Patent Application
20200157513
The enzyme Reverse Transcriptase (RT) was discovered in two different laboratories in 1970 in the virus particles of RNA tumor viruses. RT copies RNA into DNA, a reaction thought to be impossible prior to this discovery. RT is found in RNA retroviruses, some DNA viruses (called pararetroviruses), and embedded in most genomes associated with retrotransposons and some types of introns. For the past forty-five years virus-derived RT has been used extensively for many molecular biology applications, including cloning, RT-polymerase chain reaction (RT-PCR), diagnostics, RNA sequencing, and the expression of many important pharmaceuticals that were first isolated as messenger RNA. RT is sold by many biotechnology companies, and is a standard component in the toolbox for molecular biology.
One RNA substrate for RT that has been proven to be quite difficult is double-stranded (ds) RNA. RT is, in fact, a single-stranded specific enzyme, and to use it for dsRNA requires several distinct approaches to make the dsRNA into ssRNA. Several methods are available for this process; the simplest method is boiling, but many protocols call for chemicals to keep the RNA single-stranded, and without the use of very toxic chemicals such as methyl mercury, the RNA very quickly reanneals to its dsRNA form. Some protocols use much higher than normal temperatures for the RT reaction, and while this helps keep the dsRNA melted, it also dramatically reduces the fidelity of the RT enzyme, introducing mutations that can be mistaken for single nucleotide polymorphisms (SNPs) with biological significance. All this makes the reaction very inefficient, and in many cases it fails completely.
The analysis of dsRNA as a hallmark of RNA virus infection has gained importance in recent years with the rise of virus discovery work. Virus discovery has revealed the amazing diversity and abundance of viruses in all environments, and the role of viruses in the ecology of life on our planet is slowing being clarified. The significance of the virome in human and animal health also is becoming increasingly apparent, and viruses can stimulate the immune system to counteract infection by pathogens, or act as early surveillance against incoming bacterial pathogens, as well as create risk for a variety of cancers. Other important biological roles for dsRNA are in RNAi, or gene silencing, and in the CRISPR adaptive immune system of bacteria and archaea that has gained widespread use for genetic manipulation of genomes from fungi to humans.
One additional problem with all commercially available RT enzymes is a relatively low fidelity. This has been particularly problematic for studies in RNA virus populations, and transcriptome SNPs. Biotechnology companies have developed a number of mutants of the RT derived from Avian myoblastosis virus that have increased fidelity (e.g. Superscript III, INVITROGEN®/THERMO-FISHER®), but they have not overcome this problem. Thus, there is an ongoing and unmet need for new technology for use in, for example, for siRNA and CRISPR work, as well as for RNAseq, and discovery and diagnostics of RNA viruses. The present disclosure is pertinent to these needs.
Provided are improved compositions and methods for making cDNA from RNA templates, including double stranded RNA (dsRNA) and single stranded RNA (ssRNA). In certain embodiments, the disclosure provides a recombinant or purified or modified recombinant RNA dependent RNA polymerase (RdRp), wherein the RdRp has an amino acid sequence that is at least 90% identical to a contiguous segment of the amino acid sequence of a Partitiviridae virus RdRp described herein, and wherein the contiguous segment comprises a reverse transcriptase (RT) domain.
In certain embodiments, the disclosure provides a purified or recombinant or modified RdRp that has RT activity for use in producing cDNA from RNA. In embodiments, the RdRp is modified, such as by having one or more amino acids changed relative to a native sequence, or one or more amino acids deleted, or added. In an embodiment, an RdRp of this disclosure is modified by including a tag, such as a purification tag, including but not necessarily limited to a histidine tag (His-tag). In embodiments, such as in methods and kits of this disclosure, the RdRp is present in or provided with one or more buffers that comprise deoxyribonucleotide triphosphates (dNTPs). Separate buffers or a single buffer can be provided such that the dNTPs used in the method and/or provided with a kit comprise all of deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), and deoxythymidine triphosphate (dTTP). In embodiments, the method and/or kit is free of added ribose-based NTPs, such as Uridine-5′-triphosphate (UTP), since the disclosure is directed to synthesis of DNA, rather than the ordinary function of an RdRp to synthesize RNA.
All intermediates formed during reactions used to generate cDNA are included within the scope of this disclosure. In an embodiment, the disclosure thus provides an isolated or recombinant or modified RdRp in a complex with an RNA template, wherein the complex is in an in vitro reaction. In an embodiment, the complex of the RdRp and the RNA template further comprises a segment of a cDNA that is complementary to one strand of the RNA template, i.e., the cDNA that is being elongated by the RdRp. In embodiments, cDNA synthesis is performed at a temperature of less than 50° C., or less than 40° C., or less than 30° C., or not more than 25° C. In embodiments, cDNA synthesis is performed at a temperature of from 10-25° C., inclusive, and including all numbers there between. In embodiments, an RdRp of this disclosure exhibits improved fidelity (i.e., a lower error rate as determined by incorporation of mis-paired nucleotides) relative to a control RT, such as an RT from a retrovirus. In embodiments, a method of the disclosure comprises cDNA generation using a one-step RT polymerase chain (PCR) reaction, or a two-step RT PCR reaction. In embodiments, a method of the disclosure comprises separating cDNA from a reaction in which the cDNA is produced. In embodiments, the sequence of the cDNA is determined. The cDNA sequence can be used for a wide variety of purposes, such as new virus discovery, or analysis of ssRNA or dsRNA viruses from any suitable sample. Thus, the disclosure provides compositions and methods that can be used for analysis of viral outbreaks, and for vaccine design, such as in the case of influenza viruses. Accordingly, in certain aspects, the RNA that is used as a template for producing cDNA may be present in a biological sample before the cDNA is generated. The biological sample can be any suitable sample, and can be used directly, or subjected to a processing step prior to cDNA generation.
The disclosure includes expression vectors encoding any RdRp or segment thereof described herein. Any suitable expression vector can be used, and many are commercially available and can be adapted by those skilled in the art to express an RdRp described herein, when given the benefit of this disclosure. Cells comprising the expression vectors are also included, as are methods of making any RdRp described herein by expressing the RdRp in a cell culture, and separating the RdRp from the cell culture.
In another approach the disclosure provides a method of testing test compounds to determine whether or not they are candidates for use as reverse transcriptase inhibitors. The method generally comprises: a) contacting a plurality of distinct test agents divided into separate reactions chambers with an isolated or recombinant RT, an RNA template, and a reverse transcriptase reaction buffer, b) allowing the test agents to be in contact with the RT, and subsequently, c) measuring of cDNA produced, wherein determining less cDNA relative to a control indicates the test agent is a candidate for use in inhibiting reverse transcriptase activity of the RT.
Unless defined otherwise herein, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.
Every numerical range given throughout this specification includes its upper and lower values, as well as every narrower numerical range that falls within it, as if such narrower numerical ranges were all expressly written herein.
The disclosure includes all polynucleotide sequences (RNA and DNA) encoding the RT of this disclosure. Cells comprising such polynucleotides are also included, as are all methods of making the polynucleotides and cells. Every possible DNA and RNA sequence encoding polypeptides disclosed herein are encompassed by this disclosure.
The present disclosure relates generally to a discovery made by our analysis of persistent viruses that are low titer viruses found in fungi and many plants, including crop plants. They are not transmitted horizontally, and remain in their hosts for thousands of years through nearly 100% vertical transmission. Through sequence analysis of a member of the Partitiviridae infecting fungi we discovered a domain in the RNA dependent RNA polymerase (RdRp) that had similarities to an RT conserved domain. A comparison of RT domains is provided in the Table below. Our analysis revealed that all of the RdRp genes from known partitivirus sequences we examined, including those infecting plants and fungi, have these conserved domains. However, and without intending to be constrained by any particular theory, it is believed that there has been no previous analysis of the potential RT activity of these enzymes because to date they are all found associated with viruses with dsRNA genomes. It seems likely, and again without intending to be limited to any particular interpretation, that any potential RT activity in these enzymes has remained undiscovered because these understudied viruses are not usually thought to be very important, and because there is no apparent reason why a dsRNA virus would ever need an RT, let alone preserve an RT domain over such a long evolutionary history. In addition partitivirus-like sequences are found integrated into the genomes of some plants and fungi, although no mechanism for how they were converted to DNA has been demonstrated. These observations, taken together with our sequence analysis, lead to the present demonstration that a partitivirus RdRp does indeed function as an RT.
In more detail, Pepper cryptic virus 1 (PCV1) is a plant persistent virus in the family Partitiviridae. PCV1 is found in all Jalapeño peppers. Since the pepper host of PCV1 is easy to grow, and PCV1 is generally a relatively high titer persistent virus, we selected this virus for further studies. We also compared PCV1 from different cultivars of hot pepper, including the presumed progenitor of all hot peppers, chiltepin. Chiltepin can be found as a wild plant in Mexico, but it is also consumed locally as a spice, and it is grown in tended areas or in gardens. Given the lack of horizontal transmission of persistent viruses, it is most likely that PCV1 has been consistently infecting chiltepin and domestic hot peppers for thousands of years, diverging with the divergence of their hosts. For most RNA viruses, thousands of years of divergence would lead to changes in the genome that might render them too different to be confident of their common origin, but in PCV1 the difference between the virus in Jalapeño peppers and in chiltepin is only 3%. Once again without intending to be restricted to any particular interpretation, these observations indicate that the RdRp of PCV1 has unusually high fidelity (i.e., a lower error rate) which supports an expectation that RT fidelity of the PCV1 RdRp, as well as other RT's described herein, will be unusually high as well.
Additional description of the isolation, demonstration of RT activity, and recombinant production of a PCV1 protein comprising an RT is presented in examples and figures of this disclosure. Additional demonstrations of embodiments of the disclosure are also provided using Pseudogymnoascus destructans partitivirus-pa (PdPV-pa) RdRp. Further, specific and non-limiting examples of a wide variety of RdRP proteins that comprise RT domains are provided, and include members of distinct virus genera, but all in the same family (Partitiviridae). In particular, representative RdRP sequences from Group I, II, III, IV and V (genera Betapartitivirus, Alphapartitivirus, Deltapartitivirus, Gammapartitivirus, and unclassified partitiviruses, respectively) of the Partitiviridae family are provided.
Based at least in part on the demonstrations of making and using RdRPs from two distinct dsRNA viruses, it is contemplated that any RdRp that is isolated from and/or produced recombinantly and/or/derived from any dsRNA virus that has an RdRp which comprises an RT domain will be suitable for use as an RT in embodiments of this disclosure. In certain examples the RdRp that comprises the RT domain is from a dsRNA virus that infects fungi or plants. In embodiments the RdRp that comprises the RT domain is from an encapsidated dsRNA virus. In embodiments the RdRp that contains the RT is a member of one of the Amalgaviridae, Birnaviridae, Chrysoviridae, Cystoviridae, Megabirnaviridae, Partitiviridae, Picobirnaviridae, Reoviridae, or Totiviridae families of plant or fungal viruses. In embodiments, the RdRp that comprises the RT domain is from a dsRNA virus that infects pepper plants. In an embodiment the RdRp that comprises the RT domain is from a dsRNA that is a PCV-1 or Pepper cryptic Virus 1.
In one non-limiting demonstration of an embodiment of the present disclosure, an in vitro translation of an RdRp and use of the translated RT to produce cDNA from a dsRNA template is demonstrated using a PCV1 RdRp that comprises SEQ ID NO:1, which is meant to be illustrative but not limiting. SEQ ID NO:1 is:
YLWRLLTGHPPQQCHTLGDDSLVGDNSYVNPQAIEEAANKLGWHFNPD
KTQYSTVPEEITFLGRTYVGGLNKRDLTKCIRLLVYPEYPVESGRISA
This sequence is available under GenBank accession no. AEJ07890.1. Conserved RT domains in SEQ ID NO:1 are shown in bold, with highly conserved amino acids shown in enlarged font.
Conserved RT domains were identified by comparison of the PCV1 sequence to other viral proteins, as shown in the following Table:
XADTFRDLR
FVNVYLDDILIFSES
KHLDTVLERLKN
ENLIVKKKXCKFA
INYLWRLLT
QCHTLGDDSLVGDNS
VNPQAIEEAANK
LGWHFNPDKTQYS
MQEILEDWI
QFGIYMDDIYIGSDL
EIVKDLANYIAQ
YGFTLPEEKRQKG
LQEPLRQVS
LLVSYMDDILYVSPT
QCYQTMAAHLRD
LGFQVASEKTRQT
INTILGEAK
HVVCYMDDILIHSKS
KHVKDVLQKLKN
ANLIINQAKCEFH
MQESFGDLK
FALLYIDDILIASNN
EHLKIFFNRVKE
VGCVLSKKKSKMF
LDGLEALLA
NYVRYADDFIITGES
QVLPVVRRFMAE
RGLMLSPEKTKIT
EETEFLG
EEITFLG
YPAKWLG
SPVPFLG
SQVKFIG
KEVEYLG
EGFDFLG
In the Table the following abbreviations are used: Ty, gypsy-like element from yeast; PCV1, Pepper cryptic virus; CAEV, Caprine arthritis encephalitis lentivirus; BLV, Bovine leukemia virus; Tf2-1, fungal retrotransposon; RTBV, Rice tungro bacciliform virus (pararetrovirus); GrpII, Group II intron from E. coli.
In the Table, sequences for the amino acid segments for each viral RT or RdRp sequence, from left to right including consecutively the top and bottom panels for each virus are as follows: for Ty3 RT, SEQ ID NOs:4-8; for PCV1 RdRp, SEQ ID NOs: 9-13; for CAEV RT, SEQ ID NO:s 14-18; For BLV RT, SEQ ID NO:19-23; for Tf2-1 RT, SEQ ID NO:s 24-28; for RTBV RT, SEQ ID NOs: 29-33; and for GrpII RT, SEQ ID NOs: 33-38.
In another demonstration of a non-limiting embodiment, isolated virions of Pseudogymnoascus destructans partitivirus-pa (PdPV-pa) (a Group IV partitivirus), comprising an RdRp with an RT domain to produce cDNA from a dsRNA template is demonstrated. The PdPV-pa RdRp comprises SEQ ID NO:2, which is:
When given the benefit of the present disclosure, conserved RT domains in any of the RdRp amino acid sequences presented herein can be identified using for use in embodiments of the invention, using for example, a domain identification approach described in Marchler-Bauer A et al. (2017), “CDD/SPARCLE: functional classification of proteins via subfamily domain architectures.”Nucleic Acids Res.45(D)200-3, the description of which is incorporated herein by reference. In certain instances an RdRp that comprises an RT domain is referred to herein as an RT.
The disclosure includes amino acid sequences that are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% similar, or identical to amino acid sequences presented herein, and/or to the RT domain of such sequences, such as across the entire length of the RdRp protein, or the RT domain. Additional representative RdRp proteins that comprise RT domains are described below and are encompassed within this disclosure. In embodiments a protein of the disclosure has any of said similarities over a contiguous segment of the sequence that contains the RT domains. In embodiments the protein comprises or consists of any amino acid sequence described herein, or comprises or consists of an RT domain present within any such amino acid sequence. In embodiments, an RdRp and/or RT domain of this disclosure comprises one or more of its amino acid residues substituted with conserved amino acid residues. In certain examples more than one amino acid change can be included. Such changes can comprise conservative or non-conservative amino acid substitutions, insertions, and/or deletions, provided the modified sequence retains or improves on the capability to catalyze the reverse transcription process. In embodiments amino acids substitutions may be substituted with conserved amino acids identified in the RT domain alignment shown in the Table. In embodiments, the disclosure provides recombinant or isolated or modified proteins that comprise RT activity and are at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identical to the sequence:
SEQ ID NO:3 is a consensus sequence of partitivirus RdRps. Residues in gray highlighting are conserved among all members of the family of RdRps that have RT-like domains. The residues in bold are domains conserved among a wide range of RTs. In embodiments, the disclosure includes an RdRp with RT activity that is from 415 to 756 amino acids in length, inclusive. In embodiments, a protein of this disclosure is from 600-650 amino acids in length, inclusive. In embodiments, a protein of this disclosure is from 550-610 amino acids in length. Proteins of such length can be at least 90% similar to any segment of any amino acid sequence presented herein. In one embodiment an RdRp with RT activity of this disclosure has at least 90% identity with SEQ ID NO:3 over its entire length, and in embodiments, includes any one or any combination of the amino acid sequences GDD, XRPL, SXFD, PSGX, wherein X is any amino acid, within the protein. In an embodiment, the RdRp with RT comprises the GDD sequence. In embodiments, an RT of this disclosure has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identity across the contiguous segment of SEQ ID NO:3 spanning amino acids 395-465, inclusive.
Any protein provided by this disclosure can be modified such as by being engineered to include a leader or secretory sequence or a sequence which can be used for purification e.g., a His-tag, and/or to include a proteolytic cleavage site. In a non-limiting embodiment the protein is engineered to comprise a ubiquitin segment and a protease cleavage site for removal of the ubiquitin segment. Modifications can be made at the N-terminus, C-terminus, or within the protein. Such modifications can be made using known reagents and techniques, given the benefit of the present disclosure.
In certain approaches the DNA or RNA sequence encoding a protein of this disclosure can be altered from a naturally occurring sequence, such as by optimizing codons for expression in any particular expression system. In certain approaches a polynucleotide sequence encoding a protein of this disclosure is modified by incorporation into any suitable expression vector, shuttle vector, plasmid, etc., as further described below and demonstrated in non-limiting examples. In certain approaches expression vectors, such as plasmids, are used. A variety of suitable expression vectors known in the art can be adapted to produce the proteins. In general, the expression vector comprises sequences that are operatively linked with the sequences encoding the protein, such as promoters, transcription initiation and termination signals, origins of replication, sequences encoding selectable markers, etc.
The disclosure includes methods of making the RT proteins. Such methods generally comprise either isolating virus that comprises the RdRp from the host where it is found normally and using isolated particles for performing reverse transcription, or by producing the protein recombinantly. Producing the protein recombinantly generally comprises initially introducing an expression vector encoding the RdRp into any suitable host cells by any method known in the art. Methods vary depending upon the type of cellular host, and include but are not limited to transfection employing cationic liposomes, electroporation, calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances as will be apparent to the skilled artisan. In certain embodiments the cells used to produce the protein recombinantly are prokaryotes, including but not necessarily limited to E. coli, but eukaryotic cells may also be used, including but not necessarily limited to plant, fungal, insect and mammalian cells. The methods include allowing for a period of time during which the protein is expressed in the cells, and then separating the protein from the cells, after which the protein can be purified to any desired degree of purity. As discussed above, the proteins may be engineered to contain segments that improve protein expression, secretion, separation and/or purification.
In general, methods of this disclosure comprise combining incubating a fully or partially double-stranded RNA template, or a single-stranded RNA template with a purified or recombinant protein of this disclosure, such that a cDNA of at least one of the strands of the RNA template is produced. The method can optionally further comprise isolating the cDNA, and if desired determining or confirming the sequence of the cDNA. The RT and the RNA template can be combined in vitro to perform one or a plurality of cDNA assays. The RT could also be used to produce a cDNA copy of an RNA within a cell.
In certain embodiments, the disclosure includes generating a cDNA as described herein and determining the sequence of the cDNA. The sequence of the cDNA can be recorded in any tangible medium of expression. In embodiments, a plurality of cDNAs are determined and are compared to one another and/or to a database. In embodiments, the disclosure includes determining the sequence of a cDNA produced from dsRNA or ssRNA from, for example, a virus, and deducing an amino acid sequence encoded by the cDNA. In embodiments, cDNA sequences from cDNAs produced according to this disclosure are from, for example, retroviruses. In embodiments, cDNAs produced according to this disclosure are from samples comprising members of Reoviridae, such as rotaviruses, which comprise dsRNA genomes. In embodiments, cDNAs are produced from samples comprising influenza virus, including Influenza A, B or C viruses. In embodiments, cDNAs produced by amplification of all or a segment of an influenza virus single stranded RNA molecule are used to deduce the amino acid sequence of hemagglutinin (HA) and/or neuraminidase (NA) encoded by the viral genome. Thus, embodiments of this disclosure are suitable for generating genetic information that may be useful in predicting viral outbreaks, and/or for vaccine design. Furthermore, generating cDNA as described herein may be useful in diagnostic applications, such as to provide information about a viral infection in a human, or a non-human animal, or a fungus, or a plant, or a protist, or a bacteria or archaea. Further still, population studies (and other experimental analysis) of RNA viruses are limited by the error rate of the enzyme used for analysis, such as previously available RTs. These studies are critical for understanding virus evolution, including projected evolution of human pathogens such as influenza A virus, that is required for deciding on the specifics of future vaccines. Thus, the lower error rate that is expected to be produced by using an RdRp comprising an RT function as described herein provides advantages that are applicable to numerous applications.
Using an RT of this disclosure to produce a cDNA can be advantageous in a variety of techniques wherein determination of the presence, absence, amount, nucleotide sequence of a strand of an RNA, or combinations thereof would be useful. In this regard it is expected that any RNA can be used as a template for cDNA production using approaches of this disclosure, and there will be no particular upper limit to the bp length or other constraints on nucleotide composition, other than those imposed by the availability of reagents (i.e., dNTPs) to continue the reverse transcription process. In certain approaches, a fully or partially double-stranded RNA template is analyzed by cDNA production using an RT of this disclosure in a cell-free in vitro assay, or in an in vitro assay comprising cells. In the performance of such assays fully or partially double-stranded RNA templates will be in physical association with an RT of this disclosure, thus forming a complex comprising the RT and a dsRNA. The dsRNA may be in physical association with more than one RT of this disclosure at any particular time, and depending on the length of the dsRNA template distinct RTs may be concurrently generating cDNAs from the same dsRNA template in an anti-parallel direction. Those skilled in the art will recognize that a dsRNA template that is being reversed transcribed by an RT of this disclosure may include a segment that is in physical association with the RT but is not itself double-stranded due to the presence of a transcription bubble, wherein the strand being reverse-transcribed is transiently separated from its complementary strand in order to catalyze cDNA synthesis. Thus, the disclosure includes complexes comprising an RT as described herein, wherein the RT is in a physical association with a fully or partially double-stranded RNA template, and wherein the RT is also in physical association with a DNA polynucleotide (e.g., a cDNA) that is being synthesized during the process via RT activity.
In one embodiment the disclosure provides for determining the presence, absence and/or the sequence and/or the amount of a segment of RNA that is in a dsRNA configuration by exposing a sample comprising or suspected of comprising dsRNA with an RT of this disclosure, along with suitable reagents that are typically employed in a Reverse-Transcription PCR (RT-PCR) approach, and after a period of time determining the presence, absence, and/or sequence and/or amount of the cDNA. In an embodiment, by comparison to a suitable control, the absence of cDNA is a basis for inferring a lack of dsRNA in the sample, whereas the presence of cDNA indicates the presence of dsRNA in the sample. In an embodiment the method comprises comparison of ssDNA in the sample with a control reaction that is performed using a portion of the sample and a standard RT, many of which are commercially available, and include Moloney murine leukemia (M-MLV) and Avian Myeloblastosis Virus (AMV) RTs. Determining a difference in ssDNA relative to the control permits determining ssDNA production that it is attributable to the RT component of the RdRp acting on a dsRNA template, whereas the standard RT would not use the dsRNA as a template. Determining the sequence of a cDNA, if present, provides for identification of the sequence of at least one strand of dsRNA that was the template for cDNA synthesis, which also permits deduction of its complementary strand. Generating and determining the sequence of cDNAs is well known in the art, and the present disclosure includes performing this process, but with substitution of the RT of this disclosure for any of the well-known commercially available reverse transcriptase enzymes typically employed in RT-PCR reactions.
Generating cDNAs using an RT of this disclosure can be performed in a one-step RT-PCR, or a two-step RT-PCR. As will be recognized by those skilled in the art, a one-step RT-PCR reaction entails generating a cDNA using the RT, and PCR amplification of the cDNA in a single reaction container. Two-step RT-PCR entails the reverse-transcription reaction being performed in a single reaction chamber to obtain single-stranded cDNAs, which are then separated from the reverse-transcription assay and PCR-amplified in a separate reaction chamber. In certain approaches specific temperature parameters are included to avoid denaturing the RT. In certain approaches the methods of this disclosure include reverse-transcription cDNA generation at a temperature of less than 50° C. In embodiments, cDNA generation is performed at a temperature of less than 40° C. In embodiments, cDNA generation, i.e., producing cDNA by an RT of this disclosure, is performed at a temperature of from 10° C., to room temperature, wherein room temperature can range from 20-25° C. In embodiments, the only application of heat to generate a cDNA according to this disclosure is to temporarily denature dsRNA structures for the purpose of annealing primers. In this regard, those skilled in the art will recognize that at room temperature, currently commercially available and other previously characterized reverse transcriptase enzymes, would not be able to synthesize cDNA from a dsRNA template because the dsRNA renatures, which is not a suitable template for a non-RdRp reverse transcriptase. The kits of this disclosure described below can be adapted for either approach. Further, these approaches can be tailored for quantitative purposes.
In embodiments, an RT of this disclosure has a reduced error rate for nucleotide incorporation into the cDNA, compared to a control, such as a commercially available RT. In embodiments, the control comprises an error for reverse transcription by M-MLV virus reverse transcriptase. In embodiments, the control comprises an error rate for AMV reverse transcriptase. In embodiments, a control error rate ranges, from 1 error for every 17,000 bases to 1 error for every 30,000 bases. The error rate can be applied to a plurality of cDNAs. In embodiments, a cDNA of this disclosure can comprise from 500 nts to 12 kb in length. In embodiments, generation of a cDNA as described herein is performed without using any methyl mercury, such as methylmercury hydroxide. In embodiments, cDNA according to this method is performed without use of any reverse transcriptase obtained or derived from a bacteria, including but not necessarily limited to Thermus aquaticus. In embodiments, a cDNA generated according to this disclosure does not include an A-overhang. In embodiments, a cDNA according to this disclosure is generated without using any RNAase H.
It will be recognized from the foregoing that the present disclosure provides flexible approaches for detecting the presence, absence and/or amount of ssRNA or dsRNA by virtue of detecting the presence, absence and/or an amount of cDNA. Those skilled in the art will appreciate that as described above, this approach can be adapted to test any biological sample for dsRNA or single stranded RNA, which can be a critical step in detection and/or discovery of viruses, as well as other dsRNAs that may be present in a biological sample. Other dsRNAs from which cDNAs may be generated according to this disclosure can include double-stranded segments of RNA polynucleotides present in RNA secondary structures found in ssRNA virus genomes, mRNAs, tRNAs, snoRNAs, miRNAs, siRNAs, shRNAs, etc. Accordingly, in certain approaches an RT of this disclosure can be used for direct dsRNA sequencing and for instance, for RNA secondary structure mapping.
In certain approaches an RT of this disclosure can be used for screening a plurality of test agents to determine if they are candidates for use as an RT inhibitor, including but not necessarily limited to an RT that is a component of an RdRp. In general, the method comprises analyzing test agents using any system wherein the production of cDNA from dsRNA can be measured. In one embodiment, the method comprises screening a plurality of test agents to identify candidates for use in reducing RT activity by: a) contacting a plurality of distinct test agents (which may be divided into separate reactions chambers, such as in a high-throughput screen) with an isolated or recombinant RT of this disclosure in the presence of an RNA and RT-PCR reagents; b) allowing the test agent to be in contact with the RT for a period of time, and subsequently, c) measuring cDNA, wherein determining less cDNA relative to a control indicates the test agent is a candidate for use in inhibiting the RT. The presence and/or amount of the cDNA can be determined using standard approaches.
In an embodiment the disclosure provides a kit comprising an isolated or recombinant/modified RT as described herein. The RT can be provided as a single component or with other components, and can be included in or more sealed vials. The kit can include any reagents required for performing one-step or two-step RT-PCR, including but not limited to amplification buffers, one or more primers, nucleases, and dNTPs, including deoxyadenosine dATP, dCTP, dGTP, and dTTP. In an embodiment, an RdRp comprising RT activity is present in or provided with a buffer, such as a reverse transcriptase reaction buffer, which may comprise added dNTPs. The dNTPs need not include added nucleoside triphosphates that contain ribose as the sugar (NTPs). In embodiments, a buffer or other reaction component of the disclosure does not include uridine-5′-triphosphate (UTP) because the kit and other aspects of the disclosure are designed for DNA synthesis only. Thus, the kit and/or buffer includes dTTP instead of UTP. Accordingly, the disclosure includes buffers to which no UTP is added. In embodiments, the only nucleosides added to a buffer used in embodiments of this disclosure are dNTPs. In embodiments, the only nucleosides in a buffer or other reaction component of this disclosure are one or more dNTPs, and thus a buffer of this disclosure may include a nucleoside or nucleotide component which consists of, or consists essentially of, one or more dNTPs.
The dNTPs may be provided in any suitable molarity, such as from 1-20 μmol. In an embodiment, the dNTPs are provided in 8 μmol solutions. The kit may optionally include instructions for performing the RT that are either written on paper or in a computer-readable format.
In an embodiment, the RT is in a lyophilized form, and the kit further includes instructions for reconstituting the RT for use in cDNA production.
Additional representative RdRp amino acid sequences that comprise suitable RT domains are as follows:
The Following Representative Amino Acid Sequences are from RdRps from Group I Partitiviruses (genus Betapartitivirus)
Atkinsonella hypxylon virus
Ceratocystis polonica partitivirus
Ceratocystis resinifera partitivirus (SEQ ID NO: 43)
Fusarium poae virus 1
Heterobasidion partitivirus 8
Hop trefoil virus 2
Lentunula edodes partitivirus 1
Primula malacoides virus
Pleurotus ostreatus virus
Rosellinia necatrix partitivirus 1-W8
Rosellinia necatrix partitivirus 6
Sclerotinia sclerotiorum partitivirus 1
The Following Representative Amino Acid Sequences are from RdRps from Group II Partitiviruses (genus Alphapartitivirus)
Arabidopsis halleri partitivirus 1
Diuris pendunculata cryptic virus
Heterobasidion partitivirus 7
Heterobasidion RNA virus 1
Rhizoctonia fumigata partitivirus
Rosellinia necatrix partitivirus 2
Rosellinia necatrix partitivirus 7
Rhizoctonia solani dsRNA virus 2
Sophora japonica powdery mildew-associated partitivirus
Sclerotinia sclerotiorum partitivirus S
The Following Representative Amino Acid Sequences are from RdRps from Group III Partitiviruses (genus Deltapartitivirus)
The Following Representative Amino Acid Sequences are of for RdRp's from Group IV Partitiviruses (genus Gammapartitivirus):
The Following Representative Amino Acid Sequences are from RdRps from Group V Partitiviruses (Unclassified Members of the family Partitiviridae)
The following Examples are intended to illustrate but not limit embodiments of this disclosure.
The RdRp of PCV1, and all known encapsidated dsRNA viruses, is found in the virus particle so the analysis described in these Examples began by evaluating enzymatic activity using purified virus. This Example provides a description of preparing isolated virus.
The procedure was standard for plant viruses. The virus was purified from 50 g of plant tissue by homogenization of plant leaves in a blender with 50 ml of 0.1 M sodium phosphate buffer pH 7.4 containing 0.2 M KCl and 0.5% 2-mercaptoethanol, and 50 ml of chloroform. The resulting slurry was clarified by low speed centrifugation for 15 min. The aqueous portion was filtered through miracloth, and subjected to ultracentrifugation through a 10% sucrose cushion. The pellets were resuspended in sodium phosphate buffer and stirred overnight. The solution was clarified by low speed centrifugation, followed by a second ultracentrifugation as above. The final pellets were resuspended in sodium phosphate buffer and allowed to incubate at 4° C. overnight. The purified viral preparation was analyzed by 1% agarose gel in Tris-Glycine buffer (
This example provides a description of the RT activity of PCV1. Generating cDNA from dsRNA is not a straightforward reaction, because all known RT enzymes are only active on single-stranded RNA (ssRNA). Various strategies have been devised to use a dsRNA template, all of which require denaturing the dsRNA completely and adding chemicals, heat, and/or large amounts of oligonucleotides to maintain the template as ssRNA. Reactions are carried out at 42-55° C., temperatures that are well known in the art to be not optimal for RT enzymes. In particular, higher temperatures reduce the fidelity of the RT reaction (i.e. more errors are introduced in the cDNA sequence). In contrast, the present disclosure permits, as described above, cDNA generation at lower temperatures than have been previously possible, and without the use of denaturing chemicals.
To test the RT activity of PCV1, we replaced the MMuLV RT enzyme (commercially purchased from New England Biolabs) with the stored preparation of PCV1 in a cDNA reaction using the dsRNA of Zea mays chrysovirus 1 (ZMCV1) as a template. The reactions were adapted from standard protocols: dsRNA was mixed with a specific primer and boiled for 2 minutes, followed by rapid cooling on ice, addition of the enzyme, buffer, and dNTPs, and incubation on ice for 15 minutes. The MMuLV reaction was transferred to 42° C. for two hours, while the PCV1 reaction was held at room temperature for the same amount of time. At this temperature a “normal” RT enzyme would be unable to use dsRNA as a template because the RNA would have renatured. In a negative control, added water was used as the template along with PCV1 as RT enzyme.
Following the cDNA reaction, the samples were treated with 1 μl (10 mg/ml) of boiled ribonuclease A (Sigma, USA), and incubated at room temperature for 15 min, to destroy remaining RNA. The samples were heated to 85° C. for 2 min, and the primers removed using a cycle pure kit (Omega, Bio-Tek, USA) according to the manufacturer's instructions. The samples were eluted in 30 μl water. A 1.5 μl aliquot of the cDNA was amplified by PCR in a standard reaction with the same primer used in the cDNA reaction, and forward primer specific for another region of the cDNA. The amplified cDNAs were separated on a 1.2% agarose gel, stained and visualized (
A second reaction was carried with another dsRNA virus, Curvularia protuberata thermal tolerance virus. In this reaction we added an additional sample, where the starting dsRNA and primer were not boiled. We obtained the same product with and without boiling the dsRNA, further substantiating that the PCV1 enzyme is indeed able to use dsRNA as a template.
Following our initial success we prepared a much larger virus isolation, using about 1 kg of plant tissue. This is not a rapid process, because it takes some time to grow the plants, and this needs to be performed in a growth chamber to prevent the risk of exposure to environmental pathogens that would complicate the results. We also performed a virus purification procedure on an isogenic line of Jalapeno that is virus free to provide a control to ensure that the activity was completely attributable to the virus. Because of the large volume we concentrated the plant extract using polyethylene glycol, a standard procedure in plant virus purification. However, this prep had no RT activity, and an EM showed particles that were highly aggregated (
This Example demonstrates recombinant production of an enzyme derived from PCV1 RdRp that exhibits RT activity. In particular, in order to avoid having to prepare virus from plants, the DNA sequence encoding the PCV1 RdRp was optimized for expression in E. coli and was cloned into a commercially available vector sold under the tradename pSUMO Vector. The sequence adds a 6× His Tag to the protein to facilitate purification, and small ubiquitin-like modifier (SUMO) that can be removed by protease digestion of the recombinant protein. The E. coli expression optimized DNA sequence is:
The optimized sequence comprises modifications to remove tandem rare codons that can reduce the efficiency of translation or disengage ribosomes from the RNA, by changing
GC content to prolong mRNA half-life, to disrupt some predicted stem-loop structures, and to remove negative cis-acting sites.
High levels of expression of the protein in E. coli were induced and this expression was confirmed, as evidenced by
This Example is illustrated by the results presented in
This Example is illustrated by the results presented in
This Example is illustrated by the results presented in
While the disclosure has been particularly shown and described with reference to specific embodiments, it should be understood by those having skill in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the present disclosure as disclosed herein.
This application claims priority to U.S. provisional patent application No. 62/483,651, filed Apr. 10, 2017, the disclosure of which is incorporated herein by reference.
This invention was made with government support under Hatch Act Project No. PEN04480, awarded by the United States Department of Agriculture/NIFA. The Government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2018/026913 | 4/10/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62483651 | Apr 2017 | US |
