Methods for RNA processing regulation

Abstract

Aspects of the invention described herein relate to synthetic pseudoknots (PKs) and their use to modulate RNA processing. Aspects of the invention further relate to the use of these engineered RNAs comprising these synthetic PKs and methods for using the same.

Description

METHODS FOR RNA PROCESSING REGULATIONCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. 119(e) of U.S. Provisional Patent Application Serial No. 63 / 701,716 filed October 1, 2024, the contents of which are herein incorporated by reference in their entirety.GOVERNMENT SUPPORT CLAUSE

[0002] This invention was made with Government support under Grant No. EB037112, awarded by the National Institutes of Health. The Government has certain rights in the invention.FIELD OF THE INVENTION

[0003] The present invention relates generally methods for modulating RNA processing, for example, reversibly inhibiting RNA processing of a target sequence.BACKGROUND

[0004] Described herein is a novel mechanism for controlling protein expression from an mRNA transcript in eukaryotic cells. De novo frameshifting elements and engineered RNA structures are used to create an mRNA platform that is silenced in its default state. Upon the introduction of a complementary target RNA, these secondary structures are unwound, restoring translational activity. This approach allows the coupling of protein expression to the sensing of an RNA that could indicate a specific cell type or tissue within the body, or could discriminate between diseased and healthy tissues. Also presented herein is a computational approach that allows the rapid redesign of these elements to a nearly unlimited range of RNA targets. Demonstrated herein for the first time is the successful modulation of the eukaryotic ribosome using extremely simple RNA secondary structures, an effect that is commonly believed to not be possible. A subset of the elements designed using our framework additionally offer the ability to precisely control the ratio of multiple proteins delivered on the same transcript, an essential feature for many RNA therapeutics. A further subset of RNA structural elements enable disruption of the processivity of other enzymes along an RNA transcript, including RNases and RNA-dependent RNA polymerases (RdRps). These elements can be used to increase the lifetimes of RNA and modulate the replication and lifetime of self-amplifying RNAs. Introduction of complementary target RNA also leads to unwinding of these structures to modulate enzyme activity, enabling cell-type specific and diseasespecific effects.SUMMARY OF THE INVENTION

[0005] One aspect disclosed herein provides a synthetic RNA pseudoknot (PK) comprising at least two stem-loop domains, the at least two stem-loop domains separated by a loop domain, wherein formation of the PK results in overlapping base pair interactions.

[0006] In one embodiment of any aspect herein, each stem-loop domain is modified such that it comprises a nucleotide sequence lacking in-frame stop codons

[0007] In one embodiment of any aspect herein, the PK is configured to block translation in a 0 frame while permitting translation in a -1, +1, -2, or +2 frame, or the PK is configured to block translation and not permit downstream translation in any frame.

[0008] In one embodiment of any aspect herein, the PK is incorporated into a self-amplifying RNA vector, a circular RNA vector, a messenger RNA, or a non-coding RNA.

[0009] In one embodiment of any aspect herein, hybridization of a sequence complementary to the PK causes the PK to unwind.

[0010] In one embodiment of any aspect herein, the PK is chemically synthesized or recombinantly expressed.

[0011] In one embodiment of any aspect herein, the at least two stem-loop domains have a sequence selected from SEQ ID NO: 1-48.

[0012] In one embodiment of any aspect herein, the loop domain has a sequence selected from SEQ ID NO: 49-60.

[0013] In one embodiment of any aspect herein, the synthetic PK has a sequence selected from SEQ ID NO: 61-72.

[0014] Another aspect disclosed herein provides a synthetic RNA PK comprising, from 5’ to 3’: a first stem-loop region; a first spacer; a second stem-loop region; a second spacer; a reverse complement sequence to the first stem-loop region; a third spacer; and a reverse complement sequence to the second stem-loop region, wherein overlapping base pair interactions occur between loops and more distal stem regions.

[0015] In one embodiment of any aspect herein, the second stem-loop region is the loop of the stem formed between the first stem-loop region and its reverse complement, and the reverse complement sequence to the first stem-loop region is the loop of the stem formed between the second stem-loop region and its reverse complement.

[0016] In one embodiment of any aspect herein, further comprising, between the third spacer, and the reverse complement sequence to the second stem-loop region, a third stem-loop region, a fourth spacer, and a reverse complement sequence to the third stem-loop region.

[0017] In one embodiment of any aspect herein, at least one of spacer is removed.

[0018] In one embodiment of any aspect herein, the stem-loop region and its reverse complement are not fully complementary.

[0019] Another aspect disclosed herein provides an engineered RNA comprising a coding region sequence of a gene of interest and a secondary RNA structure, wherein the secondary RNA structure is located at or near the gene of interest and inhibits RNA processing at or near the gene of the interest.

[0020] Another aspect disclosed herein provides an engineered RNA comprising a coding region sequence of a gene of interest and any of the synthetic PKs disclosed herein, wherein the synthetic PK is located at or near the gene of interest and inhibits RNA processing at or near the gene of the interest.

[0021] Another aspect disclosed herein provides an engineered RNA comprising a coding region sequence of a gene of interest and any of the synthetic PKs disclosed herein, wherein the synthetic PK is located at or near the gene of interest and inhibits translation at or near the gene of the interest.

[0022] In one embodiment of any aspect herein, at or near is upstream, downstream, or within of the coding region of a gene of interest.

[0023] In one embodiment of any aspect herein, the RNA processing is the progression or processivity of an RNA-processing or RNA-utilizing enzyme. In one embodiment of any aspect herein, the RNA- processing enzyme is a selected from the group consisting of: a deadenylase, a decapping enzyme, an exonuclease, an endonuclease, a surveillance enzyme, an RNA-dependent RNA polymerase, and an RNA-dependent DNA polymerase.

[0024] In one embodiment of any aspect herein, further comprising a sequence complementary to the PK.

[0025] In one embodiment of any aspect herein, the sequence complementary to the PK hybridizes to the PK.

[0026] In one embodiment of any aspect herein, hybridization to the PK causes the PK to unwind.

[0027] In one embodiment of any aspect herein, unwinding of the PK permits translation of the gene of interest.

[0028] In one embodiment of any aspect herein, the sequence complementary to the PK is downstream of the coding region of a gene of interest.

[0029] In one embodiment of any aspect herein, the sequence complementary to the PK is located in the 3’ UTR of the gene of interest.

[0030] In one embodiment of any aspect herein, the sequence complementary to the PK wherein the sequence complementary is a trans-acting RNA present in a second strand.

[0031] In one embodiment of any aspect herein, the trans-acting RNA is an endogenous cellular transcript.

[0032] In one embodiment of any aspect herein, the trans-acting RNA is an endogenous RNA.

[0033] In one embodiment of any aspect herein, further comprising at least a second coding region of at least a second gene of interest.

[0034] In one embodiment of any aspect herein, further comprising at least a second synthetic RNA PK.

[0035] Another aspect disclosed herein provides a platform for modulating translation, the platform comprising: an engineered RNA comprising a coding region sequence of a gene of interest and any of the synthetic PKs disclosed herein, and a sequence complementary to the PK.

[0036] A Another aspect disclosed herein provides a platform for modulating RNA processing, the platform comprising: an engineered RNA comprising a coding region sequence of a gene of interest and any of the synthetic PKs disclosed herein.

[0037] In one embodiment of any aspect herein, the PK inhibits translation of the gene of interest when not bound to the sequence complementary to the PK.

[0038] In one embodiment of any aspect herein, the PK inhibits RNA processing of the gene of interest when not bound to the sequence complementary to the PK.

[0039] In one embodiment of any aspect herein, the sequence complementary to the PK hybridizes to the PK and causes the PK to unwind.

[0040] In one embodiment of any aspect herein, unwinding permits translation of the gene of interest.

[0041] In one embodiment of any aspect herein, the sequence complementary to the PK is located downstream of the coding region.

[0042] In one embodiment of any aspect herein, the sequence complementary to the PK is a trans-acting RNA present in a second strand.

[0043] In one embodiment of any aspect herein, the engineered RNA is any of the engineered RNAs disclosed herein.

[0044] Another aspect disclosed herein provides a method of modulating translation in a cell, the method comprising: expressing any of the engineered RNAs or platforms disclosed herein in a cell, wherein translation of the gene of interest is inhibited by the PK when not bound to the sequence complementary to the PK; and promoting hybridization of the sequence complementary to the PK and the PK, wherein hybridization permits translation of the gene of interest.

[0045] Another aspect disclosed herein provides a method of modulating RNA processing in a cell, the method comprising: expressing any of the engineered RNAs or platforms disclosed herein in a cell in a cell, wherein RNA processing of the transcript of interest is inhibited by the PK when not bound to the sequence complementary to the PK; and promoting hybridization of the sequence complementary to the PK and the PK, wherein hybridization permits RNA processing of the transcript of interest.

[0046] In one embodiment of any aspect herein, the sequence complementary to the PK is a trans-acting RNA present in a second strand and promoting hybridization comprising expressing the second strand in the cell.

[0047] In one embodiment of any aspect herein, the sequence complementary to the PK is downstream of the gene of interest and promoting hybridization comprising allowing for folding of the modified mRNA such that the sequence complementary to the PK can hybridize to the PK.

[0048] In one embodiment of any aspect herein, promoting hybridization is spatially and / or temporally regulated.

[0049] In one embodiment of any aspect herein, the platform is expressed in the cell by an expression plasmid or a vector.

[0050] In one embodiment of any aspect herein, the modified mRNA and the sequence complementary to the PK are expressed in the cell by the same expression plasmid or vector.

[0051] In one embodiment of any aspect herein, the modified mRNA and the sequence complementary to the PK are expressed in the cell by separate expression plasmids or vectors. In one embodiment of any aspect herein, the vector is a non-viral vector or a viral vector.

[0052] Another aspect disclosed herein provides an engineered RNA vector comprising: an RNA genome encoding at least one non-structural protein required for replication; a synthetic PKs disclosed herein located within a region encoding the at least one non-structural proteins; and a sequence complementary to the PK, wherein the PK inhibits translation of the at least one non-structural protein when not bound to the sequence complementary to the PK.

[0053] In one embodiment of any aspect herein, the PK inhibits translation of the at least one non- structural proteins when not bound to the sequence complementary to the PK, thereby inhibiting RNA replication.

[0054] In one embodiment of any aspect herein, the hybridization of the sequence complementary to the PK and the PK permits translation of the gene of interest, thereby activating RNA replication.

[0055] In one embodiment of any aspect herein, the hybridization of the sequence complementary to the PK and the PK permits degradation of the gene of interest, thereby deactivating RNA replication.

[0056] Another aspect disclosed herein provides an engineered RNA vector comprising: an RNA genome encoding at least one non-structural protein required for replication; a synthetic PKs disclosed herein located within a region encoding the at least one non-structural proteins; and a sequence complementary to the PK, wherein the PK inhibits replication or degradation of the engineered RNA vector when not bound to the sequence complementary to the PK.

[0057] In one embodiment of any aspect herein, the PK inhibits procession of the RdRp replicating the RNA, thereby inhibiting RNA replication.

[0058] In one embodiment of any aspect herein, the PK inhibits procession of the RNase degrading the RNA, thereby increasing RNA lifetime and enhancing RNA replication.

[0059] In one embodiment of any aspect herein, the hybridization of the sequence complementary to the PK and the PK permits procession of the RdRp, thereby activating RNA replication.

[0060] In one embodiment of any aspect herein, the hybridization of the sequence complementary to the PK and the PK permits procession of the RNase, thereby inhibiting RNA degradation and activating RNA replication.

[0061] Another aspect disclosed herein provides a computer-implemented method for in-silico generation of a synthetic pseudoknot RNA sequence capable of inducing a programmed translational frameshift or ribosomal stall, the method comprising: receiving, with a processor, a user-defined target secondary pseudoknot topology; decomposing the target topology into a plurality of hairpin domains; foreach hairpin domain, performing supervised nucleotide sampling that excludes in-frame stop codons in each of three translation frames to generate a plurality of candidate domain sequences; evaluating, for each candidate domain sequence, thermodynamic stability and inter-domain cross-talk with a secondarystructure prediction algorithm; concatenating a combination of the candidate domain sequences having minimized inter-domain cross-talk to form a complete pseudoknot candidate sequence; ranking the pseudoknot candidate sequence with respect to at least one of the thermodynamic stability and a predicted translational modulation score; an outputting at least one ranked synthetic pseudoknot sequence.

[0062] In one embodiment of any aspect herein, the ranking step further comprises applying a trained neural network to predict a degree of ribosomal blockage, enzymatic stalling, or firameshifting efficiency of each pseudoknot candidate sequence.

[0063] In one embodiment of any aspect herein, feedback from the trained neural network dynamically adjusts nucleotide sampling frequencies during the supervised nucleotide sampling.

[0064] In one embodiment of any aspect herein, the user-defined target secondary pseudoknot topology comprises at least two overlapping hairpin loop structures.

[0065] In one embodiment of any aspect herein, the supervised nucleotide sampling is performed according to user-specified target base-pair composition frequencies.

[0066] In one embodiment of any aspect herein, further comprising electronically storing the at least one ranked synthetic pseudoknot sequence in a database for subsequent chemical synthesis.

[0067] In one embodiment of any aspect herein, the ranking step selects a pseudoknot candidate sequence predicted to block translation in the 0 frame while permitting translation in a -1, +1, -2, or +2 frame.

[0068] In one embodiment of any aspect herein, the ranking step selects a pseudoknot candidate sequence predicted to block progression of an enzyme involved in processing or degrading RNA.

[0069] In one embodiment of any aspect herein, the PK stem domains comprises -7.5 <AG < -350 kcal / mol and a computationally predicted crosstalk <1% at 37°C at 100 nM concentration of the domains.

[0070] Another aspect disclosed herein provides an RNA-based translational regulatory element comprising: a slippery sequence configured to induce ribosomal firameshifting; and a hairpin structure positioned downstream of the slippery sequence, the hairpin structure comprising a stem and a loop, wherein the hairpin structure is configured to modulate translation of a downstream coding sequence by at least partially repressing ribosomal progression in a reading frame-dependent manner.

[0071] In one embodiment of any aspect herein, the hairpin structure is positioned between 3 and 54 nucleotides downstream of the slippery sequence.

[0072] In one embodiment of any aspect herein, the hairpin structure has a stem comprising between 6 and 75 nucleotides.

[0073] In one embodiment of any aspect herein, the slippery sequence is of the form XXXYYYZ (SEQ ID NO: 73), where X, Y, and Z represent individual nucleotides.

[0074] In one embodiment of any aspect herein, the slippery sequence is composed of modified nucleotides.

[0075] In one embodiment of any aspect herein, the hairpin structure is configured to completely block translation in the 0 reading frame and not permit translation in any downstream reading frame in the coding region sequence.

[0076] In one embodiment of any aspect herein, the hairpin structure is configured to partially repress translation in the 0 reading frame, thereby enabling tunable expression of multiple protein coding sequences from a single transcript.

[0077] In one embodiment of any aspect herein, the hairpin structure is designed such that its repression of translation is independent of intermolecular interactions.

[0078] In one embodiment of any aspect herein, the element is incorporated into a multicistronic RNA vector for the coordinated expression of multiple antigens or therapeutic proteins.

[0079] In one embodiment of any aspect herein, further comprising a complementary RNA segment configured to hybridize to at least a portion of the hairpin structure, wherein hybridization of the complementary RNA segment unwinds the hairpin structure and restores ribosomal progression.

[0080] In one embodiment of any aspect herein, the complementary RNA segment is encoded within the same RNA molecule in a non-coding region or is provided as a trans-acting RNA.

[0081] In one embodiment of any aspect herein, the hairpin structure and slippery sequence are selected such that the regulatory effect is programmable by varying the sequence, length, or position of the hairpin relative to the slippery sequence.

[0082] In one embodiment of any aspect herein, the hairpin structure is designed using a thermodynamic prediction algorithm to achieve a desired degree of translational repression.

[0083] In one embodiment of any aspect herein, the hairpin structure is designed using a neural network prediction algorithm to achieve a desired degree of translational repression.

[0084] In one embodiment of any aspect herein, the element is used in a eukaryotic cell for the regulation of protein expression.Definitions

[0085] For convenience, certain terms employed in the entire application (including the specification, examples, and appended claims) are collected here. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0086] The term “reduced” or “reduce” as used herein generally means a decrease by a statistically significant amount. However, for avoidance of doubt, “reduced” means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (i.e. absent level as compared to a reference sample), or any decrease between 10-100% as compared to a reference level.

[0087] The term “increased” or “increase” as used herein generally means an increase by a statically significant amount; for the avoidance of any doubt, “increased” means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4- fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10- fold or greater as compared to a reference level.

[0088] As used herein, "protein" is a polymer consisting essentially of any of the 20 amino acids. Although "polypeptide" is often used in reference to relatively large polypeptides, and "peptide" is often used in reference to small polypeptides, usage of these terms in the art overlaps and is varied. The terms "peptide(s)", "protein(s)" and "polypeptide(s)" are used interchangeably herein.

[0089] The term "wild type" refers to the naturally-occurring polynucleotide sequence encoding a protein, or a portion thereof, or protein sequence, or portion thereof, respectively, as it normally exists in vivo.

[0090] The term "mutant" refers to any change in the genetic material of an organism, in particular a change (i.e., deletion, substitution, addition, or alteration) in a wild-type polynucleotide sequence or any change in a wild-type protein sequence. The term "variant" is used interchangeably with "mutant". Although it is often assumed that a change in the genetic material results in a change of the function of the protein, the terms "mutant" and "variant" refer to a change in the sequence of a wild-type protein regardless of whether that change alters the function of the protein (e.g., increases, decreases, imparts a new function), or whether that change has no effect on the function of the protein (e.g., the mutation or variation is silent). The term mutation is used interchangeably herein with polymorphism in this application.

[0091] As used herein, the term "nucleic acid" refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides. The terms "polynucleotide sequence" and "nucleotide sequence" are also used interchangeably herein.

[0092] As used herein, the term "gene" or "recombinant gene" refers to a nucleic acid comprising an open reading frame encoding a polypeptide, including both exon and (optionally) intron sequences.

[0093] The term "recombinant," as used herein, means that a protein is derived from a prokaryotic or eukaryotic expression system.

[0094] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Preferred vectors are those capable of autonomous replication and / or expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors".

[0095] The terms "composition" or "pharmaceutical composition" used interchangeably herein refer to compositions or formulations that usually comprise an excipient, such as a pharmaceutically acceptable carrier that is conventional in the art and that is suitable for administration to mammals, and preferably humans or human cells. In some embodiments, pharmaceutical compositions can be specifically formulated for direct delivery to a target tissue or organ, for example, by direct injection or via catheter injection to a target tissue. In other embodiments, compositions can be specifically formulated for administration via one or more of a number of routes, including but not limited to, oral, ocular parenteral, intravenous, intraarterial, subcutaneous, intranasal, sublingual, intraspinal, intracerebroventricular, and the like.. In addition, compositions for topical (e.g., oral mucosa, respiratory mucosa) and / or oral administration can form solutions, suspensions, tablets, pills, capsules, sustained-release formulations, oral rinses, or powders, as known in the art are described herein. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, University of the Sciences in Philadelphia (2005) Remington: The Science and Practice of Pharmacy with Facts and Comparisons, 21st Ed.

[0096] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0097] The term “statistically significant" or “significantly" refers to statistical significance and generally means a two standard deviation (2SD) below normal, or lower, concentration of the marker or a 2SD change in level above or below that of a suitable comparator. The term refers to statistical evidence that there is a difference. It is defined as the probability of making a decision to reject the null hypothesis when the null hypothesis is actually true. The decision is often made using the p-value.

[0098] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages can mean ±1%. The present invention is further explained in detail by the following examples, but the scope of the invention should not be limited thereto.

[0099] It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.BRIEF DESCRIPTION OF FIGURES

[0100] Fig. 1 presents data showing de novo designed PKs adhere to a knotted secondary structure. An example secondary structure is depicted of a novel H-type pseudoknot returned by our design algorithms. Each of the paired and unpaired regions adopt the conformation specified as an input to the algorithm for design.

[0101] Fig. 2 presents data showing de novo designed PKs demonstrated a complete disruption of 0 frame expression. 12 novel PKs were designed using a custom framework for returning H-type pseudoknotted structures containing no stop codons in either the 0, -1, or -2 frames. GFP encoded downstream of the PK element was not observed in any of the library members. The sequences of these PKs were unique, offering an avenue for programmable reactivation in the presence of a target RNA.

[0102] Fig. 3 presents data showing de novo designed PKs demonstrated varied GFP expression in the - 1 frame. The same 12 de novo designed PKs expressed GFP to varying degrees when GFP was placed in the -1 frame downstream of each PK. This range of GFP expression suggests that translation can be tuned to produce a desired level of protein expression in HEK293T cells.

[0103] Fig. 4 presents data showing a slippery sequence is required for PK-induced frameshifting. The endogenous SARS-CoV-2 slippery sequence was excised from the dual reporter while the PK itself remained. The removal of the slippery sequence completely silenced -1 protein expression, indicating the requirement of this motif in sufficient proximity to the PK structure.

[0104] Fig. 5 presents data showing slippery sequences impact the function of sPK elements. Our library of sPK constructs were reexamined using different slippery sequences adhering to the X-XXY- YYZ sequence pattern. Each alteration of the slippery sequence led to reduced translation, with some examples (PK 11) completely abrogating expression.

[0105] Fig. 6 presents data showing PK reactivation. Depicted is the ON / OFF ratio of GFP fluorescence for our PK-based translational switch following different perturbations. The first three constructs on the left (A systems) represent complete deletions of individual hairpins within the three hairpin H-type structure. ON / OFF ratios were determined based on fluorescence measurements with the complete PK (low expression, OFF state) compared to the fluorescence obtained after deletion of a PK structural element (A, high expression, ON state). The four constructs on the right (cis-acting triggers) were assessed by placing a perfectly complementary sequence designed to unwind part of the PK in the 3’ UTR of the same transcript. These 3’ UTR cis-acting triggers bind to a 5’ upstream element within the PK to disrupt its structure. These experiments indicate (1) the role of PK structure specifically in the modulation of expression and (2) the ability for complementary RNA strands to shift the structural profile into a conformation amenable to standard ribosomal progression.

[0106] Fig. 7 presents data showing de novo designed hairpins can modulate translation downstream of a slippery sequence. In place of knotted structures, single hairpins were placed downstream of a slippery sequence and assessed in our 0-firame reporter. A subset of hairpins demonstrated partial modulation of downstream translation, offering programmable mechanisms for controlling the ratio of production from multiple coding sequences. Several hairpins completely halted translation altogether, in contrast to the common belief in the literature that simple hairpins would be unable to do so.

[0107] Fig. 8 presents a schematic showing sPK trans-activation. A fully formed sPK structure will by default block ribosomal progression, silencing downstream protein production. Upon the introduction of a target RNA complementary to the sPK, the knotted structure will unwind, allowing the ribosome toprogress and translation to be restored. The programmability of sPK hairpins enables this approach to function against a nearly unlimited range of target RNA molecules that could indicate cell type, disease state, or any other specific biological context.

[0108] FIGs 9A-9C present data showing pseudoknot-induced blockage of xRNl improves mRNA expression in DNA plasmid transfection. Fig. 9A, Diagram of sPK in the 5’UTR and 3’UTR. Fig. 9B, sPK elements were placed into the 5’ UTR and Fig. 9C, 3’UTR of a fluorescent mCherry reporter construct and transfected into HEK293T with fluorescence values reported 48 hours following DNA plasmid exposure. sPK elements demonstrated compared to the commercial vector (mFP09). Bars indicate the mean of each construct over three biological replicates while individual points indicate a specific replicate. Error bars denote + / - 1 standard deviation.

[0109] FIGs 10A-10D present diagrams of sPK placement in mRNA. Fig. 10A, Diagram of multiple sPK in the 5’UTR. Fig. 10B, Diagram of multiple sPKs in the 3’UTR. Fig. 10C, Diagram of sPKs in the 5’UTR and 3’UTR. Fig. 10D, Diagram of multiple sPKs in the 5’UTR and 3’UTR

[0110] FIGs 11 A and 1 IB present data showing pseudoknot-induced blockage of xRNl improves mRNA expression in DNA plasmid transfection. Fig. 11A, sPK elements were placed into the 3’ UTR of a fluorescent mCherry reporter construct and transfected into HEK293T with fluorescence values reported 48 hours following DNA plasmid exposure. Both individual sPK elements and specific combinations of multiple sPKs concatenated in series are demonstrated compared to the commercial vector (mFP09) and CO VID PK (COV). Fig. 1 IB, Individual sPK elements were inserted into the 5’ UTR and 3’ UTR of the same fluorescent reporter mRNA and transfected and assessed for resulting expression in HEK293T cells 48 hours following DNA exposure. Both individual sPK elements inserted into the 3’ UTR as well as 5’ and 3’ combinations are depicted as well as the commercial vector (mFP09). Bars indicate the mean of each construct over three biological replicates while individual points indicate a specific replicate. Error bars denote + / - 1 standard deviation.

[0111] FIGs 12A and 12B present data showing pseudoknot-induced blockage of xRNl improves mRNA expression in mRNA transfection. Fig. 12A, sPK elements in the 5’ and 3’ UTR of a fluorescent mCherry reporter transfected into HEK293T with fluorescence values reported 22 to 114 hours following mRNA exposure. sPK elements demonstrated compared to circRNA with the same mCherry reporter. Fig. 12B, sPK elements in the 3’ UTR of a fluorescent mCherry reporter transfected into HEK293T with fluorescence values reported 22 to 114 hours following mRNA exposure. sPK elements demonstrated compared to circRNA with the same mCherry reporter, a UTR length control, and a construct with MALAT1 after the mCherry reporter. C, sPK elements in the 3’ UTR of a fluorescent mCherry reporter, transcribed with N1 -methylpseudouridine, and transfected into HEK293T with fluorescence values reported 22 to 114 hours following mRNA exposure. sPK elements demonstrated compared to circRNA with the same mCherry reporter and a UTR length control. Data points indicate the mean of each construct over three biological replicates. Error bars denote + / - 1 standard deviation.

[0112] FIGs 13A-13G present diagrams showing pseudoknot placement in saRNA. Fig. 13 A, Diagram of general saRNA. Fig. 13B, Diagram of saRNA with sPK in the 5’UTR of the subgenomic strand. Fig. 13C, Diagram of saRNA with sPK in the 3’UTR of the subgenomic strand. Fig. 13D, Diagram of saRNA with sPK after nsp4 and before the SGP. Fig. 13E, Diagram of saRNA with sPK in the 5’UTR before the 5’ CSE. Fig. 13F, Diagram of saRNA with sPK in the 5’UTR after the 5’ CSE. Fig. 13G, Diagram of saRNA with sPK in the 3’UTR after the 3’ CSE.

[0113] FIGs 14A-14D present data showing Pseudoknot blockage of RdRp in saRNA. Fig. 14A, sPK elements in the 5’ and 3’ UTR of the subgenomic strand of a fluorescent mCherry reporter transfected into HEK293T with the percentage of mCherry-positive cells value reported 22 to 114 hours following saRNA exposure. sPK elements are compared to a positive control saRNA with no structures in the 5’ or 3’ UTR. Fig. 14B, sPK elements in the 5’ and 3’ UTR of a fluorescent mCherry reporter transfected into HEK293T with the mCherry fluorescent value normalized to the positive control in the live cell population and the mCherry positive cell population reported 70 hours following saRNA exposure. Fig. 14C, sPK elements in the 5’ and 3’ UTR of a fluorescent mCherry reporter transfected into HEK293T with the mCherry fluorescence value of the live cell population and the Fig. 14D, mCherry positive cell population reported 22 to 114 hours following saRNA exposure. sPK elements demonstrated compared to positive control. Data points indicate the mean of each construct over three biological replicates. Bars indicate the mean of each construct over three biological replicates while individual points indicate a specific replicate. Error bars denote + / - 1 standard deviation.DETAILED DESCRIPTION

[0114] The engineered RNA and platform comprising the same, as disclosed herein provides a generalized solution to confer cell-type specific expression to RNA vectors. This approach could be rapidly applied to nearly any clinical indication in which RNA is currently being deployed, overcoming the immunogenic effects of off-target, ubiquitous expression.

[0115] There are many existing technologies that aim to solve the problem of off-target RNA expression by coupling translation to the presence of an endogenous RNA or that control the ratio of multiple coding sequences. These include the recently developed RADAR system, miRNA induced RNA silencing, engineered IRES elements, and miRNA classification circuits using self-amplifying RNA vectors. There are two key drawbacks to these approaches that prevent the translation of these technologies out of laboratory settings. The first is the dependence on protein inputs to demonstrate a sensing effect. Systems that engineer either RADAR or utilize responsive IRES elements require the detection of an RNA target followed by a regulatory action conferred by translation initiation factors or RNA editing enzymes. The expression of these proteins varies greatly across different cell types, limiting the generalizability and dynamic range offered by these systems. The invention disclosed herein relies only on engineered secondary or tertiary structures, it offers a degree of generalizability not currently satisfied with other technologies. Omitting the dependency of protein cofactors additionally creates a farmore physically compact sensing mechanism, eliminating the engineering constraint of delivering large nucleic acids in vivo. The second key challenge of existing systems is related to their approach of selective silencing rather than activation. In a laboratory setting, using miRNA or RNA binding protein targets to selectively repress translation of an exogenously delivered mRNA is an effective approach. In dynamic living systems, however, utilizing a transcript that is fully active in its default state until the target molecule is detected creates a degree of leakiness within the sensing apparatus that can lead to immunogenic responses. The engineered RNA and platform comprising the same disclosed herein can only produce protein if the target is encountered, greatly minimizes this drawback.

[0116] The invention disclosed herein can be directly applied to any RNA system being deployed as a therapeutic or vaccination platform to confer cell-type specific expression. The extremely compact nature of our sensing mechanism and wide range of target molecules could allow the creation of a platform licensing service for bestowing improved therapeutic performance to existing RNA modalities.Synthetic RNA pseudoknot (PK)

[0117] Disclosed herein are synthetic RNA PKs useful in modulating, e.g., reversibly inhibiting, RNA processing. In one embodiment, the synthetic RNA PKs comprise at least two stem-loop domains, the at least two stem-loop domains separated by a loop domain, wherein each stem-loop domain is modified such that it comprises a nucleotide sequence lacking in-frame stop codons, wherein formation of the PK results in overlapping base pair interactions.

[0118] As used herein, the term “RNA pseudoknot” refers to a secondary RNA structure characterized by nucleotides in a loop region of a hairpin or stem-loop base-pairing with complementary sequences located outside that loop, resulting in the intercalation of one stem-loop into another. Pseudoknots may form naturally within cellular RNAs, including messenger RNAs (mRNAs), ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), and non-coding RNAs, or may be artificially engineered. The term “RNA pseudoknot” as used herein is intended to encompass all such structures, whether presently known or later discovered, that involve intramolecular or intermolecular base-pairing interactions generating a pseudoknot topology. It is noted, that in some contexts PKs are also considered tertiary motifs.

[0119] RNA pseudoknots are characterized by their ability to form base-pairing interactions that cross established helical regions, thereby generating complex three-dimensional structures. These structures can play critical roles in the regulation of translation, ribosomal frameshifting, viral replication, ribozyme catalysis, and other RNA-mediated processes.

[0120] In one embodiment, the synthetic RNA PKs disclosed herein are modified PKs known in the art, e.g., to remove endogenous stop codons. Known PKs include, without limitation: canonical pseudoknots, wherein nucleotides in a loop form Watson-Crick base pairs with a complementary region downstream of the stem; H-type pseudoknots, in which a hairpin loop base-pairs with a sequence downstream to form a second stem; kissing-loop pseudoknots, involving interactions between the loop regions of two hairpins; complex pseudoknots, including branched, extended, or multi-stranded pseudoknots with more than twostems; engineered pseudoknots, comprising synthetic or modified sequences designed to introduce, stabilize, destabilize, or control pseudoknot formation.

[0121] Exemplary viral plant and arthropod genomes that have PK include Dengue virus 1, Dengue virus 2, Dengue virus 3, Dengue virus 4, Zika virus, West Nile virus, Kunjin virus, Yellow fever virus, Japanese encephalitis virus, Tick-borne encephalitis virus, Powassan virus, Saint Louis encephalitis virus, Murray Valley encephalitis virus, Usutu virus, Ilheus virus, Langat virus, Louping ill virus, Kyasanur Forest disease virus, Omsk hemorrhagic fever virus, Alkhurma hemorrhagic fever virus, Wesselsbron virus, Spondweni virus, Modoc virus, Rio Bravo virus, Apoi virus, Montana myotis leukoencephalitis virus, Red clover necrotic mosaic virus, Sweet clover necrotic mosaic virus, Potato leafroll virus, Maize chlorotic mottle virus, Maize yellow dwarf virus-RMV, Cereal yellow dwarf virus-RPV, Beet necrotic yellow vein virus, Beet soil-borne mosaic virus, Cucumber mosaic virus, Peanut stunt virus, Tomato aspermy virus, Gayfeather mild mottle virus, Sweet potato C6 virus, Darwin betaflexivirus, Panax ginseng flexivirus 1, Potato mop-top virus, Tobacco rattle virus, Lamium mild mosaic virus, Bellflower veinal mottle virus, and Opium poppy mosaic virus.

[0122] Exemplary viral human and animal genomes that have PK include Epstein-Barr virus, Kaposi’s sarcoma-associated herpesvirus, Human papillomavirus 16, Hepatitis B virus, Human cytomegalovirus, Herpes simplex virus 1, Merkel cell polyomavirus, Trichodysplasia spinulosa polyomavirus, Marek’s disease virus, Human adenovirus 5, Murine cytomegalovirus.

[0123] In one embodiment, the synthetic RNA PK comprises, from 5’ to 3’: a first stem-loop region; a first spacer; a second stem-loop region; a second spacer; a reverse complement sequence to the first stem-loop region; a third spacer; and a reverse complement sequence to the second stem-loop region.

[0124] In one embodiment, overlapping base pair interactions occur between loops and more distal domains. For example, base pairing occurs between the first or second stem-loop region and its reverse complement, where the second stem-loop region is the loop of the stem formed between the first stemloop region and its reverse complement, and the reverse complement sequence to the first stem-loop region is the loop of the stem formed between the second stem-loop region and its reverse complement.

[0125] For example, in one embodiment, the synthetic RNA pseudoknot comprises, from 5’ to 3’, (a)- (spacerl)-(b)-(spacer2)-(a*)-(spacer3)-(b*), wherein (a) and (b) represent a first and second stem-loop region, and (a*) and (b*) represent reverse complements of the first and second stem-loop region. In such example, overlapping base pair interactions occur between loops and more distal domains, e.g. (b)-(b*) and (a)-(a*), where (b) is the loop of the (a)-(a*) stem and (a*) is the loop of the (b)-(b*) stem.

[0126] In one embodiment, the synthetic RNA PK further comprises, between the third spacer, and the reverse complement sequence to the second stem-loop region, a third stem-loop region, a fourth spacer, and a reverse complement sequence to the third stem-loop region.

[0127] In one embodiment, the synthetic RNA PK further comprises, between (spacer3) and (b*), (c)- (spacer4)-(c*), wherein (c) represents a third stem-loop region, and (c*) represents a reverse complement of the third stem-loop region.

[0128] In one embodiment, at least one of the spacers is removed from the synthetic RNA PK.

[0129] In one embodiment, the stem-loop regions and its reverse complement do not need to by fully complementary. In one embodiment, the stem-loop regions and its reverse complement must have sufficient complementary sequences to promotes its interaction, e.g., to promoter overlapping base pairing.

[0130] In one embodiment, the formation of the synthetic PK disclosed herein results in overlapping base pair interactions.

[0131] In one embodiment, the synthetic RNA PK is incorporated in a self-amplifying RNA vector, a circular RNA vector, mRNAs, or non-coding RNAs.

[0132] In one embodiment, the synthetic PK disclosed herein is configured to block translation in a 0 frame while permitting translation in a -1, +1, -2, or +2 frame, or the PK is configured to block translation and not permit downstream translation in any frame when incorporated into an engineered RNA.

[0133] In one embodiment, the synthetic RNA PK comprises at least two stem-loop regions. Exemplary stem-loop regions comprise a sequence selected from SEQ ID NO: 1-48, or a sequence that is at least 85% identical to SEQ ID NO: 1-48.

[0134] In one embodiment, each of the at least two stem-loop regions are modified such that it does not comprise an in-frame stop-codon.

[0135] In one embodiment, the synthetic RNA PK comprises a loop-region positioned in between the at least two stem-loop regions. Exemplary loop regions comprise a sequence selected from SEQ ID NO: 49- 60, or a sequence that is at least 85% identical to SEQ ID NO: 49-60.

[0136] In one embodiment, the synthetic RNA PK comprises a sequence selected from SEQ ID NO: 61-72, or a sequence that is at least 85% identical to SEQ ID NO: 61-72.

[0137] In one embodiment, the PK stem domains comprises -7.5 <AG < -350 kcal / mol and a computationally predicted crosstalk <1% at 37°C at 100 nM concentration of the domains.

[0138] RNA processing

[0139] Synthetic RNA PKs disclosed herein are used to modulate the progression or processivity of an RNA-processing enzyme or RNA-utilizing enzyme. For example, the synthetic RNA PK disclosed herein inhibits RNA processing. The term “RNA processing” refers to any event, activity, or mechanism that alters, modifies, or otherwise affects an RNA molecule after or during its transcription. RNA processingis not limited to naturally occurring mechanisms but also includes artificially induced and synthetically engineered modifications, whether performed in vitro, in vivo, or in cell-free systems.

[0140] RNA processing includes, without limitation, cleavage and trimming of precursor or primary RNA transcripts into smaller, functional units; splicing, including canonical splicing of introns and exons, alternative splicing, and engineered splicing modifications; capping, such as the enzymatic or synthetic addition of 5' cap structures or functional analogs; polyadenylation, including natural or engineered addition, extension, or modification of poly(A) tails or alternative 3' end modifications; RNA editing, including nucleotide substitution, insertion, deletion, or modification (e.g., adenosine-to-inosine conversion, cytidine deamination, pseudouridylation, or methylation); chemical or enzymatic modifications of bases, sugars, or the phosphate backbone (e.g., 2'-O-methylation, thiolation); stabilization or degradation, including mechanisms that extend or shorten the RNA half-life; transport and localization, including signals or sequences that direct the RNA to a particular cellular compartment, organelle, or extracellular environment; and quality control and surveillance pathways, such as nonsense- mediated decay, non-stop decay, or no-go decay. In certain embodiments, the synthetic RNA PK inhibits expression, stability, localization, replication efficiency, or translational efficiency of a target RNA molecule.

[0141] The present disclosure contemplates RNA processing events that are naturally occurring, induced by engineered proteins or nucleic acids, or achieved through chemical, biochemical, or mechanical manipulation. Accordingly, the scope of “RNA processing” as used herein is intended to include any transformation or handling of RNA molecules, whether presently known or later discovered, that impacts the structure, composition, function, or fate of the RNA.

[0142] In some embodiments, the RNA processing comprises splicing of a precursor RNA to remove one or more introns.

[0143] In some embodiments, the RNA processing comprises alternative splicing to generate two or more RNA isoforms from a single primary transcript.

[0144] In some embodiments, the RNA processing comprises the addition of a 5' cap or a functional analog thereof.

[0145] In some embodiments, the RNA processing comprises polyadenylation of the RNA, wherein the poly(A) tail is at least 10, 20, 50, 100, 200, or more nucleotides in length.

[0146] In some embodiments, the RNA processing comprises editing of the RNA by site-directed or enzyme-mediated conversion of a nucleotide (e.g., adenosine to inosine).

[0147] In some embodiments, the RNA processing comprises chemical modification of the RNA, such as methylation, pseudouridylation, or thiolation.

[0148] In some embodiments, the RNA processing comprises stabilizing the RNA to increase its halflife relative to an unmodified control RNA.

[0149] In some embodiments, the RNA processing comprises degradation of the RNA, either partially or completely, through natural or engineered pathways.

[0150] In some embodiments, the RNA processing comprises directing the RNA to a specific cellular location, compartment, or organelle by virtue of one or more localization signals.

[0151] In some embodiments, the RNA processing comprises surveillance mechanisms that detect and degrade defective or improperly processed RNA molecules.

[0152] In some embodiments, the RNA processing comprises replication of the RNA or use of the RNA as a template for replication of RNA, either partially or completely, through natural or engineered pathways.

[0153] In some embodiments, the RNA processing comprises use of the RNA as a template for production of RNA, either partially or completely, through natural or engineered pathways.

[0154] In one embodiment, RNA processing refers specifically to the progression or processivity of an RNA-processing or RNA-utilizing enzyme. As used herein, the term “RNA processing enzyme” refers broadly to enzymes that act upon RNA to regulate its stability, processing, modification, surveillance, or degradation. Such enzymes include, without limitation, (a) deadenylases, such as PAN2-PAN3, CCR4— NOT, PARN, Noctumin, and PDE12, which shorten or remove the poly(A) tail; (b) decapping enzymes, such as DCP2 and DXO, which remove the 5' cap structure; (c) exonucleases, such as Xml, Xm2, the exosome complex, DIS3L2, and ISG20, which degrade RNA from the 5' or 3' end; (d) endonucleases, such as RNase L, SMG6, IRE1, members of the RNase A family, and the RNA-induced silencing complex protein Ago2, which cleave RNA internally; (e) surveillance enzymes, such as TRAMP- associated nucleases and RNase P / Z, which participate in RNA quality control and processing of aberrant RNAs; and (f) RNA-dependent RNA polymerases (RdRps), including viral RdRps employed in selfamplifying RNA (saRNA) systems, which catalyze the synthesis of RNA from an RNA template.

[0155] Synthetic RNA PK disclosed herein are used to modulate, e.g., reversibly inhibit, RNA translation. “RNA translation” refers to the process by which a RNA molecule, typically messenger RNA (mRNA), is decoded to direct the synthesis of a polypeptide. Translation includes both naturally occurring and artificially engineered processes, and may take place in a cellular environment, in organelles such as ribosomes, or in cell-free or synthetic translation systems.

[0156] Translation generally involves the sequential decoding of nucleotide triplets (codons) by transfer RNAs (tRNAs) and the assembly of corresponding amino acids into a growing polypeptide chain at the ribosome. The process may be divided into initiation, elongation, and termination phases, each of which may be regulated or engineered.

[0157] Translation, as used herein, includes, without limitation: initiation, comprising recognition of the start codon, recruitment of ribosomal subunits, initiation factors, and the first charged tRNA; elongation, comprising codon-by-codon decoding of the mRNA, binding of aminoacyl-tRNAs, peptide bond formation, and ribosomal translocation; termination, comprising recognition of a stop codon, release of the nascent polypeptide, and ribosome recycling; translational regulation, including mechanisms that upregulate or downregulate the rate or efficiency of protein synthesis (e.g., via upstream open reading frames, riboswitches, microRNAs, or RNA-binding proteins); codon optimization or modification,including engineered codons, non-natural codons, or codon bias adjustments to improve expression in a given host system.; incorporation of non-natural amino acids, such as through engineered tRNAs, aminoacyl-tRNA synthetases, or orthogonal ribosomes; and synthetic translation systems, including cell- free translation extracts, reconstituted translation machinery, or artificial translation platforms.

[0158] In certain embodiments, the synthetic RNA PK inhibits RNA translation.

[0159] Complementary sequence

[0160] In one embodiment, the synthetic RNA PK disclosed herein is unraveled when bound by a sequence complementary to the RNA PK. As used herein, the term “complementary sequence” refers to a nucleic acid sequence that is capable of base-pairing, either fully or partially, with a target RNA sequence according to Watson-Crick or other non-canonical base-pairing rules. In the context of RNA PKs, complementary sequences may hybridize to one or more regions of the pseudoknot, thereby destabilizing intramolecular base-pairing interactions and promoting unfolding, rearrangement, or unraveling of the PK structure.

[0161] Complementary sequences may be naturally occurring, chemically synthesized, or computationally designed. They may be composed of ribonucleotides, deoxyribonucleotides, or chemically modified analogs, including but not limited to locked nucleic acids (LNAs), peptide nucleic acids (PNAs), morpholino oligonucleotides, phosphorothioate-modified nucleotides, or other stabilized backbones.

[0162] Complementary sequences may interact with the PK in several ways, including but not limited to direct hybridization to a stem or loop region, thereby displacing native intramolecular base-pairs; competitive binding to a single-stranded region of the pseudoknot, thereby preventing refolding; strand invasion, e.g., in which the complementary sequence intercalates into the pseudoknot and initiates unraveling through displacement of existing base pairs; stepwise disruption, e.g., wherein short complementary sequences sequentially destabilize portions of the pseudoknot; and triggered binding, e.g., wherein the complementary sequence hybridizes in response to environmental or chemical signals, providing inducible control over pseudoknot unraveling

[0163] Complementary sequences disclosed herein are used to unravel the PK, and thus, relieving its inhibitory modulation of RNA processing, e.g., RNA translation. In certain embodiments, The present disclosure contemplates complementary sequences that are fully or partially complementary, that bind stably or transiently, and that operate under physiological or engineered conditions.Accordingly, the term “complementary sequence” as used herein includes any nucleic acid capable of hybridizing to a synthetic PK disclosed herein in a manner that disrupts, destabilizes, or unravels the pseudoknot structure.

[0164] In one embodiment, the complementary sequence is at least 21 base pairs (bps) in length. In one embodiment, the complementary sequence is at least 22 bps, 23 bps, 24 bps, 25 bps, 26 bps, 27 bps, 28 bps, 29 bps, 30 bps, 31 bps, 32 bps, 33 bps, 34 bps, 35 bps, 36 bps, 37 bps, 38 bps, 39 bps, 40 bps, 41 bps, 42 bps, 43 bps, 44 bps, 45 bps, 46 bps, 47 bps, 48 bps, 49 bps, 50 bps, 51 bps, 52 bps, 53 bps, 54 bps, 55bps, 56 bps, 57 bps, 58 bps, 59 bps, 60 bps, 61 bps, 62 bps, 63 bps, 64 bps, 65 bps, 66 bps, 67 bps, 68 bps, 69 bps, 70 bps, 71 bps, 72 bps, 73 bps, 74 bps, 75 bps, 76 bps, 77 bps, 78 bps, 79 bps, 80 bps, 81 bps, 82 bps, 83 bps, 84 bps, 85 bps, 86 bps, 87 bps, 88 bps, 89 bps, 90 bps, 91 bps, 92 bps, 93 bps, 94 bps, 95 bps, 96 bps, 97 bps, 98 bps, 99 bps, 100 bps, 101 bps, 102 bps, 103 bps, 104 bps, 105 bps, 106 bps, 107 bps, 108 bps, 109 bps, 110 bps, 111 bps, 112 bps, 113 bps, 114 bps, 115 bps, 116 bps, 117 bps, 118 bps, 119 bps, 120 bps, 121 bps, 122 bps, 123 bps, 124 bps, 125 bps, 126 bps, 127 bps, 128 bps, 129 bps, 130 bps, 131 bps, 132 bps, 133 bps, 134 bps, 135 bps, 136 bps, 137 bps, 138 bps, 139 bps, 140 bps, 141 bps, 142 bps, 143 bps, 144 bps, 145 bps, 146 bps, 147 bps, 148 bps, 149 bps, 150 bps, 151 bps, 152 bps, 153 bps, 154 bps, 155 bps, 156 bps, 157 bps, 158 bps, 159 bps, 160 bps, 161 bps, 162 bps, 163 bps, 164 bps, 165 bps, 166 bps, 167 bps, 168 bps, 169 bps, 170 bps, 171 bps, 172 bps, 173 bps, 174 bps, 175 bps, 176 bps, 177 bps, 178 bps, 179 bps, 180 bps, 181 bps, 182 bps, 183 bps, 184 bps, 185 bps, 186 bps, 187 bps, 188 bps, 189 bps, 190 bps, 191 bps, 192 bps, 193 bps, 194 bps, 195 bps, 196 bps, 197 bps, 198 bps, 199 bps, 200 bps, or more in length.

[0165] In one embodiment, the complementary sequence is no longer than 200 bps in length. In one embodiment, the complementary sequence is no longer than 22 bps, 23 bps, 24 bps, 25 bps, 26 bps, 27 bps, 28 bps, 29 bps, 30 bps, 31 bps, 32 bps, 33 bps, 34 bps, 35 bps, 36 bps, 37 bps, 38 bps, 39 bps, 40 bps, 41 bps, 42 bps, 43 bps, 44 bps, 45 bps, 46 bps, 47 bps, 48 bps, 49 bps, 50 bps, 51 bps, 52 bps, 53 bps, 54 bps, 55 bps, 56 bps, 57 bps, 58 bps, 59 bps, 60 bps, 61 bps, 62 bps, 63 bps, 64 bps, 65 bps, 66 bps, 67 bps, 68 bps, 69 bps, 70 bps, 71 bps, 72 bps, 73 bps, 74 bps, 75 bps, 76 bps, 77 bps, 78 bps, 79 bps, 80 bps, 81 bps, 82 bps, 83 bps, 84 bps, 85 bps, 86 bps, 87 bps, 88 bps, 89 bps, 90 bps, 91 bps, 92 bps, 93 bps, 94 bps, 95 bps, 96 bps, 97 bps, 98 bps, 99 bps, 100 bps, 101 bps, 102 bps, 103 bps, 104 bps, 105 bps, 106 bps, 107 bps, 108 bps, 109 bps, 110 bps, 111 bps, 112 bps, 113 bps, 114 bps, 115 bps, 116 bps, 117 bps, 118 bps, 119 bps, 120 bps, 121 bps, 122 bps, 123 bps, 124 bps, 125 bps, 126 bps, 127 bps, 128 bps, 129 bps, 130 bps, 131 bps, 132 bps, 133 bps, 134 bps, 135 bps, 136 bps, 137 bps, 138 bps, 139 bps, 140 bps, 141 bps, 142 bps, 143 bps, 144 bps, 145 bps, 146 bps, 147 bps, 148 bps, 149 bps, 150 bps, 151 bps, 152 bps, 153 bps, 154 bps, 155 bps, 156 bps, 157 bps, 158 bps, 159 bps, 160 bps, 161 bps, 162 bps, 163 bps, 164 bps, 165 bps, 166 bps, 167 bps, 168 bps, 169 bps, 170 bps, 171 bps, 172 bps, 173 bps, 174 bps, 175 bps, 176 bps, 177 bps, 178 bps, 179 bps, 180 bps, 181 bps, 182 bps, 183 bps, 184 bps, 185 bps, 186 bps, 187 bps, 188 bps, 189 bps, 190 bps, 191 bps, 192 bps, 193 bps, 194 bps, 195 bps, 196 bps, 197 bps, 198 bps, 199 bps, or 200 bps in length.

[0166] In one embodiment, the complementary sequence is between 21 bps and 200 bps in length. In one embodiment, the complementary sequence is between 30 bps and 200 bps, 40 bps and 200 bps, 50 bps and 200 bps, 60 bps and 200 bps, 70 bps and 200 bps, 80 bps and 200 bps, 90 bps and 200 bps, 100 bps and 200 bps, 110 bps and 200 bps, 120 bps and 200 bps, 130 bps and 200 bps, 140 bps and 200 bps, 150 bps and 200 bps, 160 bps and 200 bps, 170 bps and 200 bps, 180 bps and 200 bps, 190 bps and 200 bps, 30 bps and 40 bps, 30 bps and 50 bps, 30 bps and 60 bps, 30 bps and 70 bps, 30 bps and 80 bps, 30 bps and 90 bps, 30 bps and 100 bps, 30 bps and 110 bps, 30 bps and 120 bps, 30 bps and 130 bps, 30 bpsand 140 bps, 30 bps and 150 bps, 30 bps and 160 bps, 30 bps and 170 bps, 30 bps and 180 bps, 30 bps and 190 bps, 30 bps and 200 bps, or any range therein.

[0167] In one embodiment, the complementary sequence is temporally or spatially regulated. In one embodiment, the complementary sequence is operatively linked to a promoter. In one embodiment, the promoter is an inducible or regulatable promoter. The term “promoter” is art-recognized and refers to a nucleic acid molecule with a sequence recognized by the cellular transcription machinery and able to initiate transcription of a downstream gene. A promoter can be constitutively active, meaning that the promoter is always active in a given cellular context, or conditionally active, meaning that the promoter is only active in the presence of a specific condition. For example, a conditional promoter may only be active in the presence of a specific protein that connects a protein associated with a regulatory element in the promoter to the basic transcriptional machinery, or only in the absence of an inhibitory molecule. A subclass of conditionally active promoters are inducible promoters that require the presence of a small molecule “inducer” for activity. Examples of inducible promoters include, but are not limited to, arabinose-inducible promoters, Tet-on promoters, and tamoxifen-inducible promoters. A variety of constitutive, conditional, and inducible promoters are well known to the skilled artisan, and the skilled artisan will be able to ascertain a variety of such promoters useful in carrying out the instant invention, which is not limited in this respect.Engineered RNA

[0168] Described herein are RNA sequences engineered to comprise a secondary RNA structure located at or near a gene of interest, wherein its position at or near the gene of interest inhibits RNA processing at or near the gene of interest.

[0169] Also described herein are RNA sequences engineered to comprise any of the synthetic RNA PK located at or near a gene of interest, wherein its position at or near the gene of interest inhibits RNA processing at or near the gene of interest.

[0170] Also described herein are RNA sequences engineered to comprise any of the synthetic RNA PK located at or near a gene of interest, wherein its position at or near the gene of interest inhibits RNA translation at or near the gene of interest.

[0171] As used herein, “at or near” means upstream of the gene of interest, downstream of the gene of interest, or within the UTR or coding region of the gene of interest.

[0172] A gene of interest described herein can be any gene that encodes a biomolecule of interest (e.g., a protein or an RN A molecule). A protein of interest can include any intracellular protein, membrane protein, or extracellular protein, e.g., a nuclear protein, transcription factor, nuclear membrane transporter, intracellular organelle associated protein, a membrane receptor, a catalytic protein, an RNA- dependent RNA polymerase, an RNA-dependent DNA polymerase, a protease, an enzyme, a therapeutic protein, a membrane protein, a membrane transport protein, a signal transduction protein, or an immunological protein (e.g., an IgG or other antibody protein), etc. The gene of interest may also encodean RNA molecule, including, but not limited to, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), antisense RN A, guide RN A, microRNA (miRNA), small interfering RNA (siRNA), and cell-free RNA (cfRNA).

[0173] In one embodiment, the engineered RN A further comprises a complementary sequence that hybridizes to the synthetic PK.

[0174] In one embodimen t, the complementary sequence is located downstream of the coding region of a gene of interest. In one embodiment, complementary sequence is located in the 3’UTR of the gene of interest, hi one embodiment, complementary sequence is located in the coding sequence of the gene of interest. In one embodiment, complementary sequence is located in the 5’UTR of the gene of interest.

[0175] In one embodiment, the complementary sequence is a tram-acting RNA present in a second, separate strand,

[0176] In one embodiment, the trans-acting RNA is an endogenous cellular transcript, hi one embodiment, the trans-acting RNA is an endogenous RNA, In one embodiment, the trans-acting RN A is an exogenous RNA.

[0177] One aspect disclosed herein is a vector comprising any of the engineered RNA disclosed herein. A vector can be an viral vector or a non-viral vector. Any suitable vector is encompassed in the embodiments of this invention, including, but not limited to, nonviral vectors (e.g., nucleic acids, minicircles, linear DNA, plasmids, poloxymers, exosomes, and liposomes), viral vectors and synthetic biological nanoparticles (BNP) (e.g., synthetically designed from different adeno-associated viruses, as well as other parvoviruses).

[0178] Suitable vectors also include virus vectors (e.g., retrovirus, alphavirus; vaccinia virus; adenovirus, adeno-associated virus, or herpes simplex virus), lipid vectors, poly-lysine vectors, synthetic polyamino polymer vectors that are used with nucleic acid molecules, such as plasmids, and the like.

[0179] Any viral vector that is known in the art can be used in the present invention. Examples of such viral vectors include, but are not limited to vectors derived from: Adenoviridae; Bimaviridae;Bunyaviridae; Caliciviridae, Capillovirus group; Carlavirus group; Carmovirus virus group; Group Caulimovirus; Closterovirus Group; Commelina yellow mottle virus group; Comovirus virus group; Coronaviridae; PM2 phage group; Corcicoviridae; Group Cryptic virus; group Cryptovirus; Cucumovirus virus group Family ([PHgr]6 phage group; Cysioviridae; Group Carnation ringspot; Dianthovirus virus group; Group Broad bean wilt; Fabavirus virus group; Filoviridae; Flaviviridae; Furovirus group; Group Germinivirus; Group Giardiavirus; Hepadnaviridae; Herpesviridae; Hordeivirus virus group; Illarvirus virus group; Inoviridae; Iridoviridae; Leviviridae; Lipothrixviridae; Luteovirus group; Marafivirus virus group; Maize chlorotic dwarf virus group; icroviridae; Myoviridae; Necrovirus group; Nepovirus virus group; Nodaviridae; Orthomyxoviridae; Papovaviridae; Paramyxoviridae; Parsnip yellow fleck virus group; Partitiviridae; Parvoviridae; Peaenation mosaic virus group; Phycodnaviridae; Picomaviridae; Plasmaviridae; Prodoviridae; Polydnaviridae; Potexvirus group; Potyvirus; Poxviridae; Reoviridae; Retroviridae; Rhabdoviridae; Group Rhizidiovirus; Siphoviridae; Sobemovirus group; SSV 1-TypePhages; Tectiviridae; Tenuivirus; Tetraviridae; Group Tobamovirus; Group Tobravirus; Togaviridae; Group Tombusvirus; Group Torovirus; Totiviridae; Group Tymovirus; and Plant virus satellites.

[0180] Protocols for producing recombinant viral vectors and for using viral vectors for nucleic acid delivery can be found, e.g., in Current Protocols in Molecular Biology, Ausubel, F. M. et al. (eds.) Greene Publishing Associates, (1989) and other standard laboratory manuals (e.g., Vectors for Gene Therapy. In: Current Protocols in Human Genetics. John Wiley and Sons, Inc.: 1997). Nonlimiting examples of vectors employed in the methods of this invention include any nucleotide construct used to deliver nucleic acid into cells, e.g., a plasmid, a nonviral vector or a viral vector, such as a retroviral vector which can package a recombinant retroviral genome (see e.g., Pastan et al., Proc. Natl. Acad. Sci. U.S.A. 85:4486 (1988); Miller et al., Mol. Cell. Biol. 6:2895 (1986)). For example, the recombinant retrovirus can then be used to infect and thereby, deliver a nucleic acid of the invention to the infected cells. The exact method of introducing the altered nucleic acid into mammalian cells is, of course, not limited to the use of retroviral vectors. Other techniques are widely available for this procedure including the use of adenoviral vectors (Mitani et al., Hum. Gene Ther. 5:941-948, 1994), adeno-associated viral (AAV) vectors (Goodman et al., Blood 84:1492-1500, 1994), lentiviral vectors (Naldini et al., Science 272:263- 267, 1996), pseudotyped retroviral vectors (Agrawal et al., Exper. Hematol. 24:738-747, 1996), and any other vector system now known or later identified. Also included are chimeric viral particles, which are well known in the art and which can comprise viral proteins and / or nucleic acids from two or more different viruses in any combination to produce a functional viral vector. Chimeric viral particles of this invention can also comprise amino acid and / or nucleotide sequence of non-viral origin (e.g., to facilitate targeting of vectors to specific cells or tissues and / or to induce a specific immune response). The present invention also provides “targeted” virus particles (e.g., a parvovirus vector comprising a parvovirus capsid and a recombinant AAV genome, wherein an exogenous targeting sequence has been inserted or substituted into the parvovirus capsid).

[0181] Another aspect disclosed herein is an expression plasmid comprising any of the engineered RNA disclosed herein.

[0182] In one embodiment, when fee complementary sequence a trans-acting RNA present on a second, separate strand, the engineered RNA and the second, separate strand are expressed from the same vector or expression plasmid.

[0183] In one embodiment, wfeen fee complementary sequence a trans-acting RNA present on a second, separate strand, the engineered RNA and the second, separate strand are expressed from separate vectors or expression plasmids. When expressed from separate vectors or expression plasmids, expression of both vectors or expression plasmids in a cell can he at substantially fee same time or at separate times. For example, a vector or expression plasmid expressing the complementary sequence can be expressed at a time when reversal of fee synthetic PK-induced inhibition of RNA processing is desired.

[0184] Also disclosed herein is a platform for modulating RNA process, e.g., inhibiting RNA processing, such as translation, comprising any of the engineered RNAs comprising the synthetic PKs disclosed herein, and a complementary sequence to the PK.

[0185] In one embodiment, the platform comprises an engineered mRNA and complementary sequence expressed from the same sequence.

[0186] In one embodiment, the platform comprises an engineered mRNA and complementary sequence expressed from separate sequences.

[0187] In one embodiment, the platform comprises a vector or expression plasmid that expresses the engineered mRNA and complementary sequence, In one embodiment, the platform comprises a single vector or expression plasmid that expresses both the engineered mRNA and complementary sequence. In one embodiment, the platform comprises a vector or expression plasmid that expresses the engineered mRNA and a vector or expression plasmid that expresses the complementary sequence (i.e., the engineered mRN A and complementary sequence are expressed from separate sequences).

[0188] Another aspect disclosed herein is an engineered RNA vector comprising an RNA genome encoding at least one non-structural protein required for replication; a synthetic PK disclosed herein located within a region encoding the at least one non-structural proteins; and a sequence complementary to the PK, wherein the synthetic PK inhibits translation of the at least one non-structural proteins when not bound to the sequence complementary to the PK.

[0189] In one embodiment, wherein inhibition of translation of the at least one non-structural proteins inhibits RNA replication.

[0190] In one embodiment, the RNA genome further encodes at least one payload. For example, a payload can be any gene of interest, e.g., as disclosed herein.

[0191] In one embodiment, hybridization of the complementary sequence and the PK permits translation of the gene of interest, thereby activating RNA replication.

[0192] An engineered RNA vector comprising: an RNA genome encoding at least one non-structural protein required for replication; a pseudoknot (PK) of any of claims 1-8 located within a region encoding the at least one non-structural proteins; and a sequence complementary to the PK, wherein the PK inhibits replication or degradation of the engineered RNA vector when not bound to the sequence complementary to the PK.

[0193] In one embodiment, the PK inhibits procession of the RdRp replicating the RNA, thereby inhibiting RNA replication.

[0194] In one embodiment, the PK inhibits procession of the RNase degrading the RNA, thereby increasing RNA lifetime and enhancing RNA replication.

[0195] In one embodiment, the hybridization of the sequence complementary to the PK and the PK permits procession of the RdRp, thereby activating RNA replication.

[0196] In one embodiment, the hybridization of the sequence complementary to the PK and the PK permits procession of the RNase, thereby inhibiting RNA degradation and activating RNA replication.Synthesis of synthetic engineered RNAs

[0197] The engineered RNAs described herein can be synthesized and / or modified by methods well established in the art, such as those described in “Current Protocols in Nucleic Acid Chemistry,” Beaucage, S.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated herein by reference in its entirety. Transcription methods are described further herein in the Examples.

[0198] In one embodiment of the aspects described herein, a template for a engineered RNAs is synthesized using “splint-mediated ligation,” which allows for the rapid synthesis of DNA constructs by controlled concatenation of long oligos and / or dsDNA PCR products and without the need to introduce restriction sites at the joining regions. It can be used to add generic untranslated regions (UTRs) to the coding sequences of genes during T7 template generation. Splint mediated ligation can also be used to add nuclear localization sequences to an open reading frame, and to make dominant-negative constructs with point mutations starting from a wild-type open reading frame. Briefly, single-stranded and / or denatured dsDNA components are annealed to splint oligos which bring the desired ends into conjunction, the ends are ligated by a thermostable DNA ligase and the desired constructs amplified by PCR. A synthetic, modified-RNA is then synthesized from the template using an RNA polymerase in vitro. After synthesis of a engineered RNAs is complete, the DNA template is removed from the transcription reaction prior to use with the methods described herein.Modulation of RNA Processing

[0199] Various aspects disclosed herein provide methods for modulating, e.g., reversibly inhibiting, RNA processing in the cell. For example, expression of a synthetic PK, engineered RNA, or platform disclosed herein can be used to inhibit RNA processing, e.g., translation, RNA replication, RNA degradation, or DNA production from RNA. This inhibition is reversed by promoting hybridization of the complementary sequence and PK, resulting in unraveling the PK.

[0200] Accordingly, one aspect disclosed herein is a method for modulating (e.g., inhibiting) translation in a cell, the method comprising expressing an engineered RNAs or platforms disclosed herein, wherein translation of the gene of interest is inhibited by the PK when not bound to the sequence complementary to the PK; and promoting hybridization of the sequence complementary to the PK and the PK, wherein hybridization permits translation of the gene of interest.

[0201] Another aspect disclosed herein is a method for modulating (e.g., inhibiting) RNA processing in a cell, the method comprising expressing an engineered RNAs or platforms disclosed herein, wherein RNA processing of the gene of interest is inhibited by the PK when not bound to the sequence complementary to the PK; and promoting hybridization of the sequence complementary to the PK and the PK, wherein hybridization permits RNA processing of the gene of interest.

[0202] In the methods described herein, the engineered RNA can initially be present in the cell in the absence of, or the absence of the expression of, the complementary sequence, the presence of which would result in unraveling the synthetic PK. In this scenario, the synthetic PK functions to inhibit RNA processing. When the complementary sequence of this invention is also present in the cell, the synthetic PK is unraveled, and the inhibition of RNA processing is released.

[0203] The complementary sequence can be introduced into the cell any time relative to the introduction of the engineered RNA of this invention. For example, the complementary sequence can be introduced into the cell before, simultaneously with and / or after introduction of the engineered RNA into the cell. Furthermore, the complementary sequence can be introduced into the cell one time or at multiple times over any time interval and can extend to throughout the lifespan of the cell.

[0204] In one embodiment, the modulation of RNA processing (i.e., reversible inhibition of RNA processing) described herein is selectively controlled, for example, under spatial control. For example, either of the components of the platform (i.e., the engineered RNA and complementary sequence), or both, can be delivered locally to a desired site, location, organ, cell type, tissue type, etc., to induce expression of the components.

[0205] It is not required that all components be delivered / administered locally. In one embodiment, at least one components is delivered systemically, and one component is delivered locally. Local delivery of a component can be achieved by direct delivery of the component to a specific location. Alternatively, local delivery can be achieved using a localization sequence that drives the component to a specific location, or specific promoters that allow for expression of the component in a specific location. In one embodiment, local delivery is achieved by direct injection, e.g., to muscle, heart, or other organ.

[0206] In one embodiment, the modulation of RNA processing (i.e., reversible inhibition of RNA processing) described herein is selectively controlled, for example, under temporal control. For example, either of the components of the platform (i.e., the engineered RNA and complementary sequence), or both, can be delivered for a given duration to control the timing in which components are expressed. For example, pulsed administration (e.g., discontinuous administration) of the complementary sequence could result in repeated unraveling of the synthetic PL.

[0207] In one embodiment, the modulation of RNA processing (i.e., reversible inhibition of RNA processing) described herein is selectively controlled, for example, is selectively controlled under both spatial and temporal control.

[0208] In one embodiment, RNA processing is inhibited by at least 10% when the synthetic PK is expressed as compared to an appropriate control. In one embodiment, RNA processing is inhibited by at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more when the synthetic PK is expressed as compared to an appropriate control. As used herein, an “appropriate control” refers to the level of RNA processing in an otherwise identical cell that does not express the synthetic PK. A person skilled in the art can assess the level of RNA processing, e.g., using standard techniques known in the art.

[0209] In one embodiment, the inhibition of RNA processing is reversed upon hybridization of the complementary sequence. In one embodiment, hybridization of the complementary sequence restores RNA processing levels to at least 10% of those in an appropriate control. In one embodiment, hybridization of the complementary sequence restores RNA processing levels to at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more to those in an appropriate control. As used herein, an “appropriate control” refers to the level of RNA processing in an otherwise identical cell that does not express the synthetic PK. A person skilled in the art can assess the level of RNA processing, e.g., using standard techniques known in the art.

[0210] In one embodiment, hybridization of the complementary sequence increases RNA processing levels by at least 10% compared to an appropriate control. In one embodiment, hybridization of the complementary sequence increases RNA processing levels by at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more compared to an appropriate control. As used herein, an “appropriate control” refers to the level of RNA processing in an otherwise identical cell that expresses the synthetic PK. A person skilled in the art can assess the level of RNA processing, e.g., using standard techniques known in the art.

[0211] Introducing engineered RNA into a cell

[0212] A synthetic, modified-RNA can be introduced into a cell in any manner that achieves intracellular delivery of the engineered RNA, such that in vivo expression of the polypeptide encoded by the engineered RNA can occur. As used herein, the term “transfecting a cell" refers to the process of introducing nucleic acids into a cell of a tissue using means for facilitating or effecting uptake or absorption into the tissue, as is understood by those skilled in the art. As the term is used herein, “transfection” does not encompass vector-mediated gene delivery, e.g., viral- or viral particle based delivery methods. Absorption or uptake of a engineered RNA into a cell in vivo can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. Further approaches are described herein below or known in the art.

[0213] In some embodiments, an engineered RNA can be introduced into a cell, for example, by transfection, nucleofection, lipofection, electroporation (see, e.g., Wong and Neumann, Biochem. Biophys. Res. Commun. 107:584-87 (1982)), microinjection (e.g., by direct injection of a synthetic, modified RNA), biolistics, cell fusion, and the like.

[0214] In an alternative embodiment, an engineered RNA can be delivered using a drug delivery system such as a nanoparticle, a dendrimer, a hydrogel, a biopolymer, a polymer, a liposome, or a cationic delivery system. Positively charged cationic delivery systems facilitate binding of a synthetic, modified RNA (negatively charged polynucleotides) and also enhances interactions at the negatively charged cell membrane to permit efficient cellular uptake. Cationic lipids, dendrimers, or polymers can either be bound to modified RNAs, or induced to form a vesicle or micelle (see e.g., Kim SH., et al (2008) Journal of Controlled Release 129(2): 107-116) that encases the modified RNA. Methods for making and using cationic-modified RNA complexes are well within the abilities of those skilled in the art (see e.g., Sorensen, DR., et al (2003) J. Mol. Biol 327:761-766; Verma, UN., et al (2003) Clin. Cancer Res. 9:1291-1300; Arnold, AS et al (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entirety).

[0215] In some embodiments, the engineered RNA described herein can be included in biodegradable polymeric hydrogels, such as those disclosed in U.S. Pat. No. 5,410,016 to Hubbell et al. These polymerichydrogels can be delivered to a subject and the active compounds released over time as the polymer degrades. Commercially available hydrogels can be supplied either as a dry powder or a partially hydrated paste intended for administration after dispersion in an appropriate amount of aqueous vehicle. These powders are formed by mechanical disruption of cross-linked matrices, such as absorbable gelatin sponges, U.S.P. (e.g., Gelfoam ®, Pfizer, Inc. or Surgifoam ™, Ethicon, Inc.), or the cakes that are formed during typical chemical or dehydrothermal cross-linking treatment ( see, e.g. , U.S. Patent No. 6,063,061; U.S. Patent application pub. No. 2003 / 0064109). These hydrogels can be based on gelatin, collagen, dextran, chitosan. Other compositions are also used, for example, alginate (US Patent No: 5,294,446) and synthetic polymers such as polyphosphazines, polyacrylates, polyanhydrides, and polyorthoesters, as well as "block copolymers" such as mixtures of polyethylene oxide and polypropylene glycol (US Patent Nos: 5,041,138; 5,709,854; 5,736,372). In addition, US 5,749,874 and 5,769,899 (both Schwartz et al, 1998) disclose two-component implants, where one component is an anchoring device, made of a relatively hard yet biodegradable material (such as polyglycolic acid, polylactic acid, or combinations thereof), which helps secure and anchor the hydrogel implants and a second component that comprises a more porous and flexible matrix.

[0216] The process for delivery of an engineered RNA to a cell will necessarily depend upon the specific approach for transfection chosen.

[0217] It is also contemplated herein that when the engineered RNA and complementary sequence are administered via a separate mechanism (i.e., from a separate vectors or expression plasmids, these are administered in temporally distinct manners. Thus, each can be administered at a separate time or at a different frequency interval to achieve the desired expression of a polypeptide.

[0218] In certain embodiments of the aspects described herein, the engineered RNA can be introduced into a cell via transfection or lipofection. Suitable agents for transfection or lipofection include, for example but are not limited to, calcium phosphate, DEAE dextran, lipofectin, lipofectamine, DIMRIE C™, Superfect™, and Effectin™ (Qiagen™), unifectin™, maxifectin™, DOTMA, DOGS™ (Transfectam; dioctadecylamidoglycylspermine), DOPE (l,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DOTAP (l,2-dioleoyl-3-trimethylammonium propane), DDAB (dimethyl dioctadecylammonium bromide), DHDEAB (N,N-di-n-hexadecyl-N,N-dihydroxyethyl ammonium bromide), HDEAB (N-n-hexadecyl- N,N-dihydroxyethylammonium bromide), polybrene, poly(ethylenimine) (PEI), and the like. (See, e.g., Baneijee et al., Med. Chem. 42:4292-99 (1999); Godbey et al., Gene Ther. 6:1380-88 (1999); Kichler et al., Gene Ther. 5:855-60 (1998); Birchaa et al., J. Pharm. 183:195-207 (1999)).

[0219] An engineered RNA can be transfected into a cell as disclosed herein as a complex with cationic lipid carriers (e.g., Oligofectamine™) or non-cationic lipid-based carriers (e.g., Transit-TKOTM™, Minis Bio LLC, Madison, WI). Successful introduction of a the engineered RNA into a cell can be monitored using various known methods. For example, transient transfection of a cell herein can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Successful transfectionof a cell with the engineered RNA can also be determined by measuring the protein expression level of the target polypeptide by e.g., Western Blotting or immunocytochemistry.

[0220] In some embodiments of the aspects described herein, the engineered RNA is introduced into a cell using a transfection reagent. Some exemplary transfection reagents include, for example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97 / 30731). Examples of commercially available transfection reagents include, for example Lipofectamine™ (Invitrogen;Carlsbad, CA), Lipofectamine 2000™ (Invitrogen; Carlsbad, CA), 293fectin™ (Invitrogen; Carlsbad, CA), Cellfectin™ (Invitrogen; Carlsbad, CA), DMRIE-C™ (Invitrogen; Carlsbad, CA), FreeStyle™ MAX (Invitrogen; Carlsbad, CA), Lipofectamine™ 2000 CD (Invitrogen; Carlsbad, CA), Lipofectamine™ (Invitrogen; Carlsbad, CA), RNAiMAX (Invitrogen; Carlsbad, CA), Oligofectamine™ (Invitrogen; Carlsbad, CA), Optifect™ (Invitrogen; Carlsbad, CA), X-tremeGENE Q2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAP Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPER Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), or Fugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega; Madison, WI), TransFast™ Transfection Reagent (Promega; Madison, WI), Tfx™-20 Reagent (Promega; Madison, WI), Tfx™-50 Reagent (Promega; Madison, WI), DreamFect™ (OZ Biosciences; Marseille, France), EcoTransfect (OZ Biosciences; Marseille, France), TransPassaDI Transfection Reagent (New England Biolabs; Ipswich, MA, USA), LyoVec™ / LipoGen™ (Invitrogen; San Diego, CA, USA), PerFectin Transfection Reagent (Genlantis; San Diego, CA, USA), NeuroPORTER Transfection Reagent (Genlantis; San Diego, CA, USA), GenePORTER Transfection reagent (Genlantis; San Diego, CA, USA), GenePORTER 2 Transfection reagent (Genlantis; San Diego, CA, USA), Cytofectin Transfection Reagent (Genlantis; San Diego, CA, USA), BaculoPORTER Transfection Reagent (Genlantis; San Diego, CA, USA), TroganPORTER™ transfection Reagent (Genlantis; San Diego, CA, USA ), RiboFect (Bioline; Taunton, MA, USA), PlasFect (Bioline; Taunton, MA, USA), UniFECTOR (B-Bridge International; Mountain View, CA, USA), SureFECTOR (B-Bridge International; Mountain View, CA, USA), or HiFect™ (B-Bridge International, Mountain View, CA, USA), among others.

[0221] In other embodiments, highly branched organic compounds, termed “dendrimers,” can be used to bind the exogenous nucleic acid, such as the engineered RNA described herein, and introduce it into a cell.

[0222] In other embodiments of the aspects described herein, non-chemical methods of transfection are contemplated. Such methods include, but are not limited to, electroporation (methods whereby an instrument is used to create micro-sized holes transiently in the plasma membrane of cells under an electric discharge), sono-poration (transfection via the application of sonic forces to cells), and optical transfection (methods whereby a tiny (~1 pm diameter) hole is transiently generated in the plasma membrane of a cell using a highly focused laser). In other embodiments, particle-based methods of transfections are contemplated, such as the use of a gene gun, whereby the nucleic acid is coupled to ananoparticle of an inert solid (commonly gold) which is then "shot" directly into the target cell's nucleus; “magnetofection,” which refers to a transfection method, that uses magnetic force to deliver exogenous nucleic acids coupled to magnetic nanoparticles into target cells; “impalefection,” which is carried out by impaling cells by elongated nanostructures, such as carbon nanofibers or silicon nanowires which have been coupled to exogenous nucleic acids.

[0223] Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols, such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes, such as limonene and menthone.

[0224] Another aspect herein comprising a composition comprising any of the engineered RNAs disclosed herein, or any vectors, expression plasmids, or platforms comprising the same. In one embodiment, the composition comprises pharmaceutically acceptable carriers or diluents. As used herein "pharmaceutically acceptable carriers or diluents" are well known to those skilled in the art. The carrier or diluent may be may be, in various embodiments, a solid carrier or diluent for solid formulations, a liquid carrier or diluent for liquid formulations, or mixtures thereof. In another embodiment, solid carriers / diluents include, but are not limited to, a gum, a starch (e.g. com starch, pregeletanized starch), a sugar (e.g., lactose, mannitol, sucrose, dextrose), a cellulosic material (e.g. microcrystalline cellulose), an acrylate (e.g. polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof. In other embodiments, pharmaceutically acceptable carriers for liquid formulations may be aqueous or nonaqueous solutions, suspensions, emulsions or oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Examples of oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, and fish-liver oil.RNA-based translational regulatory element

[0225] Also provided herein is an RNA-based translational regulatory element that comprising: a slippery sequence configured to induce ribosomal frameshifting; and a hairpin structure positioned downstream of the slippery sequence, the hairpin structure comprising a stem and a loop, wherein the hairpin structure is configured to modulate translation of a downstream coding sequence by at least partially repressing ribosomal progression in a reading frame-dependent manner.

[0226] As used herein, a “hairpin” refers to a nucleic acid secondary structure formed when a singlestranded nucleic acid molecule (e.g., RNA or DNA) contains a region of intramolecular complementarity that base-pairs to form a stem, together with an intervening non-complementary region that forms a loop. A hairpin structure is also referred to in the art as a stem-loop structure. Hairpins may vary in stem length, loop size, and stability, and may be naturally occurring (e.g., transcriptional terminators, pre-microRNA structures) or engineered de novo for use in synthetic biology, gene regulation, or therapeuticapplications. In some embodiments, a hairpin may serve as a regulatory element influencing RNA processing, stability, or translation.

[0227] In one embodiment, the hairpin structure is positioned at least 3 nucleotides downstream of the slippery sequence. In one embodiment, the hairpin structure is positioned no more than 54 nucleotides downstream of the slippery sequence. In one embodiment, the hairpin structure is positioned 3-54 nucleotides downstream of the slippery sequence.

[0228] In one embodiment, the hairpin structure has stem that is at least 6 nucleotides in length. In one embodiment, the hairpin structure has stem that is no more than 75 nucleotides in length. In one embodiment, the hairpin structure has stem that is between 6 and 75 nucleotides in length.

[0229] Slippery sequences are generally short motifs, typically 6 to 8 nucleotides in length, that conform to a consensus pattern such as X XXY YYZ (SEQ ID NO: 73), wherein “X” can be any nucleotide, “Y” represents a run of identical nucleotides (often adenosine or uridine), and “Z” is a nucleotide that disfavors stable pairing in the ribosomal A-site. When a ribosome encounters such a sequence, the pairing flexibility of the tRNAs allows them to realign in an alternative reading frame, often in conjunction with a downstream secondary structure such as a stem-loop or pseudoknot, thereby producing a programmed -1 ribosomal frameshift.

[0230] In one embodiment, the slippery sequence is of the form XXXYYYZ (SEQ ID NO: 73), where X, Y, and Z represent individual nucleotides. Representative examples of slippery sequences those presented in Table 1 herein.

[0231] In one embodiment, the slippery sequence is composed of modified nucleotides. As used herein, the term “chemically modified nucleotide” refers to a nucleotide, nucleoside, or nucleic acid analog comprising at least one chemical alteration relative to a naturally occurring ribonucleotide or deoxyribonucleotide. Such modifications may be introduced into the base, sugar, and / or phosphate backbone of the nucleotide, and may be naturally occurring or synthetic. Chemically modified nucleotides may be incorporated into RNA or DNA molecules in order to enhance stability, resist nuclease degradation, alter hybridization affinity, modulate immunogenicity, improve translational efficiency, or confer other desirable biochemical or pharmacological properties.

[0232] Representative examples of chemically modified nucleotides include, without limitation: Base modifications, such as pseudouridine, N1 -methylpseudouridine, 5-methylcytidine, 5- hydroxymethylcytidine, inosine, 2-thiouridine, or halogenated nucleobases. Sugar modifications, such as 2'-O-methyl, 2'-fluoro, 2'-O-methoxyethyl, locked nucleic acids (LNAs), and unlocked nucleic acids (UNAs). Backbone modifications, such as phosphorothioates, phosphoramidates, boranophosphates, peptide nucleic acids (PNAs), and morpholino phosphorodiamidates.

[0233] In certain embodiments, chemically modified nucleotides may be incorporated into synthetic RNA constructs to improve their performance in therapeutic, diagnostic, or research applications. For example, substitution of uridine with pseudouridine or N1 -methylpseudouridine within an mRNA may increase translational yield and reduce innate immune activation. In other embodiments, 2'-O-methyl or phosphorothioate modifications may be used to stabilize antisense oligonucleotides or small interfering RNAs (siRNAs) against nuclease degradation. Unless expressly stated otherwise, the term “nucleotide” as used herein encompasses both naturally occurring and chemically modified nucleotides, as well as combinations thereof.

[0234] In one embodiment, the hairpin structure is configured to completely block translation in the 0 reading frame and not permit translation in any downstream reading frame in the coding region sequence.

[0235] In one embodiment, the hairpin structure is configured to partially repress translation in the 0 reading frame, thereby enabling tunable expression of multiple protein coding sequences from a single transcript.

[0236] In one embodiment, the hairpin structure is designed such that its repression of translation is independent of intermolecular interactions.

[0237] In one embodiment, any of the RNA-based translational regulatory element disclosed herein is incorporated into a multicistronic RNA vector for the coordinated expression of multiple antigens or therapeutic proteins. As used herein, the term “multicistronic RNA vector” refers to an engineered RNA molecule that encodes two or more distinct open reading frames (ORFs) within a single contiguous RNA transcript. A multicistronic RNA vector may enable expression of multiple polypeptides from a single transcript, for example by including internal ribosome entry sites (IRES elements), 2A self-cleaving peptide sequences, ribosomal reinitiation signals, programmed firameshift elements (e.g., slippery sequences and PKs), or other translational control elements that allow ribosomes to initiate or continue translation at more than one site within the RNA. Such vectors can be used to coordinate the coexpression of functionally related proteins, subunits of multiprotein complexes, or therapeutic payloads in a single delivery construct.

[0238] In one embodiment, the translational regulatory element further comprises a complementary RNA segment designed to hybridize to at least a portion of the hairpin structure. In one embodiment, hybridization of the complementary RNA segment unwinds the hairpin structure and restores ribosomal progression. In one embodiment, the complementary RNA segment is encoded within the same RNA molecule in a non-coding region or is provided as a trans-acting RNA.

[0239] In one embodiment, the hairpin structure and slippery sequence are selected such that the regulatory effect is programmable by varying the sequence, length, or position of the hairpin relative to the slippery sequence.

[0240] In one embodiment, the hairpin structure is designed using a thermodynamic prediction algorithm to achieve a desired degree of translational repression.

[0241] In one embodiment, the hairpin structure is designed using a neural network prediction algorithm to achieve a desired degree of translational repression.

[0242] In one embodiment, the element is used in a eukaryotic cell for the regulation of protein expression.

[0243] In certain embodiments, the RNA-based translational regulatory elements described herein may be employed to modulate protein translation in a manner analogous to that described for synthetic RNA pseudoknots (Pks).

[0244] Also provided herein is a cell expressing the RNA-based translational regulatory element disclosed herein. When present in a cell, the RNA-based translational regulatory element functions to regulate any aspect of protein translation, e.g., regulation of protein expression. Aspects of protein translation are described herein above.

[0245] An engineered RNA comprising a slippery sequence configured to induce ribosomal frameshifting; and a hairpin structure positioned downstream of the slippery sequence, wherein the hairpin structure is configured to modulate translation of a downstream coding sequence by at least partially repressing ribosomal progression in a reading frame-dependent manner

[0246] A platform for modulating translation, the platform comprising a slippery sequence configured to induce ribosomal frameshifting; and a hairpin structure positioned downstream of the slippery sequence, wherein the hairpin structure is configured to modulate translation of a downstream coding sequence by at least partially repressing ribosomal progression in a reading frame-dependent manner.

[0247] A method of modulating translation in a cell, the method comprising expressing the RNA-based translational regulatory element, or an engineered RNA or platform comprising the same, wherein the hairpin structure is configured to modulate translation of a downstream coding sequence by at least partially repressing ribosomal progression in a reading frame-dependent manner. In certain embodiments, the RNA-based translational regulatory elements described herein may be employed to modulate protein translation in a manner analogous to that described for synthetic RNA PKs disclosed herein.In-Silico Generation of Synthetic PK Sequences

[0248] The present disclosure provides computer-implemented methods for the in-silico generation of synthetic RNA pseudoknot sequences capable of modulating translation, including programmed ribosomal frameshifting or ribosomal stalling. These methods employ computational design algorithms, supervised nucleotide sampling, secondary-structure prediction tools, and machine-learning models to generate and rank pseudoknot sequences with desired translational control properties.

[0249] General Worlflow

[0250] In some embodiments, a computer system receives, via a processor, a user-defined target secondary pseudoknot topology. The topology may be specified, for example, in terms of the number of stems and loops, overlapping arrangements, or inter-stem connectivity. In one embodiment, the topology comprises two or more hairpin domains. In another embodiment, the topology comprises at least two overlapping hairpin loop structures.

[0251] The processor decomposes the target topology into a plurality of hairpin domains. For each hairpin domain, the system performs supervised nucleotide sampling. In certain embodiments, supervised sampling is constrained such that codon combinations producing in-firame stop codons in each of three translation frames are excluded, thereby preserving translational continuity. In some embodiments, sampling is further guided by user-defined base-pair composition frequencies (e.g., specifying relative ratios of AU, GC, or GU pairs).

[0252] Thermodynamic and Cross-Talk Evaluation

[0253] For each candidate domain sequence, the system evaluates thermodynamic stability and potential inter-domain cross-talk using one or more secondary-structure prediction algorithms. Suitable algorithms include, but are not limited to, minimum firee-energy folding models, partition-function-basedpredictions, and stochastic sampling approaches. Candidate sequences predicted to form undesired alternative structures or to exhibit excessive cross-talk between domains are deprioritized.

[0254] In one embodiment, the PK stem domains comprises AG < -350 kcal / mol and a computationally predicted crosstalk <1% at 37°C at 100 nM concentration of the domains.

[0255] Sequence Concatenation and Candidate Assembly

[0256] A subset of candidate domain sequences having minimized inter-domain cross-talk is concatenated into a complete pseudoknot candidate sequence. The resulting pseudoknot candidates are then evaluated and ranked based on at least one criterion, including predicted thermodynamic stability, translational modulation score, or both. In some embodiments, the translational modulation score predicts the ability of the pseudoknot to induce ribosomal frameshifting, enzymatic stalling, or translational blockage.

[0257] Neural Network Prediction and Adaptive Sampling

[0258] In certain embodiments, the ranking step further comprises applying a trained neural network to predict the degree of ribosomal blockage, enzymatic stalling, or frameshifting efficiency associated with each pseudoknot candidate sequence. The neural network may be trained using experimental datasets correlating pseudoknot sequences with measured ribosomal pausing or frameshift frequencies. In some embodiments, feedback from the neural network dynamically adjusts nucleotide sampling frequencies during the supervised nucleotide sampling process, thereby biasing sequence generation toward motifs predicted to enhance frameshifting efficiency, enzymatic stalling, or blockage strength.

[0259] Storage and Output

[0260] The system outputs at least one ranked pseudoknot candidate sequence to a user interface. In some embodiments, the ranked sequence is electronically stored in a database for subsequent retrieval and chemical synthesis. Such storage may include associated metadata such as predicted free energy, frameshift efficiency score, or enzymatic stalling or ribosomal blockage profile.

[0261] Translational Modulation Applications

[0262] In certain embodiments, the ranking step selects pseudoknot candidate sequences predicted to block translation in the 0 frame while permitting translation in a -1, +1, -2, or +2 frame, or the PK is configured to block translation and not permit downstream translation in any frame. Such sequences may be useful in the design of viral mimetics, gene regulatory systems, ribosomal pause sites, or synthetic biology applications requiring controlled translational recoding.

[0263] The present disclosure thus provides a computational framework for designing pseudoknots with defined structural and functional features, enabling scalable in-silico discovery of novel regulatory RNA motifs.

[0264] The present invention is further illustrated by the following examples which in no way should be construed as being further limiting, The contents of all cited references, including literature references, issued patents, published patent applications, and co-pending patent applications, cited throughout this application are hereby expressly incorporated by reference.

[0265] The present invention has been described in terms of particular embodiments found or proposed by the present inventor to comprise preferred modes for the practice of the invention. It will be appreciated by those of skill in the art that, in light of the present disclosure, numerous modifications and changes can be made in the particular embodiments exemplified without departing from the intended scope of the invention. For example, due to codon redundancy, changes can be made in the underlying DNA sequence without affecting the protein sequence. Moreover, due to biological functional equivalency considerations, changes can be made in protein structure without affecting the biological action in kind or amount. All such modifications are intended to be included within the scope of the appended claims.

[0266] The present invention can further by described in the following numbered paragraphs:1. A synthetic RNA pseudoknot (PK) comprising at least two stem-loop domains, the at least two stem-loop domains separated by a loop domain, wherein formation of the PK results in overlapping base pair interactions.2. The synthetic RNA PK of paragraph 1, wherein each stem-loop domain is modified such that it comprises a nucleotide sequence lacking in-frame stop codons3. The synthetic RNA PK of any preceding paragraph, wherein the PK is configured to block translation in a 0 frame while permitting translation in a -1, +1, -2, or +2 frame, or the PK is configured to block translation and not permit downstream translation in any frame.4. The synthetic RNA PK of any preceding paragraph, wherein the PK is incorporated into a self-amplifying RNA vector, a circular RNA vector, a messenger RNA, or a non-coding RNA.5. The synthetic RNA PK of any preceding paragraph, wherein hybridization of a sequence complementary to the PK causes the PK to unwind.6. The synthetic RNA PK of any preceding paragraph, wherein the PK is chemically synthesized or recombinantly expressed.7. The synthetic RNA PK of any preceding paragraph, wherein the at least two stem-loop domains have a sequence selected from SEQ ID NO: 1-48.8. The synthetic RNA PK of any preceding paragraph, wherein the loop domain has a sequence selected from SEQ ID NO: 49-60.9. The synthetic RNA PK of any preceding paragraph, wherein the synthetic PK has a sequence selected from SEQ ID NO: 61-72.10. A synthetic RNA PK comprising, from 5’ to 3’: a first stem-loop region; a first spacer; a second stem-loop region; a second spacer; a reverse complement sequence to the first stem-loop region;a third spacer; and a reverse complement sequence to the second stem-loop region, wherein overlapping base pair interactions occur between loops and more distal stem regions. The synthetic RNA PK of any preceding paragraph, where the second stem-loop region is the loop of the stem formed between the first stem-loop region and its reverse complement, and the reverse complement sequence to the first stem-loop region is the loop of the stem formed between the second stem-loop region and its reverse complement. The synthetic RNA PK of any preceding paragraph, further comprising, between the third spacer, and the reverse complement sequence to the second stem-loop region, a third stem-loop region, a fourth spacer, and a reverse complement sequence to the third stem-loop region. The synthetic RNA PK of any preceding paragraph, wherein at least one of spacer is removed. The synthetic RNA PK of any preceding paragraph, wherein the stem-loop region and its reverse complement are not fully complementary. An engineered RNA comprising a coding region sequence of a gene of interest and a secondary RNA structure, wherein the secondary RNA structure is located at or near the gene of interest and inhibits RNA processing at or near the gene of the interest. An engineered RNA comprising a coding region sequence of a gene of interest and a synthetic PK of any preceding paragraph, wherein the synthetic PK is located at or near the gene of interest and inhibits RNA processing at or near the gene of the interest. An engineered RNA comprising a coding region sequence of a gene of interest and a synthetic PK of any preceding paragraph, wherein the synthetic PK is located at or near the gene of interest and inhibits translation at or near the gene of the interest. The engineered mRNA of any preceding paragraph, wherein at or near is upstream, downstream, or within of the coding region of a gene of interest. The engineered RNA of any preceding paragraph, wherein the RNA processing is the progression or processivity of an RNA-processing or RNA-utilizing enzyme. The engineered mRNA of any preceding paragraph, wherein the RNA-processing enzyme is a selected from the group consisting of: a deadenylase, a decapping enzyme, an exonuclease, an endonuclease, a surveillance enzyme, an RNA-dependent RNA polymerase, and an RNA- dependent DNA polymerase.The engineered mRNA of any preceding paragraph, further comprising a sequence complementary to the PK. The engineered mRNA of any preceding paragraph, wherein the sequence complementary to the PK hybridizes to the PK. The engineered mRNA of any preceding paragraph, wherein hybridization to the PK causes the PK to unwind. The engineered mRNA of any preceding paragraph, wherein unwinding of the PK permits translation of the gene of interest. The engineered mRNA of any preceding paragraph, wherein the sequence complementary to the PK is downstream of the coding region of a gene of interest. The engineered mRNA of any preceding paragraph, wherein the sequence complementary to the PK is located in the 3’ UTR of the gene of interest. The engineered mRNA of any preceding paragraph, wherein the sequence complementary to the PK wherein the sequence complementary is a trans-acting RNA present in a second strand. The engineered mRNA of any preceding paragraph, wherein the trans-acting RNA is an endogenous cellular transcript. The engineered mRNA of any preceding paragraph, wherein the trans-acting RNA is an endogenous RNA. The engineered mRNA of any preceding paragraph, further comprising at least a second coding region of at least a second gene of interest. The engineered mRNA of any preceding paragraph, further comprising at least a second synthetic RNA PK. A platform for modulating translation, the platform comprising: an engineered RNA comprising a coding region sequence of a gene of interest a synthetic PK of any preceding paragraph, and a sequence complementary to the PK. A platform for modulating RNA processing, the platform comprising: an engineered RNA comprising a coding region sequence of a gene of interest a synthetic PK of any preceding paragraph. The platform of any preceding paragraph, wherein the PK inhibits translation of the gene of interest when not bound to the sequence complementary to the PK. The platform of any preceding paragraph, wherein the PK inhibits RNA processing of the gene of interest when not bound to the sequence complementary to the PK. The platform of any preceding paragraph, wherein the sequence complementary to the PK hybridizes to the PK and causes the PK to unwind.The platform of any preceding paragraph, wherein unwinding permits translation of the gene of interest. The platform of any preceding paragraph, wherein the sequence complementary to the PK is located downstream of the coding region. The platform of any preceding paragraph, wherein the sequence complementary to the PK is a trans-acting RNA present in a second strand. The platform of any preceding paragraph, wherein the engineered RNA is an engineered RNA of any of any preceding paragraph. A method of modulating translation in a cell, the method comprising: expressing an engineered RNA of any preceding paragraph, or a platform of any preceding paragraph in a cell, wherein translation of the gene of interest is inhibited by the PK when not bound to the sequence complementary to the PK; and promoting hybridization of the sequence complementary to the PK and the PK, wherein hybridization permits translation of the gene of interest. A method of modulating RNA processing in a cell, the method comprising: expressing an engineered RNA of any preceding paragraph, or a platform of any preceding paragraph in a cell, wherein RNA processing of the transcript of interest is inhibited by the PK when not bound to the sequence complementary to the PK; and promoting hybridization of the sequence complementary to the PK and the PK, wherein hybridization permits RNA processing of the transcript of interest. The method of any preceding paragraph, wherein the sequence complementary to the PK is a trans-acting RNA present in a second strand and promoting hybridization comprising expressing the second strand in the cell. The method of any preceding paragraph, wherein the sequence complementary to the PK is downstream of the gene of interest and promoting hybridization comprising allowing for folding of the modified mRNA such that the sequence complementary to the PK can hybridize to the PK. The method of any preceding paragraph, wherein promoting hybridization is spatially and / or temporally regulated. The method of any preceding paragraph, wherein the platform is expressed in the cell by an expression plasmid or a vector. The method of any preceding paragraph, wherein the modified mRNA and the sequence complementary to the PK are expressed in the cell by the same expression plasmid or vector. The method of any preceding paragraph, wherein the modified mRNA and the sequence complementary to the PK are expressed in the cell by separate expression plasmids or vectors.The method of any preceding paragraph, wherein the vector is a non-viral vector or a viral vector. An engineered RNA vector comprising: an RNA genome encoding at least one non-structural protein required for replication; a pseudoknot (PK) of any preceding paragraph located within a region encoding the at least one non-structural proteins; and a sequence complementary to the PK, wherein the PK inhibits translation of the at least one non-structural protein when not bound to the sequence complementary to the PK. The engineered RNA vector of any preceding paragraph, wherein the PK inhibits translation of the at least one non-structural proteins when not bound to the sequence complementary to the PK, thereby inhibiting RNA replication. The engineered RNA vector of any preceding paragraph, wherein the hybridization of the sequence complementary to the PK and the PK permits translation of the gene of interest, thereby activating RNA replication. The engineered RNA vector of any preceding paragraph, wherein the hybridization of the sequence complementary to the PK and the PK permits degradation of the gene of interest, thereby deactivating RNA replication. An engineered RNA vector comprising: an RNA genome encoding at least one non-structural protein required for replication; a pseudoknot (PK) of any preceding paragraph located within a region encoding the at least one non-structural proteins; and a sequence complementary to the PK, wherein the PK inhibits replication or degradation of the engineered RNA vector when not bound to the sequence complementary to the PK. The engineered RNA vector of any preceding paragraph, wherein the PK inhibits procession of the RdRp replicating the RNA, thereby inhibiting RNA replication. The engineered RNA vector of any preceding paragraph, wherein the PK inhibits procession of the RNase degrading the RNA, thereby increasing RNA lifetime and enhancing RNA replication. The engineered RNA vector of any preceding paragraph, wherein the hybridization of the sequence complementary to the PK and the PK permits procession of the RdRp, thereby activating RNA replication. The engineered RNA vector of any preceding paragraph, wherein the hybridization of the sequence complementary to the PK and the PK permits procession of the RNase, thereby inhibiting RNA degradation and activating RNA replication.A computer-implemented method for in-silico generation of a synthetic pseudoknot RNA sequence capable of inducing a programmed translational frameshift or ribosomal stall, the method comprising: receiving, with a processor, a user-defined target secondary pseudoknot topology; decomposing the target topology into a plurality of hairpin domains; for each hairpin domain, performing supervised nucleotide sampling that excludes inframe stop codons in each of three translation frames to generate a plurality of candidate domain sequences; evaluating, for each candidate domain sequence, thermodynamic stability and inter-domain cross-talk with a secondary-structure prediction algorithm; concatenating a combination of the candidate domain sequences having minimized interdomain cross-talk to form a complete pseudoknot candidate sequence; ranking the pseudoknot candidate sequence with respect to at least one of the thermodynamic stability and a predicted translational modulation score; and outputting at least one ranked synthetic pseudoknot sequence. The method of any preceding paragraph, wherein the ranking step further comprises applying a trained neural network to predict a degree of ribosomal blockage, enzymatic stalling, or frameshifting efficiency of each pseudoknot candidate sequence. The method of any preceding paragraph, wherein feedback from the trained neural network dynamically adjusts nucleotide sampling frequencies during the supervised nucleotide sampling. The method of any preceding paragraph, wherein the user-defined target secondary pseudoknot topology comprises at least two overlapping hairpin loop structures. The method of any preceding paragraph, wherein the supervised nucleotide sampling is performed according to user-specified target base-pair composition frequencies. The method of any preceding paragraph, further comprising electronically storing the at least one ranked synthetic pseudoknot sequence in a database for subsequent chemical synthesis. The method of any preceding paragraph, wherein the ranking step selects a pseudoknot candidate sequence predicted to block translation in the 0 frame while permitting translation in a -1, +1, -2, or +2 frame. The method of any preceding paragraph, wherein the ranking step selects a pseudoknot candidate sequence predicted to block progression of an enzyme involved in processing or degrading RNA. The method of any preceding paragraph, wherein the PK stem domains comprises -7.5 <AG < -350 kcal / mol and a computationally predicted crosstalk <1% at 37°C at 100 nM concentration of the domains.An RNA-based translational regulatory element comprising: a slippery sequence configured to induce ribosomal frameshifting; and a hairpin structure positioned downstream of the slippery sequence, the hairpin structure comprising a stem and a loop, wherein the hairpin structure is configured to modulate translation of a downstream coding sequence by at least partially repressing ribosomal progression in a reading frame-dependent manner. The RNA-based translational regulatory element of any preceding paragraph, wherein the hairpin structure is positioned between 3 and 54 nucleotides downstream of the slippery sequence. The RNA-based translational regulatory element of any preceding paragraph, wherein the hairpin structure has a stem comprising between 6 and 75 nucleotides. The RNA-based translational regulatory element of any preceding paragraph, wherein the slippery sequence is of the form XXXYYYZ (SEQ ID NO: 73), where X, Y, and Z represent individual nucleotides. The RNA-based translational regulatory element of any preceding paragraph, wherein the slippery sequence is composed of modified nucleotides. The RNA-based translational regulatory element of any preceding paragraph, wherein the hairpin structure is configured to completely block translation in the 0 reading frame and not permit translation in any downstream reading frame in the coding region sequence. The RNA-based translational regulatory element of any preceding paragraph, wherein the hairpin structure is configured to partially repress translation in the 0 reading frame, thereby enabling tunable expression of multiple protein coding sequences from a single transcript. The RNA-based translational regulatory element of any preceding paragraph, wherein the hairpin structure is designed such that its repression of translation is independent of intermolecular interactions. The RNA-based translational regulatory element of any preceding paragraph, wherein the element is incorporated into a multicistronic RNA vector for the coordinated expression of multiple antigens or therapeutic proteins. The RNA-based translational regulatory element of any preceding paragraph, further comprising a complementary RNA segment configured to hybridize to at least a portion of the hairpin structure, wherein hybridization of the complementary RNA segment unwinds the hairpin structure and restores ribosomal progression. The RNA-based translational regulatory element of any preceding paragraph, wherein the complementary RNA segment is encoded within the same RNA molecule in a non-coding region or is provided as a trans-acting RNA.79. The RNA-based translational regulatory element of any preceding paragraph, wherein the hairpin structure and slippery sequence are selected such that the regulatory effect is programmable by varying the sequence, length, or position of the hairpin relative to the slippery sequence.80. The RNA-based translational regulatory element of any preceding paragraph, wherein the hairpin structure is designed using a thermodynamic prediction algorithm to achieve a desired degree of translational repression.81. The RNA-based translational regulatory element of any preceding paragraph, wherein the hairpin structure is designed using a neural network prediction algorithm to achieve a desired degree of translational repression.82. The RNA-based translational regulatory element of any preceding paragraph, wherein the element is used in a eukaryotic cell for the regulation of protein expression.EXAMPLESEXAMPLE 1

[0267] Pseudoknots (PKs) are a class of RNA secondary structured defined by intermolecular pairing of a complete stem loop motif with a complementary stretch of base pairs at a separate location within the same molecule. These nested structures create an additional layer of complexity within single RNA molecules that lead to a diverse range of regulatory mechanisms. One of the most well studied examples of a PK-induced regulatory mechanism is programmed firameshifting, where a ‘slippery’ nucleotide sequence and a downstream PK divert a translating ribosome into an alternative reading frame. These slippery sequences take the form of X-XXY-YYZ (X, Y, and Z representing unique nucleotides), while the downstream PK structures can precisely modulate the stoichiometric ratio of proteins encoded within multiple frames. This motif is present in many viral genomes and modulates translation across both prokaryotes and eukaryotes, allowing for a compact genomic region to encode multiple protein coding sequences (1-2).

[0268] The ability to precisely control protein ratios from a single transcript offers a promising starting point for engineering synthetic biology systems using PK elements. Despite this, the intercalated structural motifs present within PK molecules presents an extreme challenge for computational RNA tools. Both the prediction and inverse design of RNA secondary structure are built on recursive dynamic programming algorithms that ‘solve’ the predicted structure within local regions. This approach requires an immense degree of compute time [O(N3)], and is not conceptually amenable to considering PKs, which would require retracing solved regions to see if they form a knotted motif with distal elements. While some solutions have been proposed for predicting and designing PK structures, there is a great deal of uncertainty when employing these methods. Furthermore, building a synthetic biology system that couples PK formation to a programmable input such as a small molecule or trans-acting RNA isextremely difficult, as there are no available computational tools for considering intermolecular PK interactions.

[0269] Several studies have found a limited degree of success using high-throughput screening to modulate frameshifting behavior (3-4). These techniques have largely been applied to naturally occurring PK elements within viral genomes, with the goal of disrupting viral replication as a therapeutic modality. To the best of our knowledge, only a single study has been conducted to couple a small molecule input to the function of synthetic PK structures (3). This approach required the screening of hundreds of thousands of candidates to identify a small subset of functioning examples, and still exhibited an extremely limited dynamic range of only 7-fold in in vitro settings.

[0270] In addition to programmed frameshifting, several reports in the literature have found that PK- like structures can elicit ribosomal pausing, with some PKs completely halting ribosomal progression altogether (5-7). The specific features needed to reliably reproduce this response remain uncharacterized despite the potential utility in therapeutic and synthetic biology applications offered by a transcript that is OFF in its default conformation. Extensive research has been conducted to selectively control the expression of protein from an RNA transcript, as ubiquitous expression following injection can lead to an unwanted immunogenic response (20). Available technologies for doing so require complex ensembles of RNA and protein components or utilize transcripts that are selectively silenced rather than activated. These characteristics largely inhibit the application of these technologies outside of laboratory settings, despite the pressing need for improved selectivity of RNA expression.

[0271] In this work, we present an approach to reliably design synthetic PK (sPK) structures that can exhibit programmable frameshifting or completely block ribosomal progression. We further demonstrate the manipulation of these structures through RNA-RNA interactions and secondary structure manipulations to selectively activate or repress PK-induced modulations of translation. This system provides a broadly applicable and compact mechanism for precisely engineering translation of proteins in eukaryotic cells.

[0272] Results

[0273] An algorithm for designing pseudoknots without stop codons in multiple frames

[0274] To avoid the computational constraints of sPK design, we developed a new approach that allows for potential knotted structures to be returned without the need to reconsider nested folding. This entails several steps beginning with the determination of a target sPK secondary structure. This structural target can be generalized to allow any combination of hairpins and loops of different lengths, can include fully- or partially determined regions, and can consider additional contextual regions that are not explicitly a portion of the knotted structure. Once a target for design has been determined, the PK is decomposed into a set of individual domains consisting of the specific hairpins, loops, or conserved regions that must be present in the output. The nucleotide compositions of the variable components are determined by a supervised sampling approach. A target frequency of A-U or G-C nucleotides can be specified, at which point a random sampling is conducted to populate the target structural elements in the 5’ to 3’ direction.During this approach, the algorithm constantly supervises the previous selections for the patterns ‘UA’ and ‘UG’, indicating the first two bases of a stop codon. If these patterns are identified, the sampling frequencies are shifted to eliminate the possibility of incorporating a nucleotide that would complete the stop codon. This enables individual sPK domains to be designed without stop codons in each of the three possible translation frames, an essential step for utilizing these devices in synthetic biology settings.

[0275] This sampling process is repeated to identify many different examples of each individual structural feature that differ in their specific nucleotide compositions. Once a large catalogue is determined, the pool of components is assessed for their degree of cross talk while treating each structural element as a separate molecule. The specific combination of each structural examples that minimizes intermolecular interactions is then selected for concatenation into a complete sPK. Decomposing the knotted structure in this manner allows for classical secondary structure tools to be utilized for design without introducing the computational bottlenecks of explicit PK consideration. As a final filter, the junctions formed by the concatenation of individually designed sPK components are assessed for stop codons. While available PK structural prediction tools may be limited in their accuracy, they do reveal that sequences designed in this manner are reliably predicted to adopt the target structure initially specified (Fig. 1).

[0276] Supplementary steps that augment the sPK design process to incorporate information conferred by a trained predictive neural network have also been developed. These predictive tools can assess the degree of complete ribosomal blockage or diversion of translation into alternative frames. Feedback from the predictive models can be used to either (1) rank complete sPK candidates following thermodynamic design or (2) directly adjust the frequencies of nucleotide selection during initial generation. This approach allows for predictive experimental performance to directly influence the sPK design process, a feature that is unavailable through any other methods in the literature.

[0277] PK algorithm steps summarized1. Determination of a target secondary structure, (3 hairpin, etc.)2. Sampling of nucleotide sequences within individual hairpins3. Supervised sampling to prevent the inclusion of stop codons in multiple frames4. Pooling of individual hairpin components into a single test tube simulation5. Thermodynamic based ranking of cross-talk between individual hairpin components6. Concatenation of low-crosstalk hairpins into a single PK structure7. Ranking of the degree of blockage / firameshifting using a deep learning model8. Multi-frame translational blockage with de novo pseudoknots

[0278] We initially utilized the 3 hairpin, h-type PK structure of the SARS-CoV-2 PK as a target for design due to the extensive body of literature studying this element (8-13). We evaluated a library of 12 CoV-like sPK structures featuring hairpins ranging in length from 9-15 nucleotides (see, Tables 2 and 3). These constructs were placed into a dual reporter assay which utilized mCherry as a transfection marker and a GFP coding sequence placed into each of the three reading frames. The degree of GFP fluorescencemeasured using this reporter indicated the fraction of translational events that were diverted into each frame. As demonstrated in (Fig. 2), the sPKs uniformly resulted in no GFP expression in the 0 frame, while a subset of 5 PK structures demonstrated active -1 expression. When considering the complete deactivation of expression demonstrated by a subset of our library with the published results of PK- induced ribosomal pausing, we have concluded that sPKs returned by our algorithm can completely decouple a ribosome from an actively translating transcript.

[0279] The sPK constructs that resulted in no expression across each of the three reading frames represent an exciting new biological motif for controlling protein expression. Given the widespread success of RNA-based vaccines and therapeutics, extensive work has been conducted to develop mechanisms that control translation activation at the RNA level. The unifying objective of these approaches is to elicit precise control of the temporal or spatial profile of a therapeutic candidate in the most compact manner possible. Throughout each of these studies, one of the most glaring engineering challenges remains the design of an RNA that is deactivated in its default state and reactivated only within the specific context in which the therapeutic would be beneficial. A successful version of this design paradigm could bypass most of the hurdles presented in RNA platform development, including off-target expression and innate immune activation. The silencing effects of our sPK library, which occupy only ~ 100 nt of space within a transcript, pose an elegant potential solution to this problem. Furthermore, our algorithmic approach that confers complete control of the nucleotide composition within these structures provides an excellent starting point for engineering programmatic activation of translation using sPK elements as templates.

[0280] Slip sequences significantly impact PK-induced effects

[0281] To understand the relationship between sPK-induced modulation and the contents of the slippery sequence, a number of experiments were conducted. First, -1 expression driven by the wild-type SARS-CoV-2 PK element was assessed with the slip sequence excised from the reporter. A comparison to the natural context reveals the complete abrogation of frameshifting in the absence of the AAAUUUC slip site (Fig. 4), indicating the essential role of this motif in the diversion of the ribosome. To assess the degree of dependence of our sPK constructs on the slippery sites, the canonical, A-T rich elements were replaced with alternative motifs adhering to the X-XXY-YYZ sequence pattern. These experiments revealed a range of effects on resulting expression, with each variant decreasing overall translation (Fig. 5).

[0282] In some cases, replacement of the natural SARS-CoV-2 element demonstrated a modest reduction in expression ranging from 10-20% (Fig. 5). In contrast however, PK 11 showed a complete reduction in expression across different reading frames under the replacement of the canonical slippery site. Both results reveal that a degree of programmable expression can be conferred by considering the precise combination of both stimulatory structure and slippery sequence.

[0283] Structural perturbations can reactivate translation

[0284] Following our results highlighting the importance of the slip sequence, we next assessed how the different structural subcomponents of the H-type sPK architecture contributed to the overall blocking or frameshifting effect. The initial sPK library, which each contained 3 distinct hairpins and loops, was modified to contain different combinations of the initial hairpin sequences. Sequences were placed into our 0-frame GFP reporter to assess the degree of expression that resulted from the structurally manipulated versions compared to the full-length original sPK templates. In this context the removal (delta) of stems 1 or 3 creates a more compact 2 hairpin PK structure, while the removal of stem 2 returns a double hairpin structure lacking a pseudoknot motif. Many of our constructs manipulated under these schedules did not demonstrate a significant change in 0-frame expression, indicating the robustness of translational suppression offered through a slippery sequence and a downstream stimulatory structure. When compared to the original sPK 10 expression, however, the deletions of hairpin 1 and 3 of sPK 10 each restored expression leading to over a 200-fold increase in 0-frame expression (Fig. 6). The removal of stem 2 of sPK 8 additionally reactivated 0-frame expression by over 50-fold, indicating that removal of each of the programmable components within our constructs can lead to translational reactivation. These results highlight the degree of difficulty imposed by the design of highly dynamic sPK constructs, and the importance of a computational framework capable of rapidly returning new sPK candidates with targeted structural profiles.

[0285] RNA-mediated reactivation of translation

[0286] After demonstrating the potential impact of individual hairpin deletion on sPK performance, we sought to investigate if programmable RNA-RNA interactions could demonstrate a similar effect.

[0287]

[0288] In this setting, RNA sequences perfectly complementary to the 2nd stem of each sPK were placed into the 3’ untranslated region of our dual reporter. In cellular contexts, this complementary region would be expected to hybridize to the 3’ end of stem 2, thereby unwinding the hairpin. Importantly, unwinding of stem 2 affects the overall knotted structure of each sPK, leaving only a double hairpin structure when in the target-bound conformation. This less-complex double hairpin would not be expected to block the ribosome as effectively as the full sPK, thereby reactivating some degree of translation. In alignment with this hypothesis, four of the original 12 sPKs examined in this manner showed substantial protein reactivation, ranging from 100- to 175-fold compared to the target-free context.

[0289] While these initial experiments utilized complementary RNA targets placed within the same transcript as the sPK, this process could be generalized for trans-acting RNA targets. Our algorithm enables complete control over the nucleotide contents of the sPK hairpins, and could allow us to change the hairpins to selectively respond to any RNA input. These target sequences could be endogenous transcripts, micro RNAs, or any other form of coding- or non-coding RNA sequence, and could indicate cell type, disease state, cellular localization, or any biological state differentiable using RNA or RNA-related factors. The rich landscape of biological variation that can be described using RNA will allow the sPK system to exhibit unprecedented precision for manipulating living systems.

[0290] Many structures can modulate translation downstream of a slippery sequence

[0291] Following our experiments of sPK translational regulation, we sought to investigate if simple hairpin structures could demonstrate a similar effect. Hairpin elements have been proven to stimulate frameshifting in naturally occurring systems (14), however, a direct comparison of the effects of PK and hairpin structural elements downstream of identical slippery and spacer sequences has not been characterized. We created a library by designing hairpins ranging in length from 6 to 18 nt in size and by varying their position relative to the slippery sequence from 3 to 54 nt, encompassing the entire footprint of our sPK designs. This library was again examined in our dual reporter system, with mCherry and GFP fluorescent signals used to assess total and frameshifted translation, respectively. While a handful of examples fully repressed 0-frame expression, the remaining elements of the library showed a wide range of repressive effects (Fig. 7). Importantly, the consensus in the literature would suggest that these hairpins are insufficiently structured to disrupt the helicase activity of the ribosome (14-15). It is highly likely that these regulatory effects are driven by an essential combination of the structured element and a sufficient slippery sequence.

[0292] The subset of hairpin elements demonstrating a partial regulation of downstream 0-frame GFP expression offer an avenue for precise regulation of multiple protein coding sequences within the same transcript. This regulation is not dependent on any intermolecular interactions, offering a compact and straightforward mechanism for exhibiting this effect. Multicistronic vectors have shown utility in a range of therapeutic applications including the production of multiple antigens for vaccination and combination immunotherapy (16-19). However, available techniques for co-delivering multiple RNA vectors struggle in generalization due to cell type specific effects or a lack of a tunable molecular mechanism. These hairpins demonstrate a robust modulatory effect without either of these drawbacks, indicating their potential for immediate utility in these settings.

[0293] Those hairpins which demonstrated complete blockage of translation offer a promising alternative avenue for RNA-dependent translational control. Unlike the sPK system, which has no available methods to account for intermolecular interactions, standard thermodynamic tools can be readily deployed to predict RNA-RNA impacts on hairpin structure formation. A target RNA molecule could be designed using these tools to selectively unwind the repressing hairpin, thus restoring ribosomal progression. The applications of this system overlap directly with the proposed results for our sPK system, but could be designed using commercially available software tools. Given the higher rate of 0- frame expression demonstrated by most of the library, further investigation is warranted to identify the specific features that contribute to complete hairpin-induced blockage.

[0294] Activation of a PK / hairpin in response to an RNA for repression of translation

[0295] The subset of our sPK constructs that demonstrated active frameshifting into the -1 frame offer a programmable mechanism for selectively repressing translation. As demonstrated in our priorexperiments, the structural conformation of the sPK can be modulated by a complementary target RNA. This alternative approach will enable the construction of NOT logical operations aided by an sPK. In the default conformation the fully formed construct will divert ribosomal progression into the -1 frame, resulting in detectable translational outputs. Upon binding of the complementary target, the sPK will unwind, thus removing -1 activity and silencing output expression. The hairpins which demonstrated partial 0-frame readthrough could similarly be deployed for NOT logic. An additional structural engineering approach that could exhibit this effect would be to sequester the knot-forming stem 2 of an sPK into a downstream, larger hairpin. In the default state this would shift the structural profile into a series of hairpins rather than allowing the critical stem 2 region to form the knotted structure and likely allowing the ribosome to progress unperturbed. The nucleotide contents of the sequestering hairpin could be programmatically determined by a target RNA, allowing the selective release and reformation of the full, blocking sPK in the presence of the target. The behavior of this system would similarly invert the relationship between protein output and target demonstrated in the default sPK system.

[0296] The sPKs which demonstrated complete blockage could additionally be converted into a NOT operator by the engineering of an additional RNA-gated structure. These alternative structures will sequester the nested region of the sPK into a downstream hairpin, thereby resulting in a complex where the complete sPK is not formed. Without the complete sPK the default progression through this transcript will result in protein expression. Upon introduction of a complementary target, the sequestered portion of the sPK will be released, allowing the completed structure to form and translation to be completely blocked. These constructs will be evaluated once again in our dual-reporter system and subjected to both cis- and trans-acting complementary RNAs.

[0297] Multi-PK systems for AND logic (activator) and NOR logic (repressor)

[0298] Engineering sPK and hairpin elements to respond only to their specific target input offers the ability to concatenate several independent elements into multi-input gated logical operations. In one example, multiple sPKs would be placed directly adjacent to each other upstream of a protein of interest. Each element would independently block ribosomal progression in the absence of the unique target RNA, with both targets required to sufficiently restore translation. The presence of either individual target alone would leave the other sPK in the locked conformation, preventing the progression of the ribosome. Alternatively, a single or combination unit of sPK elements could be placed upstream of multiple coding sequences on the same transcript, offering orthogonal mechanisms for regulating multiple protein outputs. Our proposed inverse logical mechanism which couples sPKs into repressive hairpins could similarly be concatenated to offer multi-input control. Finally, the constructs that demonstrated -1 activation or partial 0-frame expression could be placed upstream of independent output proteins, expanding precise stoichiometric control beyond only 2 reporters.

[0299] Evading translational repression effects by placing the target interaction domain into the 3’ UTR

[0300] Similar technologies which couple programmable RNA targets to the activation of translation suffer from a conserved roadblock imposed by the formation of double stranded RNA. Switch-targethybrids placed within the coding region of a transcript have been shown to reduce overall translation levels (20-21), reducing the dynamic range offered by these approaches. To circumvent this limitation, the cis-acting approach could be engineered to place the RNA-RNA duplex within the 3’ untranslated region (UTR) of the switch transcript. By placing the sequence complementary to stem 2 with the loop of a repressing hairpin located in the 3’ UTR, the sPK will remain in the locked state, silencing translation. The UTR hairpin would instead be designed using thermodynamic software to unwind in the presence of the target, releasing the stem 2 complement and disrupting total sPK formation. This would place the majority of RNA-RNA interactions into the 3’ UTR of the transcript, which would mitigate the repressing effects of this complex on translation.

[0301] Modulation of saRNA vector systems.

[0302] The sPK system could be deployed in several ways to regulate the self-amplifying RNA (saRNA) platform. The replication cycle of saRNA vectors entails the propagation of an RNA cargo to produce many downstream copies, requiring the initial production of 4 non-structural proteins encoding an RNA-dependent RNA polymerase (RdRp). An sPK coupled to a specific target could regulate this initiation step, preventing the production of the replication machinery until a specific biological context is encountered. Alternatively, the transcription of the RdRp itself could be silenced by the inclusion of an sPK and sufficient pause sequence; both poly A and poly U motifs have been demonstrated to initiate RdRp slippage and could directly replace the ribosomal slip sequence. Finally, the saRNA system creates many viable copies of a complete cargo protein. Placing an sPK into the UTR of a cargo sequence would enable the propagation of this regulatory motif to the downstream replication products. The large degree of amplification demonstrated by the saRNA platform represents a promising avenue for increasing the dynamic range of the sPK system.

[0303] When sPKs are positioned in the 5' or 3' UTR in self-amplifying RNA (saRNA), they can block or partially inhibit RNA-dependent RNA polymerase (RdRp) activity, halting or reducing replication. As a result, payload expression can be selectively turned off or tuned. Similarly to sPKs placed in the 5 ’UTR of mRNA, translation can be turned on through annealing of an RNA target of interest to the saRNA to disrupt the inhibitory sPK.

[0304] Modulation of additional enzymes

[0305] While the slippery sequences covered in this work are specific to the ribosome, many other nucleic acid-affecting enzymes could be regulated using a combination of pause sequences and stimulatory secondary structures returned by this framework. One notable example of this could be the pairing of a poly ‘A’ or poly ‘U’ sequence that has been reported to initiate slippage of transcription to an sPK structure. In a similar manner to our previous reactivation experiments, the formation of these transcriptional blocking sPKs could be regulated using a target RNA molecule. This approach should be directly amenable to the modulation of either RNA- or DNA-dependent polymerases and could offer an orthogonal approach for controlling the flow of genetic information within living systems.

[0306] Referencesill, C. H. & Brierley, I. Structural and Functional Insights into Viral Programmed RibosomalFrameshifting. Annual Review of Virology 10, 217-242 (2023). hen, J. et al. Dynamic pathways of-1 translational frameshifting. Nature 512, 328-332 (2014).nzalone, A. V., Lin, A. J., Zairis, S., Rabadan, R. & Cornish, V. W. Reprogramming eukaryotic translation with ligand-responsive synthetic RNA switches. Nat Methods 13, 453-458 (2016). ikl, M., Pilpel, Y. & Segal, E. High-throughput interrogation of programmed ribosomal frameshifting in human cells. Nat Commun 11, 3061 (2020). aliskan, N., Katunin, V. I., Belardinelli, R., Peske, F. & Rodnina, M. V. Programmed -1Frameshifting by Kinetic Partitioning during Impeded Translocation. Cell 157, 1619-1631 (2014). im, H.-K. et al. A frameshifting stimulatory stem loop destabilizes the hybrid state and impedes ribosomal translocation. Proceedings of the National Academy of Sciences 111, 5538- 5543 (2014). holstrup, J., Oddershede, L. B. & Sorensen, M. A. mRNA pseudoknot structures can act as ribosomal roadblocks. Nucleic Acids Research 40, 303-313 (2012). ength-dependent motions of SARS-CoV-2 frameshifting RNA pseudoknot and alternative conformations suggest avenues for frameshifting suppression | Nature Communications.ryo-EM and antisense targeting of the 28-kDa frameshift stimulation element from the SARS-CoV-2 RNA genome | Nature Structural & Molecular Biology. Schlick, T., Zhu, Q., Jain, S. & Yan, S. Structure-altering mutations of the SARS-CoV-2 frameshifting RNA element. Biophys J 120, 1040-1053 (2021). Roman, C., Lewicka, A., Koirala, D., Li, N.-S. & Piccirilli, J. A. The SARS-CoV-2 Programmed -1 Ribosomal Frameshifting Element Crystal Structure Solved to 2.09 A Using Chaperone- Assisted RNA Crystallography. ACS Chem. Biol. 16, 1469-1481 (2021). Ritchie, D. B., Soong, J., Sikkema, W. K. A. & Woodside, M. T. Anti-frameshifting Ligand Reduces the Conformational Plasticity of the SARS Virus Pseudoknot. J. Am. Chem. Soc. 136, 2196-2199 (2014). Bhatt, P. R. et al. Structural basis of ribosomal frameshifting during translation of the SARS- CoV-2 RNA genome. Science 372, 1306-1313 (2021). Qu, X. et al. The Ribosome Uses Two Active Mechanisms to Unwind mRNA During Translation. Nature 475, 118-121 (2011). Bao, C. et al. mRNA stem-loops can pause the ribosome by hindering A-site tRNA binding. eLife 9, e55799. Tsuchihashi, Z. Translational frameshifting in the Escherichia coli dnaX gene in vitro. Nucleic Acids Res 19, 2457-2462 (1991). Shaimardanova, A. A. et al. Production and Application of Multicistronic Constructs for Various Human Disease Therapies. Pharmaceutics 11, 580 (2019).18. kgi-admin. CSL Ltd Patent Filing: Multicistronic Self-Replicating RNA Vaccine. Pharmaceutical Technology (2024).19. Fallot, S. et al. Alternative-splicing-based bicistronic vectors for ratio-controlled protein expression and application to recombinant antibody production. Nucleic Acids Res 37, el34 (2009).20. Tuschl, T., Zamore, P. D., Lehmann, R., Bartel, D. P. & Sharp, P. A. Targeted mRNA degradation by double-stranded RNA in vitro. Genes Dev 13, 3191-3197 (1999).21. Gayet, R. V. et al. Autocatalytic base editing for RNA-responsive translational control. Nat Commun 14, 1339 (2023).22. Pfeifer, B. A., Beitelshees, M., Hill, A., Bassett, J. & Jones, C. H. Harnessing synthetic biology for advancing RNA therapeutics and vaccine design, npj Syst Biol Appl 9, 1-10 (2023).EXAMPLE 2

[0307] Pseudoknot blockage of additional enzymes

[0308] Although the slippery sequences covered in this work are specific to the ribosome, many other nucleic acid-affecting enzymes could be regulated using a combination of pause sequences and stimulatory secondary structures returned by this framework. One notable example of this could be the pairing of a poly ‘A’ or poly ‘U’ sequence that has been reported to initiate slippage of transcription with an sPK structure. In a similar manner to our previous reactivation experiments, the formation of these transcriptional blocking sPKs could be regulated using a target RNA molecule. This approach should be directly amenable to the modulation of either RNA- or DNA-dependent polymerases and could offer an orthogonal approach for controlling the flow of genetic information within living systems.

[0309] Enzymes such as xml facilitate the degradation and recycling of messenger RNA transcripts by scanning sequences in a 5’ to 3’ or 3’ to 5’ direction, cleaving RNA into non-functional byproducts as they progress. Several viruses have evolved pseudoknot motifs within their genomes which sterically halt the progression of these enzymes, thereby increasing their occupancy time on an individual transcript (1- 4). Increased dwell times on individual transcripts prevent efficient xml degradation of the remaining viral genomic copies within the cellular environment, improving the global translational capacity of the viral genome. This principle has been applied in biotechnology engineering applications by inserting naturally occurring pseudoknot motifs into therapeutic payloads, demonstrating an increase in duration and clinical effectiveness (5). We hypothesized that using our same pseudoknot design framework, we could create synthetic pseudoknots with improved properties, which diverged greatly in sequence content from existing examples while conferring pronounced degradation resistance.

[0310] To this end we developed two different sPK libraries. Both algorithms used the following steps to return new sPK examples:1) Determination of a nested target secondary structure, (3 hairpin, etc.) from either a naturally occurring template or synthetic baseline2) Sampling of nucleotide sequences within individual hairpins3) Supervised sampling to prevent the inclusion of stop codons in multiple frames4) Pooling of individual hairpin components into a single test tube simulation5) Ranking the optimal combination of hairpins to assemble into a complete molecule7) Filtering the returned outputs for optimal experimental attributes under the supervision of a predictive model.

[0311] For library synA, steps 4-7 are conducted using thermodynamic tools to predict attributes such as the secondary structure, cross-talk, ensemble defect, minimum free energy, conformational entropy, or other attribute to select individual nucleotides within hairpins, assemble hairpins together, and rank final outputs. For library synB, a trained neural network predictor and data structure encoding the neural network’s preferences were utilized for the selection processes. These networks can predict similar thermodynamic attributes as synA, or can be generalized to consider experimental attributes such as translational blockage, degradation resistance, firameshifting or other properties to guide nucleotide selection, hairpin combinations, assembly, and final ranking.

[0312] We placed our sPK libraries into the 5’ and 3’ untranslated regions (UTRs) of an mRNA encoding a fluorescent protein reporter (mCherry) and measured the resulting expression observed over time to evaluate this idea (Fig. 9A). The sPKs exhibit ribosome blocking effects in the 5’UTR with a stronger blockage effect being demonstrated with the synB library (Fig. 9B). While the entire synB library showed significantly lower mRNA translation compared to the naturally occurring SARS-CoV-2 PK (COV) in the 5’UTR, the synA library showed higher mRNA translation compared to COV. This shows that the synA library provides stability to the mRNA in the 5’UTR, as was our objective. While the 5’ UTR sPKs show ribosomal blocking, particularly the synB library, this opens up the possibility of controlling translation through the sPKs. Target RNAs could bind and unwind the blocking sPK allowing for translation, but when that target RNAs are not present, translation will be silenced. Additionally in line with our objectives, the sPK library showed a pronounced improvement in mRNA translation at a time point of 48 hours post DNA transfection compared to the reporter designed by a commercial provider (Twist Biosciences) when placed in the 3 ’UTR. Additionally, both the synA and synB libraries show improvement in mRNA translation compared to a wild type structural analogue. The design objectives of our algorithm strictly enforced the nested base pairing relationships characteristic of valid pseudoknots while maximizing the conformational entropy of each design. To validate the impact of both attributes on resulting performance, we further created two distinct controls to isolate each effect. The first, ult low, was created to exhibit minimal conformational entropy, while the second, ult high, exhibited maximal entropy but did not exhibit a nested base pairing structure. Both the synA and synB libraries improve expression compared to these examples, highlighting the importance of both design objectives to achieve performance improvements.

[0313] To build on the improvement in expression sustained in the 3’UTR, we placed several copies of different sPKs into the same 3’UTR, demonstrating the synergistic enhancement achievable through theircombination (Fig. 10B and Fig. 11A). Importantly, pairing the COV PK in series with our synthetic constructs shows a dramatic improvement compared to the COV template in isolation, indicating the importance of our algorithmic design principles in demonstrating this effect. Additionally, we also assessed the combination placement of individual sPKs into the 5’ and 3’ UTRs of the same transcript, revealing a similar degree of improvement (Fig. IOC and Fig. 1 IB). These results indicate that our sPK constructs reliably halt the progression of diverse classes of RNA-processing enzymes, unlocking further engineering applications for these tools. Furthermore, expanding to using multiple sPKs in the 5’UTR could also lead to further stabilization or blockage of the ribosome as well as having multiple sPKs in the 5’UTR and 3’UTR of the same mRNA (Fig. 10A, 10D).

[0314] Circular RNA (circRNA) has emerged as one of the most robust RNA scaffolds for sustaining long-term transcript stability and payload expression (6). To directly compare the stability of our sPK designs to this standard, we constructed a circRNA to express the same mCherry reporter as the sPKs. We further benchmarked our constructs against MALAT1, a naturally occurring 3’UTR element known to enhance mRNA stability, by inserting the MALAT1 sequence downstream of the same mCherry reporter. We then quantified the fluorescence output from 22 to 114 hours (4.75 days) post RNA transfection to evaluate long-term expression dynamics (Fig. 12A, 12B). In line with our hypothesis, transcripts containing sPK elements in both the 5’ and 3’ UTRs showed improvement in stability and translational output, outperforming both circRNA and MALAT1 constructs. Interestingly, when sPK-containing transcripts were synthesized with N1 -methylpseudouridine (NlmT) in place of uridine, we observed a marked reduction in stability, consistent with disruption of proper pseudoknot structure formation (Fig. 12C). This result further validates the mechanistic basis of sPK function, demonstrating that their stabilizing effect is contingent upon correct structural folding. Collectively, these findings indicate that our sPK constructs reliably halt the progression of diverse RNA degradation pathways and represent a significant advance over current mRNA stabilization strategies.

[0315] Finally, we extended our evaluation to self-amplifying RNA (saRNA) systems to investigate the impact of sPK elements on saRNA stability and replication dynamics. We inserted sPKs into both the 5’ and 3’ UTRs of the subgenomic strand of an saRNA construct encoding the mCherry reporter and monitored fluorescence from 22 to 114 hours post-transfection (Fig. 13B, 13C). Analysis of the live-cell population revealed a reduction in overall transfection efficiency for the sPK-modified constructs relative to the positive control, as indicated by a lower percentage of mCherry-positive cells and diminished bulk fluorescence intensify (Fig. 14A-14C). Notably, when we examined the mCherry-positive population, fluorescence intensify was comparable between sPK-containing constructs and the positive control (Fig. 14B, 14D). These findings suggest that the presence of sPKs partially impedes RNA-dependent RNA polymerase (RdRp), reducing the number of transcripts undergoing successful replication. In principle, unwinding of the sPK structures using a complementary target RNA could relieve RdRp inhibition and enable cell-type-specific saRNA replication. Nonetheless, for transcripts that are successfully replicated, sPK elements appear to stabilize the resulting subgenomic RNA, enabling expression levels on par withcontrol constructs. Additionally, these data open up many configurations such as having an xml-blocking sPK downstream of the SGP in a subgenomic strand with an IRES driving the reporter in order to enhance payload expression.

[0316] Given the demonstrated sensitivity of the sPKs to modified bases, it may be possible to engineer saRNA constructs with sPKs that have different effects depending on if they are the transfected strand (i.e. one with modified bases) or a replicated strand (i.e. one without modified bases or different modified bases). For instance, an sPK could be inserted into the 3’ UTR of a saRNA transcribed with modified bases that prevent its folding and abolish its RdRp inhibitory effects. However, once additional sense saRNA and subgenomic strands are transcribed by the RdRp in the cell with unmodified bases, the sPK can regain its activity to enhance transcript lifetime and suppress antisense strand production.

[0317] The sPKs can also be shifted around the saRNA strand to provide different effects. A sPK after the non-structural protein 4 (nsp4) and prior to the SGP would act as a translational blocker in order to prevent readthrough translation after nsp4 and reduce any expression in off-target cells (Fig. 13D). Placing a sPK in the 5 ’UTR prior to the 5’CSE or after the 5’CSE and before non-structural protein 1 (nspl) could provide additional blockage of the RdRp until a target disrupts the sPK to allow for translation (Fig. 13E, 13F). Conversely, a sPK in these locations could also stabilize the initial saRNA strand in the cell by blocking xml proteins. This would also apply to having a sPK in the 3 ’UTR after the 3’CSE (Fig. 13G). Combinations of sPKs could also be added at different sites in the saRNA, and more than one sPK could be added at the same site in series to enhance the effect of the sPKs.

[0318] Several viral genomes have pseudoknotted structures which could be redesigned using this method, including, but not limited to, the following list of viruses: Exemplary viral plant and arthropod genomes that have PK include Dengue vims 1, Dengue vims 2, Dengue vims 3, Dengue vims 4, Zika vims, West Nile vims, Kunjin vims, Yellow fever vims, Japanese encephalitis vims, Tick-bome encephalitis vims, Powassan vims, Saint Louis encephalitis vims, Murray Valley encephalitis vims, Usutu vims, Ilheus vims, Langat vims, Louping ill vims, Kyasanur Forest disease vims, Omsk hemorrhagic fever vims, Alkhurma hemorrhagic fever vims, Wesselsbron vims, Spondweni vims, Modoc vims, Rio Bravo vims, Apoi vims, Montana myotis leukoencephalitis vims, Red clover necrotic mosaic vims, Sweet clover necrotic mosaic vims, Potato leafroll vims, Maize chlorotic mottle vims, Maize yellow dwarf vims-RMV, Cereal yellow dwarf vims-RPV, Beet necrotic yellow vein vims, Beet soil- borne mosaic vims, Cucumber mosaic vims, Peanut stunt vims, Tomato aspermy vims, Gayfeather mild mottle vims, Sweet potato C6 vims, Darwin betaflexivirus, Panax ginseng flexivirus 1, Potato mop-top vims, Tobacco rattle vims, Lamium mild mosaic vims, Bellflower veinal mottle vims, and Opium poppy mosaic vims. Exemplary viral human and animal genomes that have PK include Epstein-Barr vims, Kaposi’s sarcoma-associated herpesvirus, Human papillomavirus 16, Hepatitis B vims, Human cytomegalovirus, Herpes simplex vims 1, Merkel cell polyomavims, Trichodysplasia spinulosa polyomavims, Marek’s disease vims, Human adenovirus 5, Murine cytomegalovirus.

[0319] References1. Langeberg, C. J., Szucs, M. J., Sherlock, M. E., Vicens, Q. & Kieft, J. S. Tick-bome flavivirus exoribonuclease-resistant RNAs contain a double loop structure. Nat Commun 16, 4515 (2025).2. Dilweg, I. W., Gultyaev, A. P. & Olsthoom, R. C. Structural features of an Xml-resistant plant vims RNA. RNA Biol 16, 838-845 (2019).3. Chapman, E. G., Moon, S. L., Wilusz, J. & Kieft, J. S. RNA structures that resist degradation by Xml produce a pathogenic Dengue vims RNA. eLife 3, e01892 (2014).4. Chen, X. et al. Zika vims RNA structure controls its unique neurotropism by bipartite binding to Musashi-1. Nat Commun 14, 1134 (2023).5. Zhang, G. et al. Enhancement of prime editing via xrRNA motif-joined pegRNA. Nat Commun 13, 1856 (2022).6. Litke, J.L. et al. Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts. Nat Biotechnol 37, 667-675 (2019).

Claims

CLAIMS:

1. A synthetic RNA pseudoknot (PK) comprising at least two stem-loop domains, the at least two stem-loop domains separated by a loop domain, wherein formation of the PK results in overlapping base pair interactions.

2. In one embodiment, wherein each stem-loop domain is modified such that it comprises a nucleotide sequence lacking in-frame stop codons3. The synthetic RNA PK of claim 1, wherein the PK is configured to block translation in a 0 frame while permitting translation in a -1, +1, -2, or +2 frame, or the PK is configured to block translation and not permit downstream translation in any frame.

4. The synthetic RNA PK of claim 1, wherein the PK is incorporated into a self-amplifying RNA vector, a circular RNA vector, a messenger RNA, or a non-coding RNA.

5. The synthetic RNA PK of claim 1, wherein hybridization of a sequence complementary to the PK causes the PK to unwind.

6. The synthetic RNA PK of any of claims 1-5, wherein the PK is chemically synthesized or recombinantly expressed.

7. The synthetic RNA PK of any of claims 1-6, wherein the at least two stem-loop domains have a sequence selected from SEQ ID NO: 1-48.

8. The synthetic RNA PK of any of claims 1-6, wherein the loop domain has a sequence selected from SEQ ID NO: 49-60.

9. The synthetic RNA PK of any of claims 1-8, wherein the synthetic PK has a sequence selected from SEQ ID NO: 61-72.

10. A synthetic RNA PK comprising, from 5’ to 3’: a first stem-loop region; a first spacer; a second stem-loop region; a second spacer; a reverse complement sequence to the first stem-loop region; a third spacer; and a reverse complement sequence to the second stem-loop region, wherein overlapping base pair interactions occur between loops and more distal stem regions.

11. The synthetic RNA PK of claim 10, where the second stem-loop region is the loop of the stem formed between the first stem-loop region and its reverse complement, and the reverse complement sequence to the first stem-loop region is the loop of the stem formed between the second stem-loop region and its reverse complement.

12. The synthetic RNA PK of claim 10 or 11, further comprising, between the third spacer, and the reverse complement sequence to the second stem-loop region, a third stem-loop region,a fourth spacer, and a reverse complement sequence to the third stem-loop region.

13. The synthetic RNA PK of any of claims 10-12, wherein at least one of spacer is removed.

14. The synthetic RNA PK of any of claims 10-13, wherein the stem-loop region and its reverse complement are not fully complementary.

15. An engineered RNA comprising a coding region sequence of a gene of interest and a secondary RNA structure, wherein the secondary RNA structure is located at or near the gene of interest and inhibits RNA processing at or near the gene of the interest.

16. An engineered RNA comprising a coding region sequence of a gene of interest and a synthetic PK of any of claims 1-14, wherein the synthetic PK is located at or near the gene of interest and inhibits RNA processing at or near the gene of the interest.

17. An engineered RNA comprising a coding region sequence of a gene of interest and a synthetic PK of any of claims 1-14, wherein the synthetic PK is located at or near the gene of interest and inhibits translation at or near the gene of the interest.

18. The engineered mRNA of any of claims 15-17, wherein at or near is upstream, downstream, or within of the coding region of a gene of interest.

19. The engineered RNA of claims 15 or 16, wherein the RNA processing is the progression or processivity of an RNA-processing or RNA-utilizing enzyme.

20. The engineered mRNA of claim 19, wherein the RNA-processing enzyme is a selected from the group consisting of: a deadenylase, a decapping enzyme, an exonuclease, an endonuclease, a surveillance enzyme, an RNA-dependent RNA polymerase, and an RNA-dependent DNA polymerase.

21. The engineered mRNA of claims 16 or 17, further comprising a sequence complementary to the PK.

22. The engineered mRNA of claim 21, wherein the sequence complementary to the PK hybridizes to the PK.

23. The engineered mRNA of claim 22, wherein hybridization to the PK causes the PK to unwind.

24. The engineered mRNA of claim 23, wherein unwinding of the PK permits translation of the gene of interest.

25. The engineered mRNA of any of claims 21-25, wherein the sequence complementary to the PK is downstream of the coding region of a gene of interest.

26. The engineered mRNA of any of claims 21-25, wherein the sequence complementary to the PK is located in the 3’ UTR of the gene of interest.

27. The engineered mRNA of any of claims 21-25, wherein the sequence complementary to the PK wherein the sequence complementary is a trans-acting RNA present in a second strand.

28. The engineered mRNA of claim 20, wherein the trans-acting RNA is an endogenous cellular transcript.

29. The engineered mRNA of claim 20, wherein the trans-acting RNA is an endogenous RNA.

30. The engineered mRNA of any of claims 15-29, further comprising at least a second coding region of at least a second gene of interest.

31. The engineered mRNA of any of claims 15-29, further comprising at least a second synthetic RNA PK.

32. A platform for modulating translation, the platform comprising: an engineered RNA comprising a coding region sequence of a gene of interest a synthetic PK of any of claims 1-14, and a sequence complementary to the PK.

33. A platform for modulating RNA processing, the platform comprising: an engineered RNA comprising a coding region sequence of a gene of interest a synthetic PK of any of claims 1-14.

34. The platform of claim 32, wherein the PK inhibits translation of the gene of interest when not bound to the sequence complementary to the PK.

35. The platform of claim 33, wherein the PK inhibits RNA processing of the gene of interest when not bound to the sequence complementary to the PK.

36. The platform of any of claims 32-35, wherein the sequence complementary to the PK hybridizes to the PK and causes the PK to unwind.

37. The platform of claim 36, wherein unwinding permits translation of the gene of interest.

38. The platform of any of claims 32-37, wherein the sequence complementary to the PK is located downstream of the coding region.

39. The platform of any of claims 32-37, wherein the sequence complementary to the PK is a transacting RNA present in a second strand.

40. The platform of any of claims 32-39, wherein the engineered RNA is an engineered RNA of any of claims 15-31.

41. A method of modulating translation in a cell, the method comprising: expressing an engineered RNA of claim any of claims 15-31, or a platform of any of claims 32-40 in a cell, wherein translation of the gene of interest is inhibited by the PK when not bound to the sequence complementary to the PK; and promoting hybridization of the sequence complementary to the PK and the PK, wherein hybridization permits translation of the gene of interest.

42. A method of modulating RNA processing in a cell, the method comprising:expressing an engineered RNA of claim any of claims 15-31 , or a platform of any of claims 32-40 in a cell, wherein RNA processing of the transcript of interest is inhibited by the PK when not bound to the sequence complementary to the PK; and promoting hybridization of the sequence complementary to the PK and the PK, wherein hybridization permits RNA processing of the transcript of interest.

43. The method of claim 41 or 42, wherein the sequence complementary to the PK is a trans-acting RNA present in a second strand and promoting hybridization comprising expressing the second strand in the cell.

44. The method of claim 41 or 42, wherein the sequence complementary to the PK is downstream of the gene of interest and promoting hybridization comprising allowing for folding of the modified mRNA such that the sequence complementary to the PK can hybridize to the PK.

45. The method of any of claims 41-44, wherein promoting hybridization is spatially and / or temporally regulated.

46. The method of any of claims 41-45, wherein the platform is expressed in the cell by an expression plasmid or a vector.

47. The method of any of claims 41-46, wherein the modified mRNA and the sequence complementary to the PK are expressed in the cell by the same expression plasmid or vector.

48. The method of any of claims 41-46, wherein the modified mRNA and the sequence complementary to the PK are expressed in the cell by separate expression plasmids or vectors.

49. The method of claim 47 or 48, wherein the vector is a non-viral vector or a viral vector.

50. An engineered RNA vector comprising: an RNA genome encoding at least one non-structural protein required for replication; a pseudoknot (PK) of any of claims 1-14 located within a region encoding the at least one non-structural proteins; and a sequence complementary to the PK, wherein the PK inhibits translation of the at least one non-structural protein when not bound to the sequence complementary to the PK.

51. The engineered RNA vector of claim 50, wherein the PK inhibits translation of the at least one non-structural proteins when not bound to the sequence complementary to the PK, thereby inhibiting RNA replication.

52. The engineered RNA vector of claim 50, wherein the hybridization of the sequence complementary to the PK and the PK permits translation of the gene of interest, thereby activating RNA replication.

53. The engineered RNA vector of claim 50, wherein the hybridization of the sequence complementary to the PK and the PK permits degradation of the gene of interest, thereby deactivating RNA replication.

54. An engineered RNA vector comprising: an RNA genome encoding at least one non-structural protein required for replication; a pseudoknot (PK) of any of claims 1-14 located within a region encoding the at least one non-structural proteins; and a sequence complementary to the PK, wherein the PK inhibits replication or degradation of the engineered RNA vector when not bound to the sequence complementary to the PK.

55. The engineered RNA vector of claim 54, wherein the PK inhibits procession of the RdRp replicating the RNA, thereby inhibiting RNA replication.

56. The engineered RNA vector of claim 54, wherein the PK inhibits procession of the RNase degrading the RNA, thereby increasing RNA lifetime and enhancing RNA replication.

57. The engineered RNA vector of claim 54, wherein the hybridization of the sequence complementary to the PK and the PK permits procession of the RdRp, thereby activating RNA replication.

58. The engineered RNA vector of claim 54, wherein the hybridization of the sequence complementary to the PK and the PK permits procession of the RNase, thereby inhibiting RNA degradation and activating RNA replication.

59. A computer-implemented method for in-silico generation of a synthetic pseudoknot RNA sequence capable of inducing a programmed translational frameshift or ribosomal stall, the method comprising: receiving, with a processor, a user-defined target secondary pseudoknot topology; decomposing the target topology into a plurality of hairpin domains; for each hairpin domain, performing supervised nucleotide sampling that excludes in-frame stop codons in each of three translation frames to generate a plurality of candidate domain sequences; evaluating, for each candidate domain sequence, thermodynamic stability and inter-domain cross-talk with a secondary-structure prediction algorithm; concatenating a combination of the candidate domain sequences having minimized interdomain cross-talk to form a complete pseudoknot candidate sequence; ranking the pseudoknot candidate sequence with respect to at least one of the thermodynamic stability and a predicted translational modulation score; and outputting at least one ranked synthetic pseudoknot sequence.

60. The method of claim 59, wherein the ranking step further comprises applying a trained neural network to predict a degree of ribosomal blockage, enzymatic stalling, or frameshifting efficiency of each pseudoknot candidate sequence.

61. The method of claim 60, wherein feedback from the trained neural network dynamically adjusts nucleotide sampling frequencies during the supervised nucleotide sampling.

62. The method of claim 59, wherein the user-defined target secondary pseudoknot topology comprises at least two overlapping hairpin loop structures.

63. The method of claim 59, wherein the supervised nucleotide sampling is performed according to user-specified target base-pair composition frequencies.

64. The method of claim 59, further comprising electronically storing the at least one ranked synthetic pseudoknot sequence in a database for subsequent chemical synthesis.

65. The method of claim 59, wherein the ranking step selects a pseudoknot candidate sequence predicted to block translation in the 0 frame while permitting translation in a -1, +1, -2, or +2 frame.

66. The method of claim 59, wherein the ranking step selects a pseudoknot candidate sequence predicted to block progression of an enzyme involved in processing or degrading RNA.

67. The method of claim 59, wherein the PK stem domains comprises -7.5 <AG < -350 kcal / mol and a computationally predicted crosstalk <1% at 37°C at 100 nM concentration of the domains.

68. An RNA-based translational regulatory element comprising: a slippery sequence configured to induce ribosomal firameshifting; and a hairpin structure positioned downstream of the slippery sequence, the hairpin structure comprising a stem and a loop, wherein the hairpin structure is configured to modulate translation of a downstream coding sequence by at least partially repressing ribosomal progression in a reading frame-dependent manner.

69. The RNA-based translational regulatory element of claim 68, wherein the hairpin structure is positioned between 3 and 54 nucleotides downstream of the slippery sequence.

70. The RNA-based translational regulatory element of claim 68 and 69, wherein the hairpin structure has a stem comprising between 6 and 75 nucleotides.

71. The RNA-based translational regulatory element of claim 68, wherein the slippery sequence is of the form XXXYYYZ (SEQ ID NO: 73), where X, Y, and Z represent individual nucleotides.

72. The RNA-based translational regulatory element of claim 68, wherein the slippery sequence is composed of modified nucleotides.

73. The RNA-based translational regulatory element of claim 68, wherein the hairpin structure is configured to completely block translation in the 0 reading frame and not permit translation in any downstream reading frame in the coding region sequence.

74. The RNA-based translational regulatory element of claim 68, wherein the hairpin structure is configured to partially repress translation in the 0 reading frame, thereby enabling tunable expression of multiple protein coding sequences from a single transcript.

75. The RNA-based translational regulatory element of claim 68, wherein the hairpin structure is designed such that its repression of translation is independent of intermolecular interactions.

76. The RNA-based translational regulatory element of claim 68, wherein the element is incorporated into a multicistronic RNA vector for the coordinated expression of multiple antigens or therapeutic proteins.

77. The RNA-based translational regulatory element of claim 68, further comprising a complementary RNA segment configured to hybridize to at least a portion of the hairpin structure, wherein hybridization of the complementary RNA segment unwinds the hairpin structure and restores ribosomal progression.

78. The RNA-based translational regulatory element of claim 77, wherein the complementary RNA segment is encoded within the same RNA molecule in a non-coding region or is provided as a trans-acting RNA.

79. The RNA-based translational regulatory element of claim 68, wherein the hairpin structure and slippery sequence are selected such that the regulatory effect is programmable by varying the sequence, length, or position of the hairpin relative to the slippery sequence.

80. The RNA-based translational regulatory element of claim 68, wherein the hairpin structure is designed using a thermodynamic prediction algorithm to achieve a desired degree of translational repression.

81. The RNA-based translational regulatory element of claim 68, wherein the hairpin structure is designed using a neural network prediction algorithm to achieve a desired degree of translational repression.

82. The RNA-based translational regulatory element of claim 68, wherein the element is used in a eukaryotic cell for the regulation of protein expression.

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