Protecting RNAs from degradation using engineered viral RNAs

Abstract

This invention is in the field of molecular biology, gene expression, functional genomics, and bioinformatics and relates to novel RNA and related structures and methods of use thereof that enables modulation of gene expression and preservation of particular transcriptome targets. The invention contemplates various applications of RNA sequences derived from the genomic RNA of flaviviruses (FVs) and the application of such features in combination with heterologous sequences.

Claims

1. A ribonucleic acid (RNA) duplex comprising a synthetic first RNA sequence that provides exonuclease resistance to the RNA duplex when hybridized to a heterologous second RNA sequence without exonuclease resistance, wherein a region of said synthetic RNA sequence and a region of said heterologous RNA sequence hybridize to form an interwoven pseudoknot structure, wherein the synthetic first RNA sequence that provides exonuclease resistance to the RNA duplex comprises a sequence of nucleotides 10-60 of SEQ ID NO:2.

2. The RNA duplex of claim 1, wherein the interwoven pseudoknot structure comprises a conserved three-way junction.

3. The RNA duplex of claim 1, wherein the heterologous second RNA sequence comprises a naturally occurring RNA sequence.

4. The RNA duplex of claim 1, wherein the synthetic first RNA sequence further comprises a further heterologous RNA sequence ligated to the 3′ end of the synthetic RNA sequence.

5. The RNA duplex of claim 4, wherein the further heterologous RNA sequence ligated to the 3′ end of the synthetic first RNA sequence comprises a small molecule sensing riboswitch.

6. The RNA duplex of claim 4, wherein the synthetic first RNA sequence further comprises a translation initiation element.

7. The RNA duplex of claim 5, wherein the small molecule sensing riboswitch disrupts the interwoven pseudoknot structure in a presence of a small molecule sensed by the small molecule sensing riboswitch.

8. The RNA duplex of claim 4, wherein the heterologous second RNA sequence comprises an open reading frame.

9. The RNA duplex of claim 4, wherein the heterologous second RNA sequence comprises a protein binding sequence.

10. The RNA duplex of claim 4, wherein the heterologous second RNA sequence comprises a spinach sequence.

11. The RNA duplex of claim 1, wherein the RNA duplex comprises a chemical modification of either the 5′ end or the 3′ end of the synthetic first RNA sequence, and/or the heterologous second RNA sequence, in the RNA duplex.

12. The RNA duplex of claim 1, wherein the RNA duplex comprises at least one chemically modified nucleotide.

13. The RNA duplex of claim 1, wherein the synthetic first RNA sequence comprises a sequence of GGGUAAUCCGCCAUAGUACGGAAAAAACUAU GCUACCUGUGAGCCCCGUCCAAGGACGUU (SEQ ID NO:2).

14. The RNA duplex of claim 1, wherein the heterologous second RNA sequence comprises a sequence of nucleotides 1-43 of SEQ ID NO:1.

15. A ribonucleic acid (RNA) duplex comprising two ribonucleic acids (RNAs): a synthetic first RNA; and a heterologous second RNA, wherein the synthetic first RNA provides exonuclease resistance to the RNA duplex when hybridized to a heterologous second RNA, wherein a region of said synthetic first RNA and a region of said heterologous second RNA hybridize to form an interwoven pseudoknot structure comprising a conserved three-way junction, and wherein the interwoven pseudoknot structure of the RNA duplex comprises sequences that form P3/L3 stem loop and P4/L4 stem loop structures of a flavivirus (FV) xrRNA1 or xrRNA2 element.

16. The RNA duplex of claim 15, wherein the sequences that form the P3/L3 stem loop and P4/L4 stem loop structures comprise P3/L3 stem loop, P1, S4, and P4/L4 stem loop sequences found downstream of P2/L2 stem loop sequences of a FV xrRNA1 or xrRNA2 element.

17. The RNA duplex of claim 16, wherein the P3/L3 stem loop, P1, S4, and P4/L4 stem loop sequences found downstream of the P2/L2 stem loop sequences of the FV xrRNA1 or xrRNA2 element comprise a GCCA sequence at a 5′ end of the P3/L3 stem loop, P1, S4, and P4/L4 stem loop sequences.

18. The RNA duplex of claim 15, wherein the FV xrRNA1 or xrRNA2 element is from a flavivirus selected from the group consisting of Dengue (DENV), Yellow Fever (YFV), Japanese Encephalitis (JEV), West Nile (WNV), Murray Valley Encephalitis (MVE), and Zika virus.

19. The RNA duplex of claim 15, wherein the FV xrRNA1 or xrRNA2 element is from a DENV xrRNA1 or xrRNA2 element.

20. The RNA duplex of claim 15, wherein the synthetic first RNA that provides exonuclease resistance to the RNA duplex comprises a sequence of nucleotides 10-60 of SEQ ID NO:2.

Description

DESCRIPTION OF THE FIGURES

(1) The accompanying figures, which are incorporated into and form a part of the specification, illustrate several embodiments of the present invention and, together with the description, serve to explain the principles of the invention. The figures are only for the purpose of illustrating a preferred embodiment of the invention and are not to be construed as limiting the invention.

(2) FIG. 1A shows xrRNA function. Xrn1 (green) loads on a FV RNA, degrades the RNA, then reaches a discrete ‘xrRNA’ structure in the 3′ UTR and halts, leading to sfRNAs where Xrn1 appears to remain in an inactive state.

(3) FIG. 1B shows the pattern of sfRNA production in West Nile Virus (Northern blot).

(4) FIG. 1C shows 3′UTR organization, red arrows are known or putative Xrn1 halt sites. Known or putative xrRNAs are boxed. Although depicted as separate entities, their functions may be coupled. SLs=stem loops, DB=dumbbells.

(5) FIG. 2 depicts the basic architecture of a FV 3′ UTR and proposed halt sites for Xrn1 (DENV2 shown as a representative example). The halt sites and structures of the ‘SL’ motifs (solid arrows) have been characterized; the halt sites and structures of the ‘DB’ motifs remain unexplored (open arrows). Dashed lines indicate likely PK interactions.

(6) FIG. 3 depicts splicing and decay pathway in yeast. Both fully spliced mRNAs and splice defective pre-mRNA intermediates are degraded by Xrn1

(7) FIG. 4A shows the design of reporter transcript with a cricket paralysis virus (CrPV) IRES, but no xrRNAs. FIG. 4B shows version of this transcript with xrRNAs (wavy lines) inserted. Transcripts were constitutively transcribed from 2-micron plasmids in budding yeast.

(8) FIG. 5A depicts the pathway from transcription to degradation for transcripts from the non-xrRNA-containing RNAs. FIG. 5B depicts the Pathway for the transcripts containing xrRNAs (red). Although both contain an IRES (blue) and can produce protein, it was expected that the mRNAs without the xrRNA to be degraded more quickly and produce less protein, while those with the xrRNAs should be protected from degradation and produce more protein.

(9) FIG. 6 shows Protection of translationally competent mRNAs in budding yeast. Blue denotes the CrpV IRES, red denotes the tandem copies of xrRNA. The open box is the open reading frame encoding the enzyme. The lanes are explained in the text. The dashed box indicates the products that build up because Xrn1 cannot degrade past the xrRNAs.

(10) FIG. 7 shows an activity assay to measure B-galactosidase (LacZ) levels made from mRNAs produced in budding yeast. LacZ activity is reflected by an increase in ONPG hydrolysis (OD420) per cell (OD600). Including the xrRNAs leads to an increase in the amount of LacZ activity, demonstrating that the xrRNA-protected mRNA is competent for IRES-dependent translation. Mutation of the xrRNAs returns LacZ activity to the level of mRNAs without any xrRNAs. This increase in protein is due to the accumulation of the RNA products boxed in FIG. 6. These uncapped, partially degraded, but protected mRNAs are functional templates for translation.

(11) FIG. 8A depicts a secondary structure cartoon of an xrRNA. The nucleotides explicitly shown are conserved in all xrRNA we have studied so far. The secondary structural elements are labeled. The P2/L2 stem loop is variable between xrRNAs and we have mutated it without affecting function.

(12) FIG. 8B depicts a hypothesized L2 loop as a location where the xrRNA could be split into two pieces (bottom left sequence and top and right sequence).

(13) FIG. 8C depicts RNAs used to test this idea. RNA1 (SEQ ID NO:1) is the “substrate” which is recognized by the 5′ complementary sections of as second RNA sequence (RNA2), which is the “protector”. In theory, RNA 1 could be any length, because RNA2 (SEQ ID NO:2) must only base pair to it as shown. The part labeled as “variable” can be changed to recognize different sequences. The circled nucleotides limit what sequences may be recognized (at this point), but might be varied with more engineering of the RNA.

(14) FIG. 8D show the result of a resistance assay. RNA1 is processed to a shorter product, but is not fully degraded by Xrn1 when RNA2 is present.

(15) FIG. 9 shows a cartoon representation of a highly modified engineered xrRNA based RNA, The red is the portion that base pairs to any target mRNA in the cell (dark dashed line) and protects it from degradation. In light dashed lines is a plant viral RNA sequence that recruits the cap binding protein (round green ball) and drives translation of the protected RNA. In dark line on the 3′ end is another RNA structure; it could be a protein binding site, an aptamer, a Spinach-RNA for visualizing this RNA in the cell, etc.

(16) FIG. 10 shows a strategy to control Xrn 1 resistance using a small molecule. In this particular example, the “protecting RNA (solid) has a G-box riboswitch appended to its 3′ end (light dashed line), with a sequence that can compete for helix P1 that is formed when the protecting RNA hybridizes to the substrate RNA (dark dashed line). Upon binding of the small molecule (block with SM; in this case, hypoxanthine or other related ligands) the riboswitch folds and helix P1′″ forms. This disrupts the ⋅P1 helix and the protector” RNA no longer can halt Xrn1 from degrading the substrate.

(17) FIG. 11 depicts some types of chemical modification of the RNA that be included in the current invention. For example, the chemical modification may comprise inclusion of phosphorothioate linkages, boranophosphate linkages, locked nucleic acid, 2′-modifications, 4′-thio modified RNA, ribo-difluorotoluyl nucleotide, and uncharged nucleic acid mimics, as described by Corey (2007) J. Clin. Invest. 117(12), 3615-3622 [14], herein encorporated by reference.

(18) FIG. 12 shows that Xrn1 resistant RNAs from flaviviruses can resist degradation by different exonucleases. FIG. 12 shows a gel where input RNA containing an xrRNA was challenged with Xrn1, RNase J1 (from bacteria), and Dxo1 (from yeast). All enzymes were expressed recombinantly and purified to high purity. RppH was included in all reactions to convert the 5′ triphosphate to a monophosphate. This demonstrates that the xrRNA could be engineered to work in diverse hosts and have broad applicability.

DESCRIPTION OF THE INVENTION

(19) Flaviviruses are emerging human pathogens and worldwide health threats. During infection, pathogenic subgenomic flaviviral RNAs (sfRNAs) are produced by resisting degradation by the 5′.fwdarw.3′ host cell exonuclease Xrn1 through an RNA structure-based mechanism wherein Xrn1-resistant flaviviral RNA, which contains interwoven pseudoknots within a compact structure that depends on highly conserved nucleotides [15]. The RNA's three-dimensional topology creates a ring like conformation, with the 5′ end of the resistant structure passing through the ring from one side of the fold to the other. Disruption of this structure prevents formation of sfRNA during flaviviral infection. Thus, sfRNA formation results from an RNA fold that interacts directly with Xrn1, presenting the enzyme with a structure that confounds its helicase activity. The current invention envisions adapting creating similar RNA based interwoven pseudoknot structures to enable the preservation of and expression of targeted RNA transcripts.

(20) Signal Activated RNA Interference

(21) One reference, U.S. Pat. No. 9,029,524 [16], describes compositions and methods for signal activated RNA interference (saRNAi). The reference states that a pseudoknot is employed and may be resolved by the introduction of signal, such as a second polynucleotide. Unknotting of the pseudoknot may be accomplished when a signal polynucleotide binds to one of three regions on the signal-activated polynucleotide. In one example, the signal polynucleotide binds to the signal-detecting nucleotide and displaces the pseudoknotting strand from the stem-loop structure. This exposes the stem-loop structure to cleavage by Dicer and processing by the RISC complex. In a second example, the signal polynucleotide binds to the signal-detecting nucleotide at a region upstream of the pseudoknot, causing compression and steric/electrostatic repulsion between the signal-detecting strand and the stem-loop structure. The reference describes the polynucleotide structures as substantially resistant to endonucleases and exonucleases. The reference does not describe xrRNA sequences that contain a second heterologous sequence, nor does it describe the hybridization of two separate RNA molecules to form such an interwoven pseudoknot structure.

(22) One reference, Chapman, E. G. et al. (2014), Science 344(6181), 307-310 [15], describes the structure of the sfRNA which halts the advance of the Xrn1 endonuclease via interruption of the Xrn helicase. The reference describes the crystallized structure of the sfRNA as containing interwoven pseudoknots that interrupt the structure through sequence changes, and halts the effectiveness of Xrm1. The RNA sequences described are sections of naturally occurring xrRNA and those that were engineered to evaluate the sequence's effectiveness against Xrn1. The reference concludes that the xrRNA structure is braced against the enzyme, holding the leading edge of the relevant RNA duplex behind the concave ring structure and away from these helices, suggesting a mechanism for RNA structure-driven Xrn1 resistance and, by extension, sfRNA production. The reference does not describe xrRNA sequences that contain a second heterologous sequence, nor does it describe hybridization of two separate RNA molecules to form such an interwoven pseudoknot structure.

(23) One reference, Chapman, E. G. et al. (2014), eLife 3, e01892 [17], describes modification of sfRNA to evaluate the sequence and structural features of the RNA that conferred resistance to the Xrn1 endonuclease. The reference concludes that disruption of the lead sequences of sfRNA leads to the elimination of disease related sfRNAs, thus linking the RNA structure to sfRNA production. The reference states that such Xrn1-resistant activity resides in discrete and portable structural elements, the RNA sequences. The reference does not describe xrRNA sequences that contain a second heterologous sequence, nor does it describe the hybridization of two separate RNA molecules to form such an interwoven pseudoknot structure.

(24) One reference, Kieft, J. S. et al. (2015), RNA Biol. E-published Sep. 23, 2015 [18], describes that continued analysis and interpretation of the structure of sfRNAs reveals that the tertiary contacts that knit the xrRNA fold together are shared by a wide variety of arthropod-borne FVs, conferring robust Xrn1 resistance in all tested. The reference further discloses that some variability in the structures that correlates with unexplained patterns in the viral 3′ UTRs. The references' examination of these structures and their behavior in the context of viral infection leads to a new hypothesis linking RNA tertiary structure, overall 3′ UTR architecture, sfRNA production, and host adaptation. The reference does not describe xrRNA sequences that contain a second heterologous sequence, nor does it describe the hybridization of two separate RNA molecules to form such an interwoven pseudoknot structure.

(25) One reference, Moon, S. L. et al. (2012), RNA 18(11), 2029-2040 [19], describes an evaluation of sfRNAs, including several experiments introducing sfRNA sequences downstream from a reporter GFP-expressing construct (e.g., a heterologous sequence). In part, this was done to evaluate the decay curves for mRNA in correlation with the amount of sfRNA present in a cell. These experiments further establish the relationship of sfRNA with XRN1 depletion and mRNA transcript stability. The reference states that pseudoknots and related structures have been shown in the past to hinder the action of other enzymatic complexes that move directionally along RNA and that pseudoknots can serve as roadblocks for ribosomes and influence frameshifting rates. The reference does not describe sfRNA sequences upstream of heterologous RNA. The reference does not describe xrRNA sequences that contain a second heterologous sequence, nor does it describe the hybridization of two separate RNA molecules to form such an interwoven pseudoknot structure.

(26) One reference, Burke, D. H. et al. (1996), J. Mol. Biol. 264(4), 650-666 [20], describes several RNA inhibitors of HIV-1 RT that differ significantly from the pseudoknot ligands found previously, along with a wide variety of pseudoknot variants. Patterns of conserved and covarying nucleotides yielded structural models consistent with 5′ and 3′ boundary determinations for these molecules. Among the four isolates studied in detail, the first is confirmed as being a pseudoknot, albeit with substantial structural differences as compared to the canonical pseudoknots identified previously. The second forms a stem-loop structure with additional flanking sequences required for binding. The minimal fully active truncations of each of these four isolates compete with each other and with a classical RNA pseudoknot for binding to HIV RT, suggesting that they all recognize the same or overlapping sites on the protein, in spite of their apparently dissimilar structures. The reference does not describe xrRNA sequences that contain a second heterologous sequence, nor does it describe the hybridization of two separate RNA molecules to form such an interwoven pseudoknot structure.

(27) One reference, U.S. Pat. No. 5,256,775 [21], describes making 3′ and/or 5′ end-capped oligonucleotides so as to render the oligonucleotide resistant to degradation by exonucleases. The exonuclease degradation resistance is provided by incorporating two or more phosphoramidate and phosphorocmonothioate and/or phosphorodithioate linkages at the 5′ and/or 3′ ends of the oligonucleotide, wherein the number of phosphoramidate linkages is less than a number which would interfere with hybridization to a complementary oligonucleotide strand and/or which would interfere with RNAseH activity when the oligonucleotide is hybridized to RNA. The reference does not describe xrRNA sequences or interwoven pseudoknots, nor does it describe the hybridization of two separate RNA molecules to form such an interwoven pseudoknot structure.

(28) One reference, U.S. Patent Application Publication Number US 2014-0329880 A1 [22], describes exonuclease resistant polynucleotides with a 5′ end and a 3′ end and comprises a blocker domain having a non-nucleic acid polymer segment and a phosphorothioate segment. The reference also describes exonuclease resistant duplex polynucleotide having a length of about 17 to about 30 bp and comprising a guide strand complementary bound to a passenger strand, each of the guide strand and passenger strand having a 5′ end and a 3′ end, the duplex RNA having at least one configuration allowing processing of the guide strand by dicer and/or an argonaute enzyme, the passenger strand comprising the exonuclease resistant polynucleotide herein described, in a configuration in which the second end of non-nucleic acid polymer is presented at the 5′ end of the passenger strand. In some embodiments, the exonuclease resistant duplex polynucleotide is a targeting domain. The reference does not describe xrRNA sequences or interwoven pseudoknots, nor does it describe the hybridization of two separate RNA molecules to form such an interwoven pseudoknot structure.

Various Embodiments

(29) In one embodiment, the invention contemplates various applications of RNA sequences derived from the genomic RNA of flaviviruses (FVs). These derived RNA sequences have been found to be resistant to cell enzyme Xrn1, the dominant cytoplasmic 5′.fwdarw.3′ exonuclease. These discrete Xrn1-halting sequences are referred to as “Xrn1-resistant RNAs” (xrRNAs). The xrRNAs have been found to have distinct structural elements and further have certain conserved sequences to retain these resistant structural elements.

(30) In one embodiment, the invention contemplates that such sequences of RNA could be useful in a number of applications including, but not limited to: A) introduction of an xrRNA sequence “in cis” upstream of desired heterologous mRNA sequences to improve the half-life of the heterologous mRNA sequences from exonuclease degradation; and B) provide Xrn1 nuclease resistance by the “in trans” association of an RNA that provides Xrn1-halting ability to an RNA that does not have this ability, thus protecting a “target” RNA from degradation by hybridization of a “protecting” RNA. In this approach, structures similar to xrRNAs could be generated by the hybrid association of two heterologous RNAs rather than placing an xrRNA sequence on at least one heterologous RNA sequence.

(31) In one embodiment, the invention contemplates that the “in trans” configuration could be used: (1) to alter specific endogenous RNA degradation rates within cells as a “transcriptome editing” tool and (2) coupling such RNA combinations with a way to turn protected (but uncapped or decapped) mRNAs into viable translation templates as a “translatome editing” tool. It is believed by adjustment of the RNA sequence, that the “protecting RNA” could be “tuned” to achieve various protection levels and translation levels of endogenous RNAs.

DETAILED DESCRIPTION OF THE INVENTION

(32) The technology/inventions described here are based on our discoveries regarding a viral RNA that has the extraordinary ability to block a powerful cellular exonuclease. This RNA structure is thought to be critical to the virus, but the current invention has now enabled the removal of the RNA structure from that context and engineer it as a manipulator of cellular processes.

(33) Initial studies were focused on the ability of flaviviruses (FVs) to use RNA structure to resist degradation by Xrn1, a powerful cellular exonuclease. Briefly, during replication of arthropod-borne FVs, copies of the viral genomic RNA are made. Infection also leads to a set of discrete shorter flaviviral RNAs that accumulate to high levels [23-25]. These subgenomic flaviviral RNAs (sfRNAs) are −200-500 nt in length and are directly linked to viral cytopathicity in cultured cells (mammalian and insect) and for disease symptoms in fetal mice [26-28]. The sfRNAs are made by partial digestion of FV genomic RNA by host cell enzyme Xrn1, the dominant cytoplasmic 5′.fwdarw.3′ exonuclease (FIG. 1) [27]. In normal cellular metabolism, Xrn1 recognizes the 5′ monophosphate of decapped mRNAs and catalyzes their degradation in a 5′ to 3′ directional process [29]. In FV infection, Xrn1 loads on some presumably decapped copies of the viral RNA genome and degrades them in a 5′.fwdarw.3′ process until it halts at defined locations in the 3′UTR, resulting in the set of sfRNAs [26-28]. This mechanism is surprising given Xrn1's ability to degrade stable structured RNAs such as ribosomal RNA. These discrete Xrn1-halting elements are referred to as “Xrn1 resistant RNAs” (xrRNAs)[17]. An FV xrRNA has been characterized, that being the stem-loop (SL) type often found in tandem near the 5′ end of FV 3′UTRs [26-28] (FIG. 2). The functional, biochemical, structural, and virological studies included the first high-resolution three-dimensional structure of a functional SL-type xrRNA (solved by crystallography). It was found that these RNAs adopt a specific three-dimensional fold, and there is a suggested mechanism for how these RNAs stop progression of Xrn1 using a combination of thermodynamic stability coupled to a unique fold that mechanically confounds the helicase activity of Xrn1. This characterization of these RNAs has revealed how novel and remarkable they are; the structure is unlike anything previously found. These discoveries were published in 2014 in Science and eLife, the contents of which are incorporated herein by reference [15, 17].

(34) These discoveries have provided an insight into the function of these xrRNAs and also inspired the current invention ideas for how they could be used as tools, potentially as part of novel therapeutic strategies in which stabilizing RNAs within a cell is important. In other words, no way is known wherein a specific RNA within a cell can be super-stable, perhaps evading the degradation machinery completely and remaining a viable, biologically active RNA. The xrRNAs may provide the basis for creative engineering and inventions that would allow this type of precise control within living cells. The current invention engineers and explores this possibility.

(35) Demonstration that Flavivirus-Derived xrRNAs can Resist Xrn1 in Living Cells to Protect Functional mRNAs

(36) Characterization of these xrRNAs demonstrated that they appear to be modular structure elements. That is, the current invention hypothesizes that one could remove an xrRNA sequence from its native context in the viral genomic RNA and place it within any other RNA, and thus protect any downstream sequences from degradation by Xrn1. If true, one could then extend the lifetime of any RNA in the cell in which this element was installed. To test this, the Xrn1-mediated decay process in yeast was chosen as a tractable model system (FIG. 3). A vector that would drive expression of an mRNA was created containing (from 5′ to 3′): leaders sequence, the ACT1 intron, a viral internal ribosome entry site (IRES), and a LacZ reporter sequence (FIG. 4A). The viral IRES was included because partial digestion of the mRNA by Xrn1 may give rise to a stable, but uncapped message that would not be used in translation; the IRES allows translation to occur internally, downstream of the xrRNAs. Thus, in this mRNA two viral RNA structures from different viruses have been coupled. Expression of this mRNA in yeast may give rise to a spliced and capped RNA that would serve as the template for producing the reporter proteins at some level, and would also be subject to degradation at some rate (FIGS. 5A & B). To test the function of the xrRNAs, the two “tandem” xrRNAs from Dengue virus were installed into this reporter construct between the intron and the IRES (FIG. 4B). If the xrRNAs protect the message from Xrn1 degradation, one might expect an accumulation of partially degraded mRNAs that should be able to produce enzyme, driven by the action of the IRES (FIGS. 5A & B).

(37) This experiment was successful, and is shown in FIG. 6 and FIG. 7.

(38) This idea was also used to test the Dbr1/Xrn1-mediated decay of splice-defective pre-mRNA intermediates and found results that are consistent with the above.

(39) The important conclusions: 1) The xrRNAs from flaviviruses are modular elements that may be placed in a non-native contex1 where they retain function. 2) In living cells that differ from those they evolved in, these xrRNAs halt Xrn1 and protect downstream RNA. 3) The protected RNAs are competent as templates for translation, in this case using an IRES placed downstream of the xrRNAs.

(40) These results suggest that xrRNAs may be used as powerful genetic tools to artificially ex1 end the lifetime of any arbitrary RNA within a living cell. If placed within an mRNA, they may protect anything that is 3′ of them, and other RNA elements can be engineered with the xrRNAs to achieve specific outcome. This could be achieved using modern genomic editing tools such as CRISPR to install these engineered elements in any RNA in a cell. It is believed that currently, there is no existing way to extend the lifetime of a specific arbitrary RNA in cells. Although this method has only been tested in yeast, the fact that xrRNAs work in both insect and mammalian cells during flavivirus infection suggests that they may in fact be “universal” Xrn1 blockers. Furthermore, the IRES used in these studies has been shown to function in diverse systems, including human cells, yeast, rabbit reticulocyte lysate, bacteria, wheat germ extract, insects, etc. Hence, this may be a widely applicable method.

(41) Demonstration that Xrn1 Resistance can be Achieved by in Trans Association of Two RNAs.

(42) The results described above show that the xrRNAs could be a powerful genetic tool. In the manifestation described in section II, the xRNA was placed within the RNA to be protected (i.e. placed in cis). This is useful, but the power of the xrRNAs could be extended if they could somehow be used in a method in which one RNA could protect another RNA in trans. Towards this end, the published crystal structure of an xrRNA was examined and published biochemical characterization results also considered [15, 17]. These data show that loop L2 of the xrRNA structure may be mutated without functional affect, and the P2 stem that this loop caps may be shortened, lengthened, or altered without losing the ability to halt Xrn1 (FIGS. 8A & B). It was therefore reasoned that one could split the xrRNA into two pieces: a “target” strand (RNA1) and a “protecting” strand (RNA2). It was hypothesized that in the absence of RNA2, RNA would be rapidly degraded by Xrn1, but when RNA2 was added, it would anneal to RNA1, induce a fold, and protect RNA1 from degradation (FIG. 8C). This idea was tested using the established in vitro system with purified Xrn1 (FIG. 8D). As hypothesized, RNA1 (SEQ ID NO:1, GGGCCGGCAAAACUAACAUGAAAACAAGGCUAAAAGUCAGGUCGGAUUACCCUU UUGGAUCCCGACUGGCGAGAGCCA) was able to protect by addition of RNA2 (SEQ ID NO:2, GGGUAAUCCGCCAUAGUACGGAAAAAACUAUGCUACCUGUGAGCCCCGUCC AAGGACGUU), demonstrating the current invention idea of engineering the xrRNA to operate as an in trans protector is a valid approach. This result shows that the current invention may be an RNA-based on an xrRNA, which offers in trans protection to other RNAs from degradation by Xrn1. Such an RNA has not been discovered in nature.

(43) The potential uses and implications of this invention are many. Currently, it has been shown that this system works in vitro. If the RNA can be shown to work in the complex environment of living cells, one could have a way to protect endogenous, unmodified RNAs from degradation within a cell. This is exciting, because mRNA-based therapeutics are being actively pursued by several entities, but one limitation/challenge they face is the stability of the therapeutic mRNAs. In addition, one might be able to increase the levels of desirable mRNAs such as antitumor-encoding mRNAs or even noncoding RNAs with desirable effects, without having to alter the genome. As CRISPR is a genome-editing tool, the technology of the current invention could be a transcriptome-editing tool.

(44) Development of “in Trans” Protection of RNA Method

(45) The creation of an “in trans” protection system in vitro described herein is exciting, but additional value lies in:

(46) (1) Using the current invention system in more complex environments to alter specific endogenous RNA degradation rates within cells as a “transcriptome editing” tool.

(47) (2) Coupling it with a way to turn protected (but uncapped) mRNAs into viable translation templates as a “translatome-editing” tool.

(48) Steps for achieving this and for simultaneously developing additional in vitro and in vivo tools are described below.

(49) Step 1: Demonstrate that in trans protection can yield a stable message that is a substrate for translation in lysate, using an IRES installed within the mRNA. This is similar to what was done in yeast, except the xrRNA will not be installed in the message; rather protection will be provided in trans.

(50) Step 2: Develop a way to provide the signal to initiate translation in trans, coupled to the protecting RNAAn endogenous cellular mRNA has no IRES, and thus partial degradation by Xrn1 would not yield a viable template for translation. However, some plant viruses use a discrete structure in their 3′ untranslated regions that binds the cap-binding protein elF4E without using a cap. This structure is brought to the 5′ end of the plant viral RNA by base-pairing, and this leads to translation initiation (FIG. 9) [30]. Hence, nature has provided a translation initiation element that we may exploit to be used in trans. It is thought that by coupling this to the current xrRNA invention, one may produce a single RNA that can protect another RNA in trans AND drive its translation.

(51) Step 3. Test functionality in living cells. The above ideas (steps 1 and 2) can first be tested in a cell-free, translationally competent lysate using added Xrn1. Once success is achieved in these systems, the functionality of these RNAs will be tested by transfecting them or expressing them in human cell culture, targeting various endogenous mRNAs. If one could add an RNA to a cell, extend the lifetime of a specific RNA this would be very powerful (the opposite of siRNAs or shRNAs), this would be a novel method of broad usefulness. If the RNA is an mRNA, and one can drive it to be translated, this would also be very powerful. Achieving this would be a major leap, with consequences for both research tools and therapeutics.

(52) Step 4. Extend the RNA targets that can be affected by xrRNAs. Currently, the potential use of the in trans protection method is limited by the fact that the “protecting RNA” only can pair to a certain sequence in the target RNA; thus, this sequence must be naturally found in the endogenous RNA This sequence is not overly constraining (for example, the P53 5′ leader contains it) but does limit use. Thus, efforts could be made to engineer the protecting RNA through several strategies (including characterizing additional viral xrRNAs and in vitro selection methods) to expand the list of targets.

(53) Step 5. “Tune” the “protecting RNA” to achieve various protection levels and translation levels of endogenous RNAs.

(54) Although each of these steps are in progress, they represent an intellectual leap in taking an RNA found in the 3′ end of flaviviruses and using it to develop a tool that can be used to protect an arbitrary RNA in a cell from degradation by Xrn1, driving its translation (if desired), and doing so in a tunable way with no need to overexpress the target RNA or edit the genome.

(55) xrRNAs could be Controlled by Small Molecules

(56) An additional level of functionality to the above-described method could be achieved if the protecting function was controllable by the action of a small molecule. This could be achieved in both the “in cis” or “in trans” xrRNAs. To develop this, two next steps are envisioned:

(57) Step 1. Demonstrate the ability to couple an xrRNA to a riboswitch. Riboswitches are metabolite-sensing RNAs that change their fold depending on the binding off the small molecule metabolite. They could be used to create a small molecule sensing xrRNA, one way that is likely to work (a purine-sensing riboswitch [31]) is shown in FIG. 10. The use of riboswitches might allow coupling of a given mRNA's abundance to metabolite concentrations.

(58) Step 2. The use of riboswitches to control xrRNA function is useful, but for many applications is limited because ultimately there is a desire to control the xrRNA with a small molecule that is NOT an endogenous metabolite. Selection-based, directed evolution-type methods can help achieve this. The example shown in FIG. 9 is a proof of principle; long-term engineering could yield version capable of responding to a set of small molecules either positively or negatively.

(59) xrRNAs could be Used to Simultaneously Protect RNA and Deliver “Cargo”

(60) The fact that the P2/L2 stem-loop in an xrRNA can be altered extensively without functional consequence suggests that this could be a place to install specific RNA sequences as “cargo” that would be delivered with the xrRNA. Likewise, the 5′ or 3′ ends of the RNA could be used to attached additional cargo (as with the elF4E binding element mentioned above). For example, a protein-binding sequence could be placed there, or “Spinach” sequence [32, 33], or even perhaps a short open reading frame (FIG. 9). Cargo could also be attached to the 3′ end of the protecting RNA, which is not necessary for protection from Xrn1. In this way, an RNA could be protected and coupled with delivery of a specific RNA structure or functional element. In its most dramatic manifestation, an entire open reading frame could be placed as cargo, and combined with small-molecule dependent control (for example) unique tools created.

(61) xrRNAs could be Used to Block Diverse Exonucleases and Affect RNA Levels in Several Domains of Life

(62) Flaviviruses infect eukaryotes and the xrRNAs have evolved to resist progression of Xrn 1. However, it was reasoned that they might function by forming a general mechanical unfolding blocks that would stop exonucleases in addition to Xrn1. The ability of an xrRNA to block RNase J1, a 5′.fwdarw.3′ exonuclease from bacteria with no known structural homology to Xrn1, was tested. The xrRNA was able to block bacterial RNase J1 and other enzymes (see FIG. 12) suggesting that the technology developed and described herein could be broadly applied.

(63) Chemical Modification of the RNA

(64) There are many methods of chemical modification of RNA known in the art. In some embodiments, the chemical modification may comprise chemical modification of one xrRNA containing RNA sequence. In one embodiment, wherein there is a substrate strand and a protecting strand, one or both strands may be chemically modified. Although not limiting the current invention to any particular means or type of chemical modification, in one embodiment, chemical modification comprises modification of the 3′ or 5′ ends of said RNA strands. In one embodiment, the 5′ modification may include: addition of 7-methylguanosine (m7G) and other synthetic additions. In one embodiment, the 3′ modification may include: addition of a poly(A) tail and other synthetic additions

(65) In some embodiments, chemical modification of RNA may also comprise inclusion of phosphorothioate linkages, boranophosphate linkages, locked nucleic acid, 2′-modifications, 4′-thio modified RNA, ribo-difluorotoluyl nucleotide, and uncharged nucleic acid mimics, as described by Corey 2007 J. Clin. Invest. 117(12), 3615-3622 [14], herein encorporated by reference. See FIG. 11. Replacing one nonbridging oxygen atom on the backbone phosphate between two ribonucleotides with a sulphur atom creates a phosphorothioate (PS) linkage. In one embodiment, the current invention contemplates modifying the phosphate backbone of an oligonucleotide is the introduction of a boron atom in place of one of the nonbridging oxygen atoms to create a boron-phosphorous linkage. In one embodiment, the current invention contemplates the use of locked nucleic acid (LNA) nucleotides that contain a methylene bridge between the 2′ and 4′ carbons of the ribose ring. In one embodiment, the current invention contemplates the use of substitutions for the hydroxyl group on the 2′ carbon atom of the ribose ring of particular nucleotides. In one embodiment, the current invention contemplates the use of 4′-Thio modified nucleotides, which contain a sulphur atom in place of oxygen attached to the 4′ carbon of the ribose ring. In one embodiment, the current invention contemplates introduction of ribo-difluorotoluyl (rF) nucleotides (65). In one embodiment, single-stranded uncharged nucleic acid mimics, such as peptide nucleic acids (PNAs) (66, 67) and morpholino oligomers (68) may be useful as partial or full replacement s for nucleotides in the RNA.

(66) Thus, specific compositions and methods of protecting RNAs from degradation using engineered viral RNAs have been disclosed. It should be apparent, however, to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. Moreover, in interpreting the disclosure, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms “comprises” and “comprising” should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced.

(67) Although the invention has been described with reference to these preferred embodiments, other embodiments can achieve the same results. Variations and modifications of the present invention will be obvious to those skilled in the art and it is intended to cover in the appended claims all such modifications and equivalents. The entire disclosures of all applications, patents, and publications cited above, and of the corresponding application are hereby incorporated by reference.

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