Patent Application
20240076748
The instant application contains a Sequence Listing which has been submitted in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Aug. 21, 2023, is named IPDM9PCT_SL.xml and is 18,625 bytes in size.
This invention relates to methods of enzymatically repairing nicked or partially cleaved RNAs and detecting RNAs.
Nucleic acids-based disease monitoring (including diagnosis and prognosis) based on minimally invasive liquid biopsies has the potential to impact on the patients' survival rates by detecting diseases, relapse, or resistance to treatment in the patients at an early stage. However, this has been mostly studied in the context of extracellular cell-free DNA (cfDNA).
Extracellular RNAs (exRNA) can be more informative than cfDNAs because they also reflect gene expression changes across tissues and cell types. ExRNAs circulating in human bodily fluids can predict disease before the onset of clinical symptoms. For instance, exRNAs have been recently used successfully to predict preeclampsia in pregnant women at a pre-symptomatic stage (Moufarrej et al., 2022).
However, exRNAs are less studied and there still lacks fundamental knowledge on the types of exRNAs and their carriers in bodily fluids. When studying the exRNA world, a population composed of non-vesicular ribosomes and full-length tRNAs was uncovered (Tosar et al., 2020). Studies show that exRNA profiles may also contain from tRNA halves to full-length tRNAs (Nechooshtan et al., 2020). Moreover, stress can induce the formation of intracellular tRNA halves, potentially upregulating extracellular tRNA halves in EVs (Li et al., 2022; Tosar and Cayota, 2020). When encapsulated in extracellular vesicles (EVs), these fragments can be transferred to recipient cells (Gambaro et al., 2020), where they can trigger pattern recognition receptor-mediated signaling (Pawar et al., 2020; Xiao et al., 2020). However, most non-vesicular tRNA halves are generated directly in the extracellular space by endonucleolytic cleavage of extracellular full-length tRNAs (Nechooshtan et al., 2020; Sanadgol et al., 2022; Tosar et al., 2020). Extracellular ribosomes can induce dendritic cell activation in vitro in an exRNA-dependent manner (Tosar et al., 2020).
The studies on exRNAs have been more focused on the context of extracellular vesicles (EVs), which protect exRNAs from enzymatic degradation, because biofluids contain high quantities of enzymes that degrade RNA. Nonetheless, it has been known for years that the majority of exRNAs circulating in human blood plasma or in cell-conditioned medium are not associated with EVs (Arroyo et al., 2011; Turchinovich et al., 2011; Tosar et al., 2015). As a consequence of the strong ribonuclease activity that characterizes the extracellular space (Sorrentino, 2010), these non-vesicular exRNAs are expected to be rapidly degraded unless protected by RNA-binding proteins. It thus remains unknown on how these non-vesicular exRNAs resist degradation, diffuse to recipient cells, and trigger downstream effects, or even remain measurable and may serve as potential disease biomarkers. Explanations for some of these questions are provided in this invention, and the inventors have discovered a population of intrinsically stable non-vesicular extracellular RNAs, characterized by the presence of broken phosphodiester bonds in their structure (“nicked exRNAs”).
The problem is that nicked exRNAs cannot be identified by conventional or currently available sequencing and/or amplification methods. When reverse transcriptases are exposed to the broken phosphodiester bonds present in these RNAs, they fall off. As a result, these RNAs are either not represented in the sequencing or RT-qPCR assays, or they appear as RNA fragments, loosing important structural information and the physical association between 5′ and 3′ fragments.
There thus remains a need in the art to develop an effective method to more accurately characterize these exRNAs to enable their detection and/or subsequent applications (e.g., reverse transcription, amplification, analysis, hybridization, sequencing, etc.) that would have been undetectable in the biological sample due to the limits of the currently available RNA detection technology. This disclosure answers that need.
One aspect of the invention relates to a method of enzymatically repairing RNAs that are nicked or at least partially cleaved. The method comprises providing a biological sample containing RNAs that are nicked or at least partially cleaved; purifying the nicked or at least partially cleaved RNAs contained in the biological sample, under a non-denaturing condition, to remove non-RNA components; and treating the purified RNAs with at least one of the following: (i) one or more enzymes that exhibit the activity of an RNA 3′ phosphatase or cyclic phosphatase and the activity of an RNA 5′ kinase, and an RNA ligase, or (ii) a 3′-5′ RNA ligase, thereby forming repaired RNAs from the nicked or at least partially cleaved RNAs.
Another aspect of the invention relates to a method for detecting RNAs from a biological sample. The method comprises providing a biological sample containing RNAs that are nicked or at least partially cleaved; purifying the nicked or at least partially cleaved RNAs contained in the biological sample, under a non-denaturing condition, to remove non-RNA components; treating the purified RNAs with at least one of the following: (i) one or more enzymes that exhibit the activity of an RNA 3′ phosphatase or cyclic phosphatase and the activity of an RNA 5′ kinase, and an RNA ligase, or (ii) a 3′-5′ RNA ligase, thereby repairing at least a portion of the nicked or partially cleaved RNAs; and detecting the repaired RNAs.
Additional aspects, advantages and features of the invention are set forth in this specification, and in part will become apparent to those skilled in the art on examination of the following, or may be learned by practice of the invention. The inventions disclosed in this application are not limited to any particular set of or combination of aspects, advantages and features. It is contemplated that various combinations of the stated aspects, advantages and features make up the inventions disclosed in this application.
Studying the nicked or at least partially cleaved RNAs remains a challenge because standard protocols for RNA extraction, northern blotting, and RNA sequencing induce their artefactual denaturation. In this disclosure, the inventors have developed an approach to enable reverse transcription, amplification, analysis, and/or sequencing of stable extracellular RNAs, without missing highly structured, nicked or partially cleaved exRNAs. The nicked or partially cleaved RNAs are purified under a non-denaturing condition (e.g., with a phenol-free method; without heating) and then enzymatically repaired to enable subsequent reverse transcriptions and/or various detections.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Allen et al., Remington: The Science and Practice of Pharmacy 22nd ed., Pharmaceutical Press (Sep. 15, 2012); Hornyak et al., Introduction to Nanoscience and Nanotechnology, CRC Press (2008); Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology 3rd ed. revised ed., J. Wiley & Sons (New York, NY 2006); Smith, March's Advanced Organic Chemistry Reactions, Mechanisms and Structure 7th ed., J. Wiley & Sons (New York, N.Y. 2013); Singleton, Dictionary of DNA and Genome Technology 3rd ed., Wiley-Blackwell (Nov. 28, 2012); and Green and Sambrook, Molecular Cloning: A Laboratory Manual 4th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, N.Y. 2012), provide one skilled in the art with a general guide to many of the terms used in the present application.
One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present disclosure. Other features and advantages of the disclosure will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various features of embodiments of this invention. Indeed, the present disclosure is in no way limited to the methods and materials described herein. For purposes of the present disclosure, certain terms are defined below.
“Treated” or “treatment” as used herein in the context of an assay means applying an effective amount of a substance under conditions that allow for the action of the substance. For example, “treating a sample with an enzyme” means applying a sufficient amount of an enzyme and under the appropriate conditions (buffers, temperature, etc.) to allow for an enzymatic reaction, as would be recognized by one of skill in the art.
A “nicked or partially cleaved RNA” or “nicked or at least partially cleaved RNA” generally refers to an RNA molecule having a discontinuity between adjacent nucleotides and/or having a portion removed from the parent, full-length RNA. For instance, a nicked or partially cleaved RNA may refer to an exRNA such as a tRNA having a cleaved phosphodiester bond in the anticodon loop. A nicked or partially cleaved RNA may also include a scenario where the RNA has a region (e.g., a loop or overhang or a portion thereof) removed from the parent, full-length RNA. A nicked or partially cleaved RNAs would include those artificially-generated RNA fragments, i.e., those generated by human interventions such as some experimental and/or treatment methods that would dissociate or completely cleave the nicked or partially cleaved RNAs into fragments. However, if an RNA fragment is not generated by human intervention, i.e., if different parts of the nicked or partially cleaved RNAs are already fragmented and separated in a biological sample that is used as an input, before the collection of said sample and before any human intervention such as experiment and/or treatment methods to biological sample, then these RNA fragments would typically not be considered as a nicked or partially cleaved RNA.
A “repaired RNA” refers to an RNA that is regenerated by ligating at least two portions or fragments (e.g., 5′ fragment and 3′ fragment) of a nicked or partially cleaved RNA, produced by cleaving the parent RNA, and reconstitutes the parent RNA's full-length or substantial full-length. For instance, for a nicked or partially cleaved tRNA, after the enzymatic repairing disclosed herein, the repaired RNA can refer to an RNA that is regenerated by ligating the 5′ half and the 3′ half of the nicked or partially cleaved tRNA, which are produced by cleaving the parent tRNA (or the nicked or partially cleaved tRNA), thereby reconstituting the parent RNA in full-length or substantial full-length. The repaired RNA can have the same length as the parent RNA, i.e., it is reconstituted to the parent RNA's full-length. Or the repaired RNA can have a length that is substantially the same as the parent RNA, i.e., it is reconstituted to the parent RNA's substantial full-length. By “substantial” full-length, the length of the repaired RNA may be at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or virtually 100% of the full length of the parent RNA. The repaired RNA can comprise a nucleotide sequence identical or substantially identical to the parent RNA. By “substantially identical,” the sequence of the repaired RNA may be at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or virtually 100% identical to sequences of the parent RNA.
Extracellular (exRNAs) circulating in human bodily fluids can predict disease before the onset of clinical symptoms. Beyond translational applications as disease biomarkers in liquid biopsies (Heitzer et al., 2019), exRNAs are also involved in intercellular communication pathways between cells in different tissues (Thomou et al., 2017) and in host-pathogen interactions (Buck et al., 2014; Cai et al., 2018; Garcia-Silva et al., 2014).
One key aspect governing both exRNA functionality and utility as biomarkers is their stability against ubiquitous extracellular RNases (Tosar et al., 2021). This can be achieved by RNA encapsulation inside extracellular vesicles (EVs) such as exosomes and microvesicles (Skog et al., 2008; Valadi et al., 2007). However, despite the functional relevance (Mateescu et al., 2017) and biotechnological applications (O'Brien et al., 2020) of EV-encapsulated RNA, the majority of exRNAs in cell culture (Tosar et al., 2015; Turchinovich et al., 2011; Wei et al., 2017; Zhang et al., 2021) and in human plasma (Arroyo et al., 2011; Geekiyanage et al., 2020; Turchinovich et al., 2011; Vickers et al., 2011) are not transported as part of EV cargo, and are non-vesicular.
Cells can release ribosomes and full-length tRNAs into the extracellular space, where they can trigger immune cell activation. However, because non-vesicular exRNAs are prone to the action of extracellular ribonucleases, it has been considered unlikely that they could act as mediators of intercellular communication.
Contrary to common belief, the inventors have discovered that certain extracellular, non-vesicular RNAs are intrinsically stable against enzymatic degradation and show very high half-lives (from minutes to hours) when incubated in human biofluids at physiological condition (e.g., at 37° C.). These intrinsically stable non-vesicular RNAs circulate in human bodily fluids and are not associated with EVs. These intrinsically stable non-vesicular RNAs are very abundant inside most cells, but some RNAs can have tissue-specific expression. Cell damage or death may be the main source of non-vesicular RNAs in extracellular samples. Thus, intrinsically stable non-vesicular RNAs can be elevated in biofluids in situations where non-physiological quantities of cell damage or death are happening in the body (e.g., trauma, ischemia-reperfusion, cancer, autoimmunity, etc.).
Without being bound by theory, characteristics shared by the intrinsically stable non-vesicular RNAs include but are not limited to: a) they are highly structured and contain high % of nucleobases in double-stranded form, even for the ones that are single-stranded; b) they bear modified nucleotides; and c) they bear broken phosphodiester bonds (i.e., they are “nicked RNAs”) due to partial RNA cleavage by extracellular ribonucleases (RNases). This partial cleavage is not sufficient to break the structure of these molecules, which remain as dsRNAs stabilized by several internal base-pairing interactions.
tRNAs
Transfer RNA (tRNA) is a small RNA molecule that plays a key role in protein synthesis. Transfer RNA serves as a link (or adaptor) between the messenger RNA (mRNA) molecule and the growing chain of amino acids that make up a protein. tRNA typically has 70-100 (e.g., 76-90) nucleotides in length (in eukaryotes). tRNAs can be cleaved at the anticodon loop to produce “tRNA halves” that are 30-35 nucleotides in length in a process facilitated by the enzyme, Angiogenin, following induction of stress.
A tRNA fragment can be used to refer to functional short non-coding RNAs generated from a tRNA locus. tRNA fragments (tRFs) have lengths that range from 10 to 40 or more nucleotides. The term “tRNA locus” refers to the genomic region that includes a tRNA gene and gives rise to the tRNA transcript.
Transfer RNA-derived RNAs (tDRs) are among the most abundant nonvesicular small RNAs in cell culture (Tosar et al., 2015; Wei et al., 2017). Inside cells, tRNA cleavage and the consequent upregulation of specific tDRs is a conserved response to stress in all kingdoms of life (David et al., 1982; Li et al., 2008; Thompson et al., 2008). In humans, RNase A superfamily members, such as RNase 5 (Angiogenin), are responsible for stress-induced tRNA cleavage at the anticodon, generating stress-induced tRNA halves or tiRNAs (Fu et al., 2009; Yamasaki et al., 2009). tiRNAs can regulate gene expression at various levels, including global inhibition of translation initiation (Ivanov et al., 2011), by sequestering eIF4G (Lyons et al., 2021). Other, shorter tDRs can bind to mRNAs and regulate their translation (Kim et al., 2017) or silence genes by a miRNA-like, Argonaute-dependent mechanism (Kuscu et al., 2018).
Extracellular tDRs were first reported in EVs from murine immune cells (Nolte'T Hoen et al., 2012) but were later shown to be present mainly outside vesicles in mouse serum (Dhahbi et al., 2013; Zhang et al., 2014). In human cancer cell lines, tDRs could be detected in vesicular fractions but were overwhelmingly more abundant in EV-depleted ultracentrifugation supernatants (Tosar et al., 2015). While a heterogeneous population of tDRs can be detected inside cells, extracellular non-vesicular tDRs are mainly 5′ tRNA halves of 30 or 31 nucleotides, derived from tRNAGly and tRNAGlu. These specific fragments are also ubiquitous in human biofluids (Srinivasan et al., 2019). The extracellular enrichment of these fragments may be due to their enhanced stability against degradation, because tRNAGlyGCC 5′ halves of 30-31 nt can form RNase-resistant homodimers in vitro (Tosar et al., 2018). However, the inventors have found that nicked tRNAs are probably the form in which tDRs are transported in biofluids. The high stability of nicked tRNAs against degradation therefore contributes to explain the abundance of extracellular non-vesicular tDRs.
One aspect of the invention relates to a method of enzymatically repairing RNAs that are nicked or at least partially cleaved. The method comprises providing a biological sample containing RNAs that are nicked or at least partially cleaved; purifying the nicked or at least partially cleaved RNAs contained in the biological sample, under a non-denaturing condition, to remove non-RNA components; and treating the purified RNAs with at least one of the following: (i) one or more enzymes that exhibit the activity of an RNA 3′ phosphatase or cyclic phosphatase and the activity of an RNA 5′ kinase, and an RNA ligase, or (ii) a 3′-5′ RNA ligase, thereby forming repaired RNAs from the nicked or at least partially cleaved RNAs.
The biological sample containing RNAs that are nicked or at least partially cleaved are purified to remove non-RNA components, such as proteins, lipids, salts, etc. that could interfere with downstream analysis. The purification is carried out under a non-denaturing condition.
In some embodiments, the non-denaturing condition comprises a solid phase extraction (SPE), chromatographic methods (such as size exclusion chromatography (SEC) or ion exchange chromatography), RNA precipitation (e.g., precipitation with a polar solvent, such as acetone or an alcohol (e.g., ethanol or isopropanol)), or a combination thereof. In one embodiment, the non-denaturing condition comprises SPE, such as a silica-based solid phase extraction columns.
The method may also contain one or more separation steps to extract one or more specific types of RNA, prior to the enzymatic treatment. In one embodiment, the method further comprises density gradient separation to isolate non-vesicular RNAs. In one embodiment, the method further comprises chromatographic methods to isolate non-vesicular RNAs. In one embodiment, the method further comprises native electrophoresis to fractionate RNAs based on their sizes.
The method may also contain one or more enrichment steps, prior to the enzymatic treatment. In one embodiment, the method further comprises gel-purifying to enrich one or more RNAs or fragments having 25-100 nucleotides.
In some embodiments, the purification of the biological sample and enzymatic treatment of the purified RNAs are carried out in the absence of a condition that results in denaturation of an RNA molecule. The condition that can result in denaturation of an RNA molecule can comprise heating, an adapter ligation, a chemical denaturant, or combinations thereof. In one embodiment, the condition that can result in denaturation of an RNA molecule comprises employing a chemical denaturant, such as phenol. In one embodiment, the condition that can result in denaturation of an RNA molecule comprises heating. The purification used herein avoids denaturing conditions that can create an artefact of missing certain highly structured, nicked or partially cleaved exRNAs and instead detecting an increased population of degraded/denatured RNAs that are in fact caused by the denaturing conditions.
In some embodiments, the method of enzymatically repairing nicked or at least partially cleaved RNAs is carried out in absence of a condition that results in denaturation of an RNA molecule. In one embodiment, the method is carried out in absence of a condition comprising heating, an adapter ligation, a chemical denaturant, or combinations thereof.
As discussed above, the nicked or partially cleaved RNA may be an RNA molecule having a discontinuity between adjacent nucleotides and/or having a portion removed from the parent, full-length RNA. In one embodiment, the nicked or partially cleaved RNA comprises a tRNA having a discontinuity between adjacent nucleotides (e.g., a cleaved phosphodiester bond) in the anticodon loop of the tRNA. In one embodiment, the nicked or partially cleaved RNA comprises an RNA having a region (e.g., a loop or overhang or a portion thereof) removed from the parent, full-length RNA.
The biological sample can contain various types of RNAs and various types of nicked or partially cleaved RNA. All the nicked or partially cleaved RNAs may be enzymatically repaired, e.g., simultaneously, by this method. Thus, by this method, after the enzymatic treatment disclosed herein, the final product would contain all RNAs originally contained in the biological samples, including those nicked or partially cleaved RNAs that have now been repaired.
In some embodiments, at least a portion of the nicked or partially cleaved RNAs are formed from extracellular RNAs. In some embodiments, at least a portion of the nicked or partially cleaved RNAs are formed from intracellular RNAs.
In some embodiments, at least a portion of the nicked or partially cleaved RNAs are formed from non-vesicular RNAs. In some embodiments, at least a portion of the nicked or partially cleaved RNAs are formed from vesicular RNAs.
In some embodiments, the nicked or at least partially cleaved RNAs comprise one or more RNAs selected from the group consisting of tRNA (e.g., nicked and full length), rRNA, YRNA, 7SL RNA, 7SK RNA, snRNA, snoRNA, vaultRNA, Alu RNA, transposable element-derived RNA, pri-microRNA, pre-microRNA, mRNA exons, mRNA introns, 5′ UTR, 3′ UTRs, and fragments and combinations thereof. These RNAs can be codified by either the nuclear genome or the genome of an organelle (e.g., mitochondria). These RNAs are examples of intrinsically stable non-vesicular RNAs. These RNAs are usually longer than 40 nucleotides in length, but sometimes appear in biofluid as relatively short RNA fragments due to the use of denaturing methods (sRNA-seq and/or northern blotting) applied to nicked exRNAs.
In one embodiment, the nicked or at least partially cleaved RNAs comprise tRNA, tRNA fragment (tRFs), tRNA-derived RNAs (tDRs), and/or tRNA half.
In one embodiment, at least a portion of the nicked or at least partially cleaved RNAs comprise single stranded tRNA half.
For the enzymatic repair treatment, one or more enzymes can be used. Without being bound by theory, the enzyme(s) used would perform the functions of dephosphorylating at 3′ end of the nicked or at least partially cleaved RNA (forming a 3′-OH), phosphorylating at 5′ end of the nicked or at least partially cleaved RNA (forming a 5′-phosphate), and ligating the 5′-phosphate RNA to the 3′-OH RNA. The enzymatic treatment can be carried out by one enzyme that has all these functions, two enzymes with the combination of both of which having all these functions, or three enzymes each having a different function and the combination of all of which having all these functions.
In some embodiments, the purified RNAs is treated with (i)(a) a polynucleotide kinase (PNK) and an RNA ligase. In one embodiment, the polynucleotide kinase is added together with the RNA ligase. In one embodiment, the polynucleotide kinase is added first, followed by the addition of the RNA ligase. In one embodiment, the polynucleotide kinase used is T4 PNK. In one embodiment, the RNA ligase is T4 RNA ligase 1.
In some embodiments, the purified RNAs is treated with (i)(b) an RNA 3′ phosphatase or an RNA 2′,3′ cyclic phosphatase, an RNA 5′ kinase, and an RNA ligase, added together, or in sequential order. In one embodiment, the RNA 3′ phosphatase or RNA 2′,3′ cyclic phosphatase, the RNA 5′ kinase, and the RNA ligase are all added together. In one embodiment, the RNA 3′ phosphatase or RNA 2′,3′ cyclic phosphatase, and the RNA 5′ kinase are added first, followed by the addition of the RNA ligase. In one embodiment, the RNA 3′ phosphatase or RNA 2′,3′ cyclic phosphatase is added first, followed by the RNA 5′ kinase and the RNA ligase. In one embodiment, the RNA 3′ phosphatase or RNA 2′,3′ cyclic phosphatase is added first, followed by the addition of the RNA 5′ kinase, and followed by the addition of the RNA ligase. In one embodiment, the RNA ligase is T4 RNA ligase 1.
In some embodiments, the purified RNAs is treated with (ii) a 3′-5′ RNA ligase. In one embodiment, the 3′-5′ RNA ligase is RtcB ligase.
In some embodiments, the treatment of the purified RNAs according to (i) (inclusive of (i)(a) and (i)(b)) or (ii) is carried out at least in part in the presence of a cofactor. In one embodiment, the cofactor is an adenosine triphosphate (ATP) or guanosine-5′-triphosphate (GTP).
The nicked or at least partially cleaved RNAs contained in the biological sample are repaired to reconstitute the parent RNA's full-length or substantial full-length. Having longer reads (and more complete reads of these nicked or at least partially cleaved RNAs) provides more important structural information and confers more discriminative power (especially in the case of tRNAs, that contain a high number of very similar isoacceptors and isodecoders that can have tissue-specific and cancer-specific expression).
In some embodiments, the repaired RNA comprises a nucleotide sequence identical or substantially identical to the parent RNA from which the nicked or at least partially cleaved RNA was formed, in full-length or in substantial full-length.
In one embodiment, the repaired RNA comprises a nucleotide sequence identical or substantially identical to the parent RNA from which the nicked or at least partially cleaved RNA was formed, in full-length.
In some embodiments, the repaired RNA comprises a nucleotide sequence identical or substantially identical to the parent RNA from which the nicked or at least partially cleaved RNA was formed, in substantial full-length. For instance, the length of the repaired RNA may be at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or virtually 100% of the full length of the parent RNA.
In one embodiment, the repaired RNA is 1-11 nucleotides shorter compared to its parent RNA from which the nicked or at least partially cleaved RNA was formed.
In one embodiment, the repaired RNA is 3-5 nucleotides shorter compared to its parent RNA from which the nicked or at least partially cleaved RNA was formed, losing a single-stranded overhang.
In one embodiment, the repaired RNA is 3-7 nucleotides shorter compared to its parent RNA from which the nicked or at least partially cleaved RNA was formed, losing an anticodon loop or a portion thereof.
The source of the biological sample may be a biofluid, a cell, a tissue, an organ, or any combination thereof.
In some embodiments, the biological sample is from a cell. In one embodiment, the biological sample is from a cancerous cell. In one embodiment, the nicked or at least partially cleaved RNAs are the precursors of stress-induced tRNA halves (tiRNAs).
In some embodiments, the biological sample is from an extracellular biofluid. For instance, the extracellular biofluid may be blood, blood serum, blood plasma, urine, lymph, saliva, synovia, milk, cerebrospinal fluid, or a combination thereof.
In some embodiments, the method comprises further purifying the enzyme-treated RNAs.
The method described herein can be used to detect RNAs such as extracellular RNAs in a biological sample, with a particular focus on providing important information on the highly structured RNAs, such as those nicked or partially cleaved RNAs, that are typically not identified by the conventional or currently available sequencing and/or amplification methods. The method can monitor gene expression changes in tissues by sequencing extracellular samples and to monitor disease (diagnosis, prognosis, relapse, response to treatment, etc.) based on the analysis of liquid biopsies.
Accordingly, another aspect of the invention relates to a method for detecting RNAs from a biological sample. The method comprises providing a biological sample containing RNAs that are nicked or at least partially cleaved; purifying the nicked or at least partially cleaved RNAs contained in the biological sample, under a non-denaturing condition, to remove non-RNA components; treating the purified RNAs with at least one of the following: (i) one or more enzymes that exhibit the activity of an RNA 3′ phosphatase or cyclic phosphatase and the activity of an RNA 5′ kinase, and an RNA ligase, or (ii) a 3′-5′ RNA ligase, thereby repairing at least a portion of the nicked or partially cleaved RNAs; and detecting the repaired RNAs.
All above descriptions and all embodiments discussed in the above aspect relating to the method of enzymatically repairing RNAs that are nicked or at least partially cleaved, including various aspects of the biological sample, nicked or at least partially cleaved RNAs, various RNA purification, separation, and/or enrichment methods, non-denaturing conditions, various enzyme treatment protocols, and the structure and sequences of the repaired RNA are applicable to this aspect of the invention relating to a method for detecting RNAs from a biological sample.
The method described herein can be applied to vesicular sample, non-vesicular sample, or total extracellular sample. It can be applied to patients suffering from various disease or conditions or to healthy donors. It can be applied to humans or to animals in veterinary medicine.
After the enzymatic repairing of the RNAs that are nicked or at least partially cleaved in the biological sample, the RNAs can be further reverse transcribed with a conventional reverse transcriptase, or a thermostable reverse transcriptase. In one embodiment, the RNAs can be further reverse transcribed with a thermostable reverse transcriptase. A typical thermostable reverse transcriptase is one that can work at high temperatures, to break the internal structure of the RNAs, and is more tolerant to modified bases. Exemplary thermostable reverse transcriptase suitable for applications herein are retroelement-derived reverse transcriptases such as TGIRT III and retrovirus-derived reverse transcriptases such as Superscript IV, among others.
After the enzymatic repairing of the RNAs that are nicked or at least partially cleaved in the biological sample, the RNAs can be detected by various detection methods known in the art.
In some embodiments, the method for detecting RNAs from a biological sample is carried out in absence of a condition that results in denaturation of an RNA molecule. In one embodiment, the method is carried out in absence of a condition comprising heating, a chemical denaturant, or combinations thereof.
Alternatively, in some embodiments, a condition that results in denaturation of an RNA molecule, such as heating, chemical denaturants, or combinations thereof, may be introduced, but is introduced after the enzymatic repair step(s) (i.e., the enzymatic treating step(s)) described herein.
In some embodiments, the detecting comprises sequencing, amplification, nucleic acid hybridization, or a combination thereof.
In one embodiment, the detecting comprises quantitative RT-PCR (RT-qPCR).
In one embodiment, the detecting comprises a form of high-throughput sequencing, which can be any form of a high-throughput sequencing including but not limited to sequencing by synthesis, nanopore sequencing, or any related technology that is available to obtain nucleic acid sequences.
In one embodiment, the biological samples are treated with RNase inhibitors before or after the purifying the nicked or at least partially cleaved RNAs contained in the biological sample.
The following examples are for illustrative purposes only and are not intended to limit, in any way, the scope of the present invention. To the extent that specific materials are mentioned, it is merely for illustrative purpose and is not intended to limit the invention. One skilled in the art may develop equivalent methods or reactants without the exercise of inventive capacity and without departing from the scope of the invention.
U2-OS cells were cultured in DMEM (Gibco) with 4.5 g/L D-glucose and 110 mg/L sodium pyruvate, supplemented with 10% fetal bovine serum (FBS) (Gibco), without antibiotics.
Blood and urine samples were collected at the Pasteur Institute of Montevideo. Blood samples were obtained by venipuncture from a healthy donor in serum blood collection tubes and centrifuged at 2,500 rpm for 15 minutes to separate serum, which was stored at −20° C. until use.
20 mL of a human urine sample was retrieved within 1 hour of collection from a healthy donor in a sterile container. The sample was transferred to a 10 mL Falcon tube and centrifuged at 300×g and 4° C. for 10 minutes, followed by centrifugation at 2000×g and 4° C. for 20 minutes. Supernatant was then stored at −20° C. until use.
SPE was carried out using Monarch RNA Cleanup Kits (10 μg binding capacity columns, New England Biolabs, NEB) except for the purification of stress-induced tRNA-derived fragments from U2-OS cells, where the Total RNA Miniprep Kit (NEB) was used. Purification was carried out following manufacturer's instructions, but in several experiments, the volume of ethanol added to the binding buffer was doubled to avoid small RNA loss.
Generation of Stress-Induced tRNA Halves and Intracellular RNA Purification
For the generation of stress-induced tRNA halves (tiRNAs), 500 μM of freshly made sodium arsenite (Sigma-Aldrich) were added to U2-OS cells grown to 90% confluence in DMEM+10% FBS. After 2 hours at 37° C., the medium was removed and cells were washed with warm 1×PBS prior to RNA extraction using a Total RNA Miniprep Kit (NEB). Manufacturer's instructions were followed, except for two modifications: i) cell lysis was performed by adding 2 mL of lysis buffer directly to the cell monolayer, followed by a short (5-minute) incubation at room temperature; and ii) twice the indicated volume of ethanol was added to the RNA sample after elution from the RNA-binding column.
For determination of RNA half-lives in biofluids, 1 μg of heated and refolded (1 minute at 90° C. followed by 30 minutes at room temperature) U2-OS total RNA was added to 50 μL undiluted human urine, undiluted human serum, diluted human serum, or FBS (both 10% in 1×PBS). Additionally, 1 μg of heated and refolded total RNA was incubated at 37° C. for variable amounts of time with recombinant human RNase 1 at 0.5 μg/mL (r-RNase 1; Bon Opus Bio). Reactions were stopped by the addition of SPE binding buffer and RNA cleanup.
For in vitro generation of nicked tRNAs and/or tDRs, 1 μg heated and refolded U2-OS total RNAs was mixed with 40 μL of r-RNase 1 (diluted in PBS at 0.0625 μg/mL) for 15, 30 or 60 minutes at 37° C.
For ligation assays involving T4 RNA ligases, 5 μL of in vitro-digested RNA (or RNA purified from CCM) were incubated for 1 h at 37° C. in a 10 μL reaction containing 20 U of RI, 1 mM ATP, 1×T4 PNK reaction buffer, and desired enzyme combinations. Enzymatic cocktails included: 10 U T4 RNA ligase 1 (Rnl1, NEB) or T4 RNA ligase 2 (Rnl2, NEB) and/or 10 U of T4 PNK (wild-type or 3′ phosphatase minus, NEB).
As a control, RNA was heat-denatured before enzymatic treatment (1 minute at 90° C. and immediately placed on ice).
For RtcB ligation, reaction mixtures (10 μL; 1 hour at 37° C.) contained 5 μL of in vitro-digested digested RNA (with or without previous heat denaturation), 20 U of RI, 1×RtcB ligase buffer, 1 mM GTP, 1 mM Mn2+, and 1 μM RtcB ligase from E. coli (NEB). The reactions were stopped by addition of 2×RNA Loading Dye and analyzed by northern blot.
Identification of Nonvesicular Nicked tRNAs in Biofluids
A 200 μL healthy donor human serum sample was thawed and centrifuged at 2,000×g and 4° C. for 10 minutes and diluted in 12 mL of PBS. EV depletion was performed by ultracentrifugation at 256,000×g and 4° C. for 1 hour in an Optima XPN ultracentrifuge (Beckman Coulter) with a SW 40 Ti rotor. The supernatant was then concentrated by ultrafiltration to 200 μL, using 10.000 MWCO Amicon Ultra-15 Centrifugal filters (Merck). Then, 440 μL of the RNA Binding Buffer included in RNA Cleanup kits (NEB) was added to the concentrated supernatant and treated with 20 μL Proteinase K (QIAgen) for 30 minutes at 37° C. Nucleic acids were then purified by SPE and eluted in 50 μL nuclease free H2O (Invitrogen). Samples were analyzed by size-exclusion chromatography using an FPLC system.
Nonvesicular samples from human serum, or total RNA from U2-OS cells stressed with sodium arsenite (with or without heat denaturation and refolding) were diluted in 1×PBS (500 μL) and centrifuged at 10.000×g and 4° C. for 10 minutes prior to being injected into a Superdex 75 10/300 column (GE).
Size-exclusion chromatography (SEC) was performed at 0.5 mL/minute in 0.2 μm-filtered 1×PBS with an Äkta Pure FPLC system and 0.2 mL fractions were collected while monitoring the absorbance at 260 and 280 nm. Nucleic acids from selected fractions were ethanol precipitated (700 μL absolute anhydrous ethanol; 100 μL 3M NaAc, pH=5.2; 0.5 μL Glycogen Blue) overnight at −20° C. and centrifuged at 12.000×g and 4° C. for 15 minutes. The pellet was washed with 500 μL 75% ethanol, centrifuged at 12.000×g and 4° C. for 15 minutes, and resuspended in 10 μL of nuclease free water (Invitrogen) before northern blot or stem-loop RT-qPCR.
Northern blot was performed with DIG-labelled DNA probes as described in Tosar (Tosar et al., 2020). Band intensities were obtained by densitometry using GelQuant 2.0 software.
For denaturing northern blots, 5 μL of RNA were mixed with 5 μL of loading buffer containing 95% formamide, 1 mM EDTA, 0.02% SDS, 0.02% bromophenol blue and 0.01% xylene cyanol, heated to 65° C. for 5 minutes and ran in 10×10 cm 10% polyacrylamide gels containing 7M urea in 1×Tris-borate EDTA (TBE, pH 8.4).
For non-denaturing northern blots, the RNA samples were mixed with 1 μL of 6× native loading buffer, and run in gels containing 1×Tris-borate (pH 8.3) and 10 mM MgCl2 (TB+Mg2+) gels. Gels were run for 80 minutes in 0.5×TBE or 0.5×TB+Mg2+ running buffer at room temperature, stained with 1×SYBR gold (Invitrogen), and then transferred to positively charged nylon membranes (Roche) using a semi-dry Trans-Blot Turbo Transfer System (Bio-Rad) in 0.5×TBE at constant I=0.3 A for 30 minutes.
The membranes were UV cross-linked and hybridized for 16 hours at 42° C. with digoxigenin-labeled DNA probes in DIG Easy Hyb solution (Roche). After hybridization, membranes were washed for 5 minutes at room temperature with low stringency wash buffer (twice, 2×SSC/0.1% SDS), 5 minutes at 42° C. with high stringency wash buffer (1×SSC/0.1% SDS), blocked for 30 minutes at room temperature with 1×blocking solution (Roche) and probed for 30 minutes with an alkaline phosphatase-labeled anti-digoxigenin antibody (Roche). The membranes were washed twice with 1×TBS-T for 5 minutes, and then incubated in detection buffer (Roche). Signals were then visualized with CDP-Star, ready-to-use (Roche) and detected using an Amersham ImageQuant 800 imager (GE Healthcare/Cytiva).
All oligonucleotides were obtained from Integrated DNA Technologies (IDT, USA), and labeled with DIG Oligonucleotide Tailing Kit, 2nd generation (Roche), following manufacturer's instructions.
Stem-loop RT-qPCR was used to amplify and quantify small RNAs following protocols used in Tosar et al. (Tosar et al., 2015, 2018). Briefly, specific cDNA of tRNAGlyGCC 5′ halves and miR-21-5p was obtained with SuperScript II (ThermoScientific). Purified RNAs were heated at 90° C. for 1 minute and immediately placed on ice before reverse transcription. qPCR was performed using a QuantStudio 3 Real Time PCR System (ThermoScientific) with FastStart Universal SYBR Green Master (Rox; Roche). 2−Cq values were obtained and normalized against the fraction containing the highest signal.
Stem-loop RT primer (“X” denotes assay-specific 3′ overhangs):
All primers were obtained from Integrated DNA Technologies (IDT, USA). Lower case letters in these primers indicate the existence of added bases to increase the melting temperature.
RT-PCR for Full-Length tRNAs.
2 μL of 10 ng/μL U2-OS total RNA (input) or RNase 1-treated RNAs (starting from an equivalent amount of input RNA) were mixed with 1 μL of 2 μM gene-specific RT primer and 1 μL of 10 mM (each) dNTP mix in a reaction volume of 11 μL, incubated for 5 min at 65° C., and chilled on ice for at least 1 min. Annealed RNAs were mixed with 4 μL 5× SuperScript IV (SSIV, Thermo) buffer, 1 μL 0.1M DTT, 20 U RI and 20 U SSIV RT. The reaction was incubated for 10 minutes at 65° C. and then for 10 minutes at 80° C. The cDNA was diluted ½ prior to qPCR or 1/10 for endpoint PCR.
Gene-specific RT primer for tRNAGlyGCC1-4:
Identification of Naked RNAs that are Stable in the Presence of Serum
To search for intrinsically stable RNAs in extracellular samples, abundant cellular transcripts were screened to determine if they can resist degradation in serum-containing media in the absence of their protein counterparts (
Naked full-length tRNALysUUU is intrinsically stable in biofluids
To precisely measure tRNA half-lives, the previous assay with better temporal resolution was repeated (
These assays were then repeated in human biofluids including urine, diluted and undiluted serum (
Glycine tRNA Halves Produced in Biofluids are Extremely Stable
Full-length tRNAGlyGCC was almost completely degraded in less than one minute in 10% FBS (
Closer inspection of the data in these figures shows the strong differences in the stability of fragments derived from the same parental tRNAs, but with slightly different lengths. tRNAGlyGCC was first cleaved at the anticodon loop, generating 34-35 nt 5′ halves that rapidly disappeared. These fragments were subsequently replaced by highly stable shorter fragments of approximately 30-31 nt, with a cleavage site at the start of the anticodon loop. An additional cleavage site in the TΨC loop (position 54) could be identified and exposed by using lower r-RNase1 concentrations (
Overall, tRNAs appeared to be more resistant to degradation than other longer noncoding RNAs, but differences in stability among tRNAs were also substantial (>50-fold). Also, naked tRNAGlyGCC 5′ halves of 30-31 nt could be accumulated at higher RNase concentrations or after longer incubations in biofluids.
Nicked tRNAs are a Source of 5′ and 3′ tRNA Halves
The relative stabilities of 5′ and 3′ tRNAGlyGCC-derived fragments were then compared, and the results are shown in
Without being bound by theory, tRNAGlyGCC 5′ and 3′ halves could remain physically associated with each other after RNase cleavage, representing a full-length tRNA bearing a cleaved phosphodiester bond at the anticodon loop (i.e., in the form of “nicked tRNAs”). This would explain the similar decay kinetics of each half among different biofluids. However, the introduction of any irreversible denaturation steps, such as those used in standard molecular biology approaches, would induce dissociation of nicked tRNAs into single-stranded tRNA halves. This can be further tested by new assays discussed herein capable of interrogating oligomeric RNA complexes under native conditions.
Nicked tRNAs are the natural substrates of T4 polynucleotide kinase (PNK) and T4 RNA ligase 1 (Rnl1) (Schwer et al., 2004). When Escherichia coli is infected by the T4 Phage, a bacterial anticodon nuclease called PrrC is activated and cleaves the host's tRNALys in an attempt to prevent the translation of viral proteins (Kaufmann, 2000). This would result in a nicked tRNA bearing a 3′ cyclic phosphate (3′ cP) and a 5′-OH adjacent to the cleavage site. However, the phage evolved two enzymes capable of performing end-healing (PNK) and tRNA repair (Rnl1). These enzymes can be used to investigate the native structure of human tRNA halves in extracellular samples.
Total RNAs from cells were purified and incubated with r-RNase1 for 0, 15 or 60 minutes (
To demonstrate that the tRNA-sized re-ligated products are indeed repaired tRNAs, a third probe (termed ACL, for anticodon loop) bridging both sides of the anticodon of tRNAGlyGCC (
Strikingly, the treatment of RNase 1-treated RNA with PNK and Rnl1 regenerated tRNA-sized products observable with the 5′, the 3′, or the ACL probes (
In summary, nicked tRNAs were produced in vitro using different tRNAs as substrates. These nicked tRNAs were repaired enzymatically to regenerate an almost full-length tRNA, presumably lacking the 3′ NCCA overhang in in vitro settings (Akiyama et al., 2022). Additional bases can be trimmed after prolonged exposure to extracellular RNases, but these shorter forms would still be reparable using the assay discussed herein.
Nicked tRNAs are Irreversibly Melted by Popular RNA Extraction Methods
Whereas heating RNase-treated RNA before tandem incubation with PNK and Rnl1 prevented the formation of a tRNA-sized band, this also affected the detection of monomeric 5′ and 3′ tRNA halves (
The size cut-off of SPE RNA cleanup columns can be reduced by doubling the amount of ethanol added to the sample. When two volumes of ethanol were used, the amount of eluted tRNA halves was increased, while the effect of heat was lost. This indicates that nicked tRNAs (>70 nt) can bind to the SPE columns when one volume of ethanol is used, but monomeric 30-35 nt tRNA halves are lost in the flow-through. In contrast, doubling the ethanol allows a highly efficient capture of both nicked tRNAs and heat-induced monomeric tRNA halves. The enzymatic repair assays were then repeated under conditions desirable for small RNAs (2×EtOH) where the monomeric tRNA halves were still detectable in the control (i.e., heat) reaction (
Given that heating irreversibly affected nicked tRNAs, the effects of various commonly used RNA extraction methods were studied, many of which include agents (e.g., phenol) known to disrupt base-pairing interactions. A recent study of different RNA purifications methods for liquid biopsies found that the miRNeasy kit (Qiagen) recovered a broad spectrum of exRNAs associated with different carrier subclasses (Srinivasan et al., 2019). However, when RNase1-treated RNA was purified with this kit following the manufacturer's instructions, tRNA halves were recovered at acceptable yields but were no longer amenable to enzymatic repair (
Nicked tRNAs are Resistant to RNase 1 Cleavage In Vitro
Having observed that nicked tRNAs can be purified by SPE, nicked tRNAs were generated and purified, and were treated again with r-RNase 1 (
In conclusion, nicked tRNAs are stable reservoirs of tRNA halves, which are degradation-prone once being dissociated from their 3′ counterparts.
In Vitro Generated tRNA Halves are Predominantly Nicked tRNAs
A comparison of the intensities of enzymatically repaired (light gray arrows) and untreated (NT) tRNAGlyGCC 5′ halves in RNase1-treated samples purified under optimized conditions (
A northern blot assay was also conducted after running the RNAs on a native (TB-Mg2+) polyacrylamide gel to further confirm this conclusion (
Native northern blots also confirmed that heating or standard RNA purification methods such as TRIzol or miRNeasy, unlike phenol-free RNA cleanup columns (SPE), induced the irreversible dissociation of nicked tRNAs (
The 3′-5′ RNA Ligase RtcB can Also Ligate Nicked tRNAs
RtcB is a ligase involved in tRNA splicing and RNA repair in all domains of life (Englert et al., 2011; Popow et al., 2011; Tanaka and Shuman, 2011). Unlike the 5′-3′ T4 Rnl1, RtcB seals broken RNAs with 2′,3′ cyclic phosphate (2,3-cP) and 5′-OH ends in the presence of GTP and Mn2+ (Chakravarty et al., 2012). This enzyme actually catalyzes a two-step process, where 2,3-cP is hydrolyzed to a 3′-monophosphate and subsequently ligated to the 5′-OH (Tanaka and Shuman, 2011).
Nicked tRNAs as a Source of Intracellular Stress-Induced tRNA Halves
The enzymatic repair assays may not be as effective to detect the existence of nicked tRNAs inside cells when a large excess of full-length tRNAs are present in intracellular samples. A different strategy based on intracellular RNA fractionation under nondenaturing conditions by size-exclusion chromatography (SEC) (
U2-OS cells were exposed to 500 μM sodium arsenite for two hours based on methods described in Yamasaki et al. (Yamasaki et al., 2009) and the presence of stress-induced tRNA halves was verified by northern blot (
A tRNA peak was consistently eluted at Ve=9.80 mL, evidenced by the registered absorbance at 260 nm (
Dimerization of tRNAGlyGCC 5′ halves could also explain their elution at Ve=9.80 mL in these chromatographic columns (Tosar et al., 2018). However, the transfection of cells with 30 nt tRNAGlyGCC 9 GG/AA mutants, which can dimerize in vitro (Tosar et al., 2018), showed a chromatographic elution consistent with their presence in monomeric form (
In sum, dimerization would not explain the elution of intracellular tRNA halves in chromatographic peaks corresponding to RNAs with twice their predicted size. The results thus suggest that at least some intracellular tiRNAs were predominantly present in the form of nicked tRNAs. Alternatively, intracellular tiRNAs might be degraded upon cell lysis, leaving only the more stable nicked tRNAs.
Nonvesicular Nicked tRNAs Circulate in Human Biofluids
Having validated a method capable of separating nicked tRNAs from single-stranded tRNA halves, this method was implemented to further address the question of whether nicked tRNAs circulate in human biofluids.
200 μL human serum was diluted in PBS, and EVs were pelleted by ultracentrifugation. RNA was then isolated from Proteinase K-treated supernatants and fractionated by SEC. Surprisingly, 30 nt tRNAGlyGCC 5′ halves could be amplified by SL-RT-qPCR (
Nicked tRNAs are Reverse-Transcribed Inefficiently Unless Repaired.
Broken phosphodiester bonds could act as roadblocks inhibiting reverse transcription (RT) of nicked tRNAs in their native state, preventing the analysis of nicked tRNAs by RT-PCR or sequencing. This effect was evaluated using a thermostable retroviral reverse transcriptase primed by a gene-specific RT primer aligning to the 3′ end of tRNAGlyGCC (
It was widely believed that all RNAs are intrinsically unstable and cannot circulate in extracellular samples unless in the context of RNPs, lipoproteins and/or EVs. Although this holds true for rRNA-derived fragments (
Importantly, the differences in extracellular stabilities were observed among tRNAs. At one extreme, the full-length tRNALysUUU was processed at slower rates than any other tested RNA, irrespective of the sample type. This was in sharp contrast with, for example, the rapidly processed full-length tRNAGlyGCC. One explanation is that post-transcriptionally modified bases are at least partially responsible for these different behaviors. Based on the modomics database, tRNAGlyGCC does not contain modified bases in the anticodon loop, except for m5C at position 37. In contrast, at least in yeast, tRNALysUUU contains mcm5s2U and t6A at positions 34 and 37, respectively. Although these modifications have been shown to facilitate cleavage by bacterial and yeast anticodon ribonucleases (Bacusmo et al., 2018; Lentini et al., 2018), mammalian RNases are often inhibited by modified bases present in the anticodon (Lyons et al., 2018).
The difference in the stability between tRNAs is less pronounced when considering that nicked tRNAs, and not tDRs, are the stable degradation intermediates dictating the abundance of nonvesicular glycine tRNA halves. Although the full-length tRNAGlyGCC was efficiently cleaved at several positions by extracellular RNases in human biofluids, the result of these cleavage events is a molecule that probably still resembles a tRNA, even if it bears some broken phosphodiester bonds. Thus, although tRNALysUUU and tRNAGlyGCC show completely different behaviors when analyzed by northern blot after being exposed to RNases, this difference could have been exaggerated because the standard northern blotting forced nicked tRNAs to denature. This would be also the case when purifying RNA using phenol and when RNA is heated at any stage in a protocol (e.g., RNA-seq). The important conclusion is the true, native form of RNA is not always being apprehended because most available analytical techniques contain denaturation steps at some point.
The case of tRNALysUUU is also interesting. Although it is highly resistant to degradation, once being cleaved by r-RNase 1 or by FBS-derived RNase A, it did not survive as a nicked tRNA (
Nicked tRNAs were not considered as stable degradation intermediates in the literature (Chen et al., 2021). Stress-induced tRNA cleavage at the anticodon loop is thought to be an irreversible process, whereas reversibility of stress-induced nicked tRNA formation in cellulo arises as an exciting possibility. In the clinical setting, double-stranded RNAs usually require encapsulation in lipid nanoparticles or GalNAc conjugation for efficient uptake, but single-stranded oligonucleotides are spontaneously endocytosed (Levin, 2019). Thus, the mechanism of the enzymatic repairing process disclosed herein where the nonvesicular nicked tRNAs are carriers of tRNA halves that can transfer information to recipient cells is at least feasible (
Disclosed herein is a new analytical technique that can be used to analyze stable nonvesicular RNAs circulating in biofluids. Although this example focused mostly on tRNAs, close inspection of gels stained with SYBR gold shows additional bands that could be restored by combined treatment with PNK and Rnl1 and that were lost in heated samples (
There are sequencing methods that can efficiently handle highly structured RNAs (Behrens et al., 2021; Qin et al., 2016). However, nicked RNAs are more challenging because they contain roadblocks of reverse transcriptases (i.e., the broken phosphodiester bonds;
In this example, protein-free RNAs were incubated in human biofluids, and their decay kinetics were measured by northern blot. Certain specific naked tRNAs were found to be intrinsically stable. The half-lives of several naked RNAs were measured. Although rapid clearance was the norm, specific full-length tRNAs decayed comparatively slowly in biofluids. Furthermore, the stability of 5′ tRNA halves generated from the endonucleolytic cleavage of unstable tRNAs was extremely high. However, these ultra-stable tRNA-derived fragments (tDRs) are not present as real fragments in most extracellular samples. On the contrary, they circulate mainly as full-length tRNAs containing a few broken phosphodiester bonds. Also surprisingly, these ultra-stable RNAs were not single-stranded.
Studying these nicked tRNAs remains a challenge because standard protocols for RNA extraction, northern blotting and RNA-seq induce their artefactual denaturation, even though their presence is supported by native gels. Different strategies of enzymatic repair and electrophoretic or chromatographic separation under native conditions were developed and employed in this example. A two-step method was employed based on enzymatic repair with T4 PNK and Rnl1 (or a one-step ligation with recombinant RtcB) after phenol-free RNA purification. Heating the samples before the enzymatic treatment abrogated nicked tRNA repair. Nicked tRNAs were separated from tDRs by chromatographic methods under native conditions. These protocols were used to identify nicked tRNAs inside stressed cells and in vesicle-depleted human biofluids.
The results also show that these RNAs natively exist as full-length tRNAs containing broken phosphodiester bonds. This is surprising and important, because commonly used RNA purification methods, sequencing, and northern blotting do not detect these nicked or partially cleaved forms, forcing them to melt into single-stranded tRNA halves. In addition,
The method shown in this example is useful to uncover a hidden layer of the extracellular RNAome composed of intrinsically stable, nick-containing, highly structured RNAs.
All documents and references cited herein, including but are not limited to, journal articles or abstracts, published or corresponding U.S. or international patent applications, issued U.S. or foreign patents, or any other documents, are each incorporated herein by reference in their entirety, including all data, tables, figures, and text presented in the cited documents and references.
This application claims benefit of priority to U.S. Provisional Application No. 63/402,523, filed Aug. 31, 2022, which is herein incorporated by reference in its entirety.
This invention was made with government support under grant UG3/UH3CA241694 (Novel Separation Methods for exRNA Carriers: Extracellular Vesicles, Lipoprotein Particles, and Protein Aggregates) by the National Institutes of Health (National Cancer Institute and Office of the Director). The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63402523 | Aug 2022 | US |
