM, preferablyabout2mM to about6mM, Dithiothreitol (DTT) with the range ofabout0.5mM to about5mM,preferablyabout 0.5mM to about2mM, adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP) within the concentration ofabout1mM to about25mM, preferably about 5mM toabout 15mM, cap analogue can be any one or more of the following: m7(3’OMeG)pppG, m7GpppG, GpppG, m7GpppA, m7GpppA, ApppG, m7GpppAmG, m7GpppAmU, m7Gpppm6ApG, m7(3’OMeG)ppp(2’OMeA)pG, m7Gppp(2’OMeA)pG, witha range ofabout 1mM to about 10mM, preferably about5mM to about15mM, made-detectable-NTP within the range ofabout0.5mM to about10mM, preferably about1mM to about5mM, and DNA-dependent RNA polymerase with a concentration of about10U to about200U, preferably about 50U to about 100U.The mixture should be incubated at temperaturesof about 20°C to about50°C, preferably about30°C toabout 40°C,and the reaction should last for about0.5hours to about24 hours, preferably for about10 hours toabout 20 hours.

Method 2: the fluorescein-labeled intermediate RNA probe was in-vitro transcribed by DNA-dependent RNA polymerase. The DNA template sequence is designed to have one or more target sites in the transcription product region, which allows the DNA-dependent RNA polymerase to incorporate modified NTP, or fluorescent-NTP, or other made-detectable NTP, into the capped RNA probe. The synthesis reaction can be set up by mixing the following components: DNA template within the range ofabout 1µg to about10µg, preferably about0.5µg toabout 2µg, Tris-HCl with a concentration withinabout 1mM to about100mM, preferably about10mM toabout 50mM, pH value ofabout6 to about10, preferably about7 to about9,MgCl ₂, or other divalent metal ions in water-soluble salt form, including but not limited to Ca ²⁺, Cu ²⁺, Fe ²⁺, Co ²⁺, Ni ²⁺, Mn ²⁺, Zn ²⁺, Sn ²⁺, Cd ²⁺, Ba ²⁺, Hg ²⁺, Sr ²⁺,with the concentration ofabout1mM to about50mM, preferably in the range of about 2mM to about 10mM, NaCl within the range ofabout5mM to about500mM, preferably about20mM to about100mM,  spermidine concentration with range ofabout0.5mM to about10mM, preferablyabout2mM to about6mM, Dithiothreitol (DTT) with the range of about0.5mM to about5mM, preferablyabout 0.5mM to about2mM, made-detectable-NTP within the range ofabout0.5mM to about10mM, preferably about1mM to about 5mM, and DNA-dependent RNA polymerase with a concentration of about10U toabout200U, preferably about50U to about100U. The mixture should be incubated at temperaturesofabout 20°C toabout 50 °C, preferably about30°C toabout 40°C, and the reaction should last about0.5 hours to about24 hours, preferably about10 hours toabout 20 hours.

 The intermediate RNA probe was then column or High-performance liquid chromatography (HPLC)purified and added to anenzymatic capping reaction. The reaction contains the intermediate RNA probe from about 10 µg toabout 200 µg, preferably about20 µg toabout 100µg, S-adenosylmethionine with concentration of about 0.1mM to about 5mM, preferably about0.2 mM toabout 1mM, guanosine triphosphate (GTP) of about 0.1 mM to about5mM, preferably about0.2mM to about1mM, Tris-HCl with a concentration within about 1mM to about100mM, preferably about10mM to about50mM, pH value ofabout6 to about10, preferably about7 to about9, KCl with a concentration ofabout 1mM to about20mM, preferably about2mM to about10mM, MgCl ₂with the concentration of about0.1mM to about10mM, preferably in the range of about 0.5mM to about 2mM,Dithiothreitol (DTT) with the range ofabout0.5mM to about 5mM, preferablyabout0.5mM toabout 2mM, 0.02% Poloxamer 188with the range ofabout0.005% toabout 0.5%, preferablyabout0.01% to about0.1%, aboutCap 2´-O-methyltransferasewith the range ofabout50U toabout500U, preferablyabout100U toabout300U, and either Faustovirus Capping Enzyme with the range of about10U toabout 200U, preferablyabout20U toabout100U,or Vaccinia Capping Enzyme with the range of about1U toabout200U, preferablyabout10U toabout100U.The mixture should be incubated at temperatures ofabout20°C to about50 °C, preferably about30°C toabout 40°C, and the reaction should last forabout15 minutes to 3 hours, preferably from 30 minutes to 2 hours.

The reaction product from either Method 1or Method 2was then subjected to 20% Urea denaturing PAGE, and the band with the correct size against the marker was excised under a gel imager. The gel slice was then ground in a microcentrifuge tube in 200 µL water. Gel debris was then removed by a 0.22 µm centrifuge filter, and the probe in the clarified liquid was loaded into a silica columnor High-performance liquid chromatography (HPLC) column and eluted in water. The final purified probe was aliquoted and stored in a -80°C freezer for at least one year.Subject to the need, one can vacuum dry the probe solution for even longer storage in a -80°C freezer. The length of the probe used in detecting the activity of influenza viral polymerase is about 10 to about 15-nucleotidewith the modified NTP to be in the 5 ^th, 6 ^th, or 7 ^thposition. 

In the format of a plate assay, the fluorescent RNA probe was added to the reaction together with acap-dependent viral RNA polymerase or a cap-binding proteinin areaction buffer containing 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)with a concentration ofabout 5mM to about200mM, preferably about10mM to about50mM, pH value ofabout6 to about10, preferably about7 to about9, NaCl within the range of about50mM to about500mM, preferably about20mM to about100mM,Dithiothreitol (DTT) with the range of about0.5mM to about5mM, preferablyabout 0.5mM toabout 2mM, MgCl ₂, or other divalent metal ions in water-soluble salt form, including but not limited to, Ca ²⁺, Cu ²⁺, Fe ²⁺, Co ²⁺, Ni ²⁺, Mn ²⁺, Zn ²⁺, Sn ²⁺, Cd ²⁺, Ba ²⁺, Hg ²⁺, Sr ²⁺, with the concentration ofabout1mM to about20mM, preferably in the range of about 2mM to about 10mM,ribonucleotide triphosphate (rNTP)with the concentration ofabout0.1mM to about10mM, preferably in the range of about 0.5mM to about 5mM, RNA templatewith the concentration ofabout0.1µM to about10µM, preferably in the range of about 0.5µM to about 5µM,and RNAse inhibitor mix with the concentration ofabout0.1 U to about10U, preferably in the range of about 0.5 U to about 5U. The reaction should last for about 10 minutesto about120minutes, preferably for about 15 minutestoabout 60 minutes, at about 15°C to about45°C, preferably at about20°C toabout 40°C.The real-time fluorescent polarization readouts representing kinetics informationwere measured in a plate reader equipped with a fluorescent polarization module.

In the format of a gel assay, the fluorescent RNA probe was added to a reaction together witha cap-dependent viral RNA polymerase or a cap-binding protein in a reaction buffer containing 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)with a concentration of about 5mM toabout 200mM, preferably about10mM to about50mM, pH value of about6 toabout 10, preferably  about7 toabout 9, NaCl within the range ofabout50mM to about 500mM, preferably  about20mM toabout 100mM,Dithiothreitol (DTT) with the range of about0.5mM to about5mM, preferablyabout 0.5mM to about2mM, MgCl ₂, or other divalent metal ions in water-soluble salt form, including but not limited to, Ca ²⁺, Cu ²⁺, Fe ²⁺, Co ²⁺, Ni ²⁺, Mn ²⁺, Zn ²⁺, Sn ²⁺, Cd ²⁺, Ba ²⁺, Hg ²⁺, Sr ²⁺, with the concentration ofabout1mM to about20mM, preferably in the range of about 2mM to about 10mM, ribonucleotide triphosphate (rNTP)with the concentration of about0.1mM to about10mM, preferably in the range of about 0.5mM to about 5mM, RNA templatewith the concentration of about0.1µM to about10µM, preferably in the range of about 0.5µM to about 5µM, and RNAse inhibitor mix with the concentration ofabout0.1 U toabout10U, preferably in the range of about 0.5 U to about 5U for about 30 to about 60 minutes, at about 30 to about 37°C. The reaction mix is then subjected to the denaturing PAGE separation, and the fluorescent signal from the extended products in the gel is scanned in a gel imager.

In the in vitro mutant screening reaction, the fluorescent probe RNA was added to the reaction together with a mutant cap-dependent viral RNA polymerase or a cap-binding protein. The reaction is then set up in a plate format or gel format, asdescribed above. A mutant viral RNA polymerase or a cap-binding protein will show different signals or real-time kinetics compared to wild-type viral RNA polymerase or cap-binding protein.

In the invitro inhibitor screening reaction, the fluorescent probe RNA was added to the reaction together with a compound and a cap-dependent viral RNA polymerase or a cap-binding protein. The reaction is then set up in the plate or gel format, as described above.The suppression in the signal or real-time kinetics of the RNA binding or extension with the addition of the compound suggests the existence of inhibitory effects of the compound.

All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

Following are examples that illustrate procedures for practicing the invention.These examples should not be construed as limiting.All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

EXAMPLE 1—SYNTHESIZING THE FLUORESCENT-CAPPED RNA PROBER

We successfully synthesized an 11-nucleotide long fluorescein-labeled  RNA probewith a cap-1 structure. The probe contains a fluorescein-UTP in the 6 ^thposition in the following sequence m7G(5')ppp(5')(2'OMeA)pGGC/fluorescein-U/ACCAAG (SEQ ID NO: 1) and purified the primer with PAGE purification. A 20 µL probe synthesis reaction was set up with 1 µg DNA template, 40 mM Tris-HCl, pH 7.5,  4 mM MgCl ₂, 50 mM NaCl,  2 mM spermidine, 1mM DTT, 10 mM ATP, 10 mM GTP, 10 mM CTP, 8 mM Cap analogue m7G(5')ppp(5')(2'OMeA)pG, 2.5 mM fluorescein-UTP and 100 U T7 RNA polymerase, at 37°C for 16 hours.  The reaction product was then subjected to 20% urea denaturing PAGE, and the band with the correct size against the marker was excised under a gel imager. The gel slice was then ground in a microcentrifuge tube in 200 µL water. Gel debris was then removed by a 0.22 µm centrifuge filter, and the probe in the clarified liquid was loaded into a silica column or High-performance liquid chromatography (HPLC) column and eluted in water. The final purified probe was aliquoted and stored in a -80°C freezer for at least one year. Subject to the need, one can vacuum dry the probe solution for even longer storage in a -80°C freezer. The length of the probe used in detecting the activity of influenza viral polymerase is 11-nucleotide with the modified NTP to be in the 5 ^th, 6 ^th, or 7 ^thposition. 

The representative preparationof the probe before and after purification is shown in FIG.1.

EXAMPLE 2—VIRAL RNA POLYMERASEACTIVITIES MEASURED BY FLUORESCENT CAPPED RNA PROBE

The fluorescent capped RNA probe was tested for the ability to measure the transcription activity of the influenza A virus RNA-dependent RNA polymerase in the denaturing PAGE assays. Briefly, the fluorescent probe RNA was added to the reaction together with influenza A virus polymerase in a 10 µL reaction of a 200 µL reaction tube containing 25mM HEPES pH7.4, 50mM NaCl, 1mM DTT, 5mM MgCl ₂, 1mM rNTP, 1 µM RNA template, and one unit of RNAse inhibitor mix for 60 minutes at 30°C. The reaction mix was subject to the denaturing PAGE separation, and the gel was scanned in a gel imager. Our result shows that mostprobes have been successfully extended ( FIG. 2).

EXAMPLE 3— VIRAL RNA POLYMERASE KINETICS MEASURED BY FLUORESCENT CAPPED RNA PROBE

The fluorescent capped RNA probe was tested for the ability to measure the transcription kinetics of the influenza A virus RNA-dependent RNA polymerase in the 384-well plate assays. Briefly, the fluorescent probe RNA was added to the reaction together with influenza A virus polymerase in a 10 µL reaction of one 384-well plate containing 25mM HEPES pH7.4, 50mM NaCl, 1mM DTT, 5mM MgCl ₂, 1mM rNTP, 1 µM RNA template, and one unit of RNAse inhibitor mix 30°C. The fluorescent polarization readouts were captured every minute for a total of 60 minutes in a plate reader equipped with a fluorescent polarization module. The fluorescent polarization readouts were plotted into a curve against time. Our result shows that the reaction with rNTP ascended to 220 mP in the first 15 minutes and then started to descendto 120 mP till 60 minutes, while the reaction without rNTP reached a plateauat 220 mP for the first 15 minutes without descending till 60 minutes. The reaction without viral polymerase remained at 0 mP throughout the 60 minutes reaction ( FIG. 3). This result suggests our probe is able to, for the first time, record the detailed kinetics information, including cap binding and elongation,of the viral RNA polymerase.

EXAMPLE 4—VIRAL RNA POLYMERASE MUTANT SCREENING PERFORMED BY FLUORESCENT CAPPED RNA PROBE

The fluorescent capped RNA probe was tested for the ability to screen the two mutant influenza A virus RNA-dependent RNA polymerases in the denaturing PAGE assays. Briefly, the fluorescent probe RNA was added to the reaction together with the mutant influenza A virus polymerase in a 10 µL reaction of a 200 µL reaction tube containing 25mM HEPES pH7.4, 50mM NaCl, 1mM DTT, 5mM MgCl ₂, 1mM rNTP, 1 µM RNA template, and one unit of RNAse inhibitor mix for 60 minutes at 30°C. The reaction mix was subjected to the denaturing by PAGE separation, and the gel was scanned in a gel imager. Our result shows that mutant 1 has no activity, while mutant 2 has 40 % activity remaining compared to wild-type polymerase( FIG. 4).

EXAMPLE 5—VIRAL RNA POLYMERASE INHIBITOR SCREENING PERFORMED BY FLUORESCENT CAPPED RNA PROBE

The fluorescent capped RNA probe was tested for the ability to screeninfluenza A virus polymerase inhibitor VX-787 in the denaturing PAGE assays. Briefly, the fluorescent probe RNA, as well as different concentrations of VX-787, was added to the reaction together with the influenza A virus polymerase in a 10 µL reaction of a 200 µL reaction tube containing 25mM HEPES pH7.4, 50mM NaCl, 1mM DTT, 5mM MgCl ₂, 1mM rNTP, 1 µM RNA template, and one unit of RNAse inhibitor mix for 60 minutes at 30°C. The reaction mix was subjected to the denaturing by PAGE separation, and the gel was scanned in a gel imager. Our result shows thatthe extended products were reduced when the inhibitor concentration increased. Our probe is able to detect the inhibitory effects of the viral polymerase inhibitorVX-787( FIG. 5).

EXAMPLE 6—CAP-BINDING PROTEIN ACTIVITY MEASURED BY FLUORESCENT CAPPED RNA PROBE

The fluorescent capped RNA probe was tested for the ability to measure the activity of the cap-binding protein eIF4E in the denaturing PAGE assays. Briefly, the fluorescent probe RNA was added to the reaction with or without the EIF4Ein a 10 µL reaction of one 384-well plate containing PBST buffer and one unit of RNAse inhibitor mix for 30minutes at 30°C. The fluorescent polarization readouts were captured at the end of 30 minutes of incubation in a plate reader equipped with a fluorescent polarization module. The fluorescent polarization readouts were plotted into a curve against time. Our result shows that the reaction with eIF4E reached a polarization reading of100 mP, while the reaction without eIF4E remained at 0 mP ( FIG. 6). This result suggests our probe is able tomeasure the activity of cap-binding protein.

The examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. In addition, any elements or limitations of any invention or embodiment thereof disclosed herein can be combined with any and/or all other elements or limitations (individually or in any combination) or any other invention or embodiment thereof disclosed herein, and all such combinations are contemplated with the scope of the invention without limitation thereto.

EXEMPLARY EMBODIMENTS

Embodiment 1.     A method for measuring the activity of a capped-RNA dependent polymerase, the method comprising:

a) providing a sample comprising the capped-RNA dependent polymerase and an RNA sequence;

b) mixing a fluorescent-labelled capped RNA probe with the sample; and

c) measuring the activity of the capped-RNA dependent polymerase.

Embodiment 2.     The method of embodiment 1, wherein the fluorescent-labelled capped RNA probe comprises a fluorescein-labelled uridine-5'-triphosphate (UTP). 

Embodiment 3.     The method of embodiment 1, wherein the measuring the activity of the capped-RNA dependent polymerase comprises detecting binding of the capped-RNA dependent polymerase to the RNA sequence. 

Embodiment 4.      The method of embodiment 3, wherein the detecting binding of the capped-RNA dependent polymerase to the RNA sequence occurs using fluorescent polarization changes. 

Embodiment 5.      The method of embodiment 1, wherein the measuring the activity of the capped-RNA dependent polymerase comprises measuring primer extension by base-pair resolution.

Embodiment 6.      The method of embodiment 1, wherein the sample comprising the capped-RNA dependent polymerase and an RNA sequence is derived from an influenza A virus or a yellow fever virus.

Embodiment 7.      The method of embodiment 1, wherein the fluorescent-labelled capped RNA probe is about 10 nucleotides to about 15 nucleotides long.

Embodiment 8.     The method of embodiment 7, wherein the fluorescent-labelled capped RNA probe is 11 nucleotides long.

Embodiment 9.      The method of embodiment 1, wherein the fluorescent-labelled capped RNA probe comprises a fluorescein-labeled UTP in the 5 ^th, 6 ^th, 7 ^th, or any combination thereof positions.

Embodiment 10.   The method of embodiment 1, wherein the fluorescent-labelled capped RNA probe comprises m7G(5')ppp(5')(2'OMeA)pGGC/fluorescein-U/ACCAAG  (SEQ ID NO: 1), wherein the m7G(5')ppp(5')(2'OMeA) is a cap-1 structure.

Embodiment 11.   A composition comprising a capped RNA probe, wherein the capped RNA probe is about 10 nucleotides to about 15 nucleotides long, and wherein the capped RNA probe is labeled by a fluorescent label at the 5 ^th, 6 ^th, and/or 7 ^thposition of the capped RNA probe.

Embodiment 12.   The composition of embodiment 11, wherein the fluorescent label is fluorescein.

Embodiment 13.   The composition of embodiment 12, wherein the capped RNA probe comprises a fluorescein-labelled UTP.

Embodiment 14.   The composition of embodiment 11, wherein the capped RNA probe is 11 nucleotides long.

Embodiment 15.    The composition of embodiment 13, wherein the capped RNA probe comprises m7G(5')ppp(5')(2'OMeA)pGGC/fluorescein-U/ACCAAG (SEQ ID NO: 1), wherein the m7G(5')ppp(5')(2'OMeA) is a cap-1 structure.

References

1.    Xu, X., Zhang, L., Chu J.T.S., Wang, Y. Chin, A.W.H., Dai, Z., Poon, L.L.M., Cheung, P.P.-H., Huang, X. A Novel Mechanism of Enhanced Transcription Activity and Fidelity for Influenza A Viral RNA-dependent RNA Polymerase,  Nucleic Acids Research, 49 (15), 8796 (2021) [Research Article; Impact Factor: 19.160]

2.    Wang, Y., Yuan, C., Xu, X., Chong, T.H., Zhang, L., Cheung P.P.H., Huang, X. The mechanism of action of T-705 as a unique delayed chain terminator on influenza viral polymerase transcription,  Biophysical Chemistry, 277, 106652 (2021) [Research Article; Impact Factor: 3.628]

3.    Zhang, L., Zhang, D., Wang X., Yuan, X., Li, Y., Jia, X., Gao, X., Yen, H.L., Cheung, P.P., Huang, X., 1’-Ribose Cyano Substitution Allows Remdesivir to Effectively Inhibit both Nucleotide Addition and Proofreading during SARS-CoV-2 Viral RNA Replication,  Physical Chemistry Chemical Physics, 23, 5852 (2021) [Research Article; Impact Factor: 3.945] 2021PCCP HOT Articles

Claims (15)

1. A method for measuring the activity of a capped-RNA dependent polymerase, the method comprising:

   a) providing a sample comprising the capped-RNA dependent polymerase and an RNA sequence;

   b) mixing a fluorescent-labelled capped RNA probe with the sample; and

   c) measuring the activity of the capped-RNA dependent polymerase.
2. The method of claim 1, wherein the fluorescent-labelled capped RNA probe comprises a fluorescein-labelled uridine-5'-triphosphate (UTP). 
3.     The method of claim 1, wherein the measuring the activity of the capped-RNA dependent polymerasecomprises detecting binding ofthe capped-RNA dependent polymerase to the RNA sequence. 
4.      The method of claim 3, wherein the detecting binding ofthe capped-RNA dependent polymerase to the RNA sequence occurs using fluorescent polarization changes. 
5.     The method of claim 1, wherein the measuring the activity of the capped-RNA dependent polymerase comprises measuring primer extension by base-pair resolution.
6.     The method of claim 1, wherein the sample comprising the capped-RNA dependent polymerase and an RNA sequence is derived from an influenza A virus or a yellow fever virus.
7.     The method of claim 1, wherein the fluorescent-labelled capped RNA probe is about 10 nucleotides to about 15 nucleotides long.
8.     The method of claim 7, wherein the fluorescent-labelled capped RNA probe is 11 nucleotides long.
9.     The method of claim 1, wherein the fluorescent-labelled capped RNA probe comprises a fluorescein-labeled UTP in the 5 ^th, 6 ^th, 7 ^th, or any combination thereof positions.
10.   The method of claim 1, wherein the fluorescent-labelled capped RNA probe comprises m7G(5')ppp(5')(2'OMeA)pGGC/fluorescein-U/ACCAAG  (SEQ ID NO: 1), wherein the m7G(5')ppp(5')(2'OMeA)is a cap-1 structure.
11.   A composition comprisinga capped RNA probe, wherein the capped RNA probe is about 10 nucleotides to about 15 nucleotides long, and wherein the capped RNA probe is labeled by a fluorescent label at the 5 ^th, 6 ^th, and/or 7 ^th position of the capped RNA probe.
12.   The composition of claim 11, wherein the fluorescent label is fluorescein.
13.   The composition of claim 12, wherein the capped RNA probe comprises a fluorescein-labelled UTP.
14.   The composition of claim 11, wherein the capped RNA probe is 11 nucleotides long.
15.   The composition of claim 13, wherein the capped RNA probe comprises m7G(5')ppp(5')(2'OMeA)pGGC/fluorescein-U/ACCAAG (SEQ ID NO: 1), wherein the m7G(5')ppp(5')(2'OMeA) is acap-1 structure.