Systems and Methods for Producing RNA Constructs with Increased Translation and Stability

Info

Publication number: 20230159915
Type: Application
Filed: Nov 7, 2022
Publication Date: May 25, 2023
Applicant: The Board of Trustees of the Leland Stanford Junior University (Stanford, CA)
Inventors: Maria Barna (Stanford, CA), Kathrin Leppek (Stanford, CA), Gun Woo Byeon (Stanford, CA)
Application Number: 18/053,324

Abstract

Systems and methods for enhancing RNA translatability and stability are disclosed. Some embodiments describe RNA molecules exhibiting increased translatability and/or stability. Additional embodiments describe methods for screening RNA molecules for increased translatability and/or stability. Various embodiments utilize screening methods, including degenerative sequences to identify sequences or regions that increase the translatability and/or stability of RNA molecules.

Description

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

The current application is a divisional of U.S. patent application Ser. No. 17/463,466, filed Aug. 31, 2021, which claims priority to U.S. Provisional Patent Application No. 63/072,669, filed Aug. 31, 2020; the disclosures of which is hereby incorporated by reference in its entirety.

FIELD OF THE DISCLOSURE

The present invention relates to ribonucleic acid (RNA). More specifically, the present invention relates to systems and methods to enhance RNA translatability and assessment thereof.

INCORPORATION OF SEQUENCE LISTING

This application hereby incorporates by reference the material of the electronic Sequence Listing filed concurrently herewith. The material in the electronic Sequence Listing is submitted as an XML file entitled “06753 DIV_Seq_List.xml” created on Nov. 7, 2022, which has a file size of approximately 412 KB, and is herein incorporated by reference in its entirety.

BACKGROUND OF THE DISCLOSURE

There are multiple problems with prior methodologies of effecting protein expression. For example, introduced DNA can integrate into host cell genomic DNA at some frequency, resulting in alterations and/or damage to the host cell genomic DNA. Alternatively, the heterologous deoxyribonucleic acid (DNA) introduced into a cell can be inherited by daughter cells (whether or not the heterologous DNA has integrated into the chromosome) or by offspring.

In addition, assuming proper delivery and no damage or integration into the host genome, there are multiple steps which must occur before the encoded protein is made. Once inside the cell, DNA must be transported into the nucleus where it is transcribed into RNA. The RNA transcribed from DNA must then enter the cytoplasm where it is translated into protein. Not only do the multiple processing steps from administered DNA to protein create lag times before the generation of the functional protein, each step represents an opportunity for error and damage to the cell. Further, it is known to be difficult to obtain DNA expression in cells as DNA frequently enters a cell but is not expressed or not expressed at reasonable rates or concentrations. This can be a particular problem when DNA is introduced into primary cells or modified cell lines.

SUMMARY OF THE DISCLOSURE

This summary is meant to provide examples and is not intended to be limiting of the scope of the invention in any way. For example, any feature included in an example of this summary is not required by the claims, unless the claims explicitly recite the feature. Also, the features described can be combined in a variety of ways. Various features and steps as described elsewhere in this disclosure can be included in the examples summarized here.

In one embodiment, a method to determine RNA translatability includes obtaining a pool of RNA molecules, where each RNA molecule is uniquely encoded with a barcoding sequence and each barcoding sequence is flanked by at least one profiling sequence, transfecting a cell or cell lysate with the pool of RNA molecules, performing polysome profiling on the pool of RNA molecules to segregate RNA molecules based on the number of ribosomes bound to the RNA molecule, and isolating a first fraction from the polysome profile to generate a first set of RNA molecules showing a first level of ribosomes bound to the RNA molecules in the set of RNA molecules.

In a further embodiment, the method further includes sequencing the barcode sequence of each RNA molecule in the first set of RNA molecules to identify the presence of each RNA molecule in the first set of RNA molecules.

In another embodiment, the method further includes determining translatability of the RNA molecules associated with each barcode sequence in the fraction by identifying the prevalence of each barcode in the fraction.

In a still further embodiment, the RNA molecules are transfected into a collection of cells.

In still another embodiment, the collection of cells is selected from mammalian cells, yeast cells, bacteria cells, and plant cells.

In a yet further embodiment, the RNA molecules are added to a cell lysate.

In yet another embodiment, polysome profiling comprises adding a cell lysate to a sucrose gradient and centrifuging the sucrose gradient to segregate the RNA molecules.

In a further embodiment again, the barcoding sequence is selected from SEQ ID NOs: 115-1380.

In another embodiment again, the profiling sequence is selected from SEQ ID NOs: 1381-1382.

In a further additional embodiment, the method further includes isolating a second fraction from the polysome profile to generate a second set of RNA molecules showing a second level of ribosomes bound to the RNA molecules in the set of RNA molecules, where the first level and second level represent different amounts of bound ribosomes.

In another additional embodiment, the method further includes sequencing the barcode sequence of each RNA molecule in the first set of RNA molecules and the second set of RNA molecules to identify the presence of each RNA molecule in the first set of RNA molecules and the second set of RNA molecules.

In a still yet further embodiment, isolating a first fraction from the polysome profile includes isolating a plurality of fractions of the polysome profile, where each fraction in the plurality of fractions generates a set of RNA molecules showing a different level of ribosomes bound to the RNA molecules in that set of RNA molecules.

In still yet another embodiment, the method further includes sequencing the barcode sequence of each RNA molecule in each set of RNA molecules to identify the presence of each RNA molecule in each set of RNA molecules.

In a still further embodiment again, the method further includes generating a distribution for each RNA molecule based on the prevalence of each RNA molecule in each fraction.

In still another embodiment again, isolating a first fraction further comprises introducing a known amount of spike-in RNA molecule, wherein the spike-in RNA molecule serves as an internal reference to allow for quantification of the first set of RNA molecules.

In a still further additional embodiment, an RNA molecule for increased translation includes a 5′ untranslated region, a 3′ untranslated region, and a coding sequence, where the 5′ untranslated region is located 5′ of the coding sequence and the 3′ untranslated region is located 3′ of the coding sequence.

In still another additional embodiment, wherein the coding sequence codes for a peptide of interest.

In a yet further embodiment again, the 5′ untranslated region is selected from SEQ ID NOs: 1-55 and SEQ ID NOs: 81-111.

In yet another embodiment again, the 3′ untranslated region is selected from SEQ ID NOs: 56-80.

In a yet further additional embodiment, the RNA molecule further includes a barcode sequence located 3′ of the coding sequence and at least one profiling sequence adjacent to the barcode sequence.

In yet another additional embodiment, the barcode sequence is selected from SEQ ID NOs: 115-1380 and the profiling sequence is selected from SEQ ID NOs: 1381-1382.

In a further additional embodiment again, a method to determine RNA stability includes obtaining a pool of RNA molecules, where each RNA molecule is uniquely encoded with a barcoding sequence and each barcoding sequence is flanked by at least one profiling sequence, treating the pool of RNA molecules under an experimental condition, and isolating the pool of RNA molecules at a specified timepoint to generate a fraction of RNA molecules showing stability under the experimental condition for the specified timepoint.

In another additional embodiment again, the method further includes sequencing the barcode sequence of each RNA molecule in the fraction to identify the presence of each RNA molecule in the fraction of RNA molecules.

In a still yet further embodiment again, the method further includes determining stability of the RNA molecules associated with each barcode sequence in the fraction by identifying the prevalence of each barcode in the fraction.

In still yet another embodiment again, the treating step includes transfecting the pool of RNA molecules into a collection of cells.

In a still yet further additional embodiment, the collection of cells is selected from mammalian cells, yeast cells, bacteria cells, and plant cells.

In still yet another additional embodiment, the treating step includes adding the pool of RNA molecules to a cell lysate.

In a yet further additional embodiment again, the treatment condition is selected from temperature, pH, presence of certain molecules, presence of certain ions, concentration of certain molecules, concentration of certain ions, irradiation, buffer type, and buffer concentration.

In yet another additional embodiment again, the method further includes size selecting for full-length RNA molecules.

In a still yet further additional embodiment again, size selecting includes performing reverse transcription PCR to transcribe a region from each into cDNA, wherein the region is selected from a full-length mRNA, a full-length CDS, a 5′UTR-CDS, a 3′UTR-CDS, and the barcode.

In still yet another additional embodiment again, the isolating step further includes isolating the pool of RNA molecules at a second specified timepoint to generate a second fraction of RNA molecules showing stability under the experimental condition for the specified timepoint.

In another further embodiment, isolating the pool of RNA molecules further includes introducing a known amount of spike-in RNA molecule, where the spike-in RNA molecule serves as an internal reference to allow for quantification of the fraction of RNA molecules.

In still another further embodiment, a method for identifying RNA molecules possessing increased translatability and stability includes obtaining a pool of RNA molecules, where each RNA molecule is uniquely encoded with a barcoding sequence and each barcoding sequence is flanked by at least one profiling sequence, assessing translatability of the pool of RNA molecules by transfecting a cell or cell lysate with a first subset of the pool of RNA molecules, performing polysome profiling on the first subset of the pool of RNA molecules to segregate RNA molecules based on the number of ribosomes bound to the RNA molecule, and isolating a fraction from the polysome profile to generate a first set of RNA molecules showing a first level of ribosomes bound to the RNA molecules in the set of RNA molecules, and assessing stability of the pool of RNA molecules by treating a second subset of the pool of RNA molecules under an experimental condition, and isolating a fraction from the second subset the pool of RNA molecules at a specified timepoint to generate a second set of RNA molecules showing stability under the experimental condition for the specified timepoint.

In yet another further embodiment, the method further includes sequencing the barcode sequence of the first set of RNA molecules and the second set of RNA molecules to identify the presence of each RNA molecule in each fraction of RNA molecules.

In another further embodiment again, the method further includes determining translatability and stability of the RNA molecules associated with each barcode sequence in the first set of RNA molecules and the second set of RNA molecules by identifying the prevalence of each barcode in each fraction of RNA molecules.

In another further additional embodiment, the barcoding sequence is selected from SEQ ID NOs: 115-1380.

In yet another further additional embodiment, the profiling sequence is selected from SEQ ID NOs: 1381-1382.

In yet again another further additional embodiment, a method to select for RNA elements includes obtaining a library of RNA molecules, where each RNA molecule comprises a coding sequence, a 5′ untranslated region (5′UTR), and a 3′ untranslated region (3′UTR), where one of the coding sequence, the 5′UTR, or the 3′UTR comprises a degenerate region, assessing a property of the library of RNA molecules, where the property is selected from translatability, in vivo stability, and in vitro stability, and selecting an RNA molecule from the library of RNA molecules showing increase in the property over other RNA molecules in the library of RNA molecules.

In yet another further additional embodiment again, the method further includes sequencing the selected RNA molecule.

In a yet further additional embodiment, the selected RNA molecule is a pool of RNA molecules.

In yet again another further embodiment, the method further includes reassessing the property of the pool of RNA molecules, and selecting an RNA molecule from the pool of RNA molecules showing increase in the property over other RNA molecules in the pool of RNA molecules.

In again another yet further additional embodiment, the method further includes sequencing the selected RNA molecule from the pool of RNA molecules.

In yet again another yet further additional embodiment, the property is translatability.

In yet another yet further additional embodiment again, the degenerate region is selected from a deletion, a random sequence, an ambiguous sequence, and a truncation.

The foregoing and other objects, features, and advantages of the disclosed technology will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

The description and claims will be more fully understood with reference to the following figures and data graphs, which are presented as exemplary embodiments of the invention and should not be construed as a complete recitation of the scope of the invention.

FIG. 1A illustrates a generalized structure of RNA molecules in accordance with various embodiments of the invention.

FIG. 1B illustrates a method for performing iterative selection of RNA elements to enhance translatability and/or stability in accordance with various embodiments of the invention.

FIG. 2 illustrates a method to screen RNAs for increased translatability in accordance with various embodiments of the invention.

FIG. 3 illustrates a method to screen RNAs for increased in vivo stability in accordance with various embodiments of the invention.

FIG. 4 illustrates a method to screen RNAs for increased in vitro stability in accordance with various embodiments of the invention.

FIG. 5A illustrates a method to screen a pool of RNAs for stability and/or translatability in accordance with various embodiments of the invention.

FIG. 5B illustrates a method to identify RNAs possessing increased translatability and/or stability in accordance with various embodiments of the invention.

FIGS. 6A-6C illustrate exemplary data of a heatmap showing RNA presence in various fractions after polysome profiling. FIG. 6A illustrates a full view of the heatmap; while FIGS. 6B-6C illustrate enlarged views of the heatmap of FIG. 6A.

FIGS. 7A-7C illustrate exemplary data in the form of box and whisker plots showing ribosome load (FIG. 7A), in cell half-life (FIG. 7B), and in solution half-life (FIG. 7C).

FIGS. 8A-8C illustrate exemplary data of correlations between in cell half-life and ribosome load (FIG. 8A), monosome-to-free-subunit ratio (FIG. 8B), and polysome-to-monosome ratio (FIG. 8C).

FIGS. 9A-9B illustrate exemplary data of in cell half-life (FIG. 9A) and ribosome load (FIG. 9B).

FIG. 9C illustrates an exemplary demonstration of how to determine or predict protein expression based on in cell half-life and ribosome load.

DETAILED DESCRIPTION OF THE DISCLOSURE

Turning now to the drawings, systems and methods to enhance RNA translatability and uses thereof, and systems and methods to quantify RNA stability and translatability and uses thereof are provided. Many embodiments provide nucleic acid molecules (e.g., RNA molecules (including messenger RNA (mRNA)), DNA molecules, DNA/RNA hybrid molecules) that allow for an assessment of in vitro, in vivo, in cell, in solution, in storage, and/or any other form of molecular stability. Some embodiments are directed to RNA molecules, including mRNA, with increased translatability and/or stability. Certain embodiments provide RNA molecules used for RNA therapeutics, including vaccines, where one or more of 1) high and sustained expression of RNA (e.g., mRNA), 2) high stability of RNA inside of cells (e.g., in vivo), and 3) high stability of RNA in solution (e.g., in vitro) is desired.

Further embodiments provide methods that provide RNA molecules with increased translatability and/or stability, while additional embodiments provide methods to test translatability and/or stability of RNA molecules. Certain embodiments provide a multiplexed workflow to generate RNA molecules, including mRNA, having increased translatability and/or stability in a single assay. In many embodiments, the RNA molecules of various embodiments are generated via rational design, while certain embodiments generate RNA molecules via iterative selection.

RNA Molecules and Design

As noted above, some embodiments generate RNA molecules via rational design, while others utilize iterative selection. Rational design is a methodology that combines sequence components, such as a 5′ UTR, a 3′ UTR, and/or a coding region that exist in nature or are synthetically engineered for specific objective (e.g., increased stability or translatability). However, certain embodiments utilize iterative selection to generate RNA molecules, where various sequence components, such as a 5′ UTR, a 3′ UTR, and/or a coding region, comprise random sequences. Certain embodiments utilizing iterative selection optimize RNA molecules for translatability and/or stability over several rounds of sequence selection (e.g., selecting for sequences showing increased translation or stability).

Turning to FIG. 1A, an exemplary structure for an embodiment of an RNA molecule in accordance with various embodiments is illustrated. Certain embodiments of an RNA molecule possess a 5′ cap moiety. Some embodiments utilize a 7-methyl guanosine triphosphate as the cap moiety, but various additional cap sequences are known in the art for a 5′ cap moiety. Additional embodiments possess a cap-proximal sequence for an mRNA region located at the 5′ end of the mRNA at the 3′end of the 5′ cap moiety. Various cap sequences are known in the art for a 5′ cap-proximal sequence. Certain embodiments use a small triplet, such GGG as the cap-proximal sequence.

Additional embodiments of an RNA molecule possess a 5′ untranslated region (5′UTR) sequence and/or a 3′UTR sequence. Certain embodiments place the 5′UTR near the 5′ end of the RNA molecule, while the 3′UTR is located near the 3′ end of the molecule. In some embodiments, the 5′UTR is located at the 3′ end of a 5′ cap moiety, while additional embodiments place the 5′UTR directly at the 5′ end without a 5′ cap moiety or cap sequence. Similarly, a 3′UTR can be placed at the 3′ end of a molecule, while additional embodiments may have a tailing sequence placed 3′ of the 3′UTR. Certain embodiments select a 5′UTR and/or a 3′UTR for a variety of factors to increase RNA translatability, stability, and/or other property based on an innate sequence, while others select a 5′UTR and/or a 3′UTR for that may pose improved translatability, stability, and/or other property based on a particular coding sequence of interest. Many possible 5′UTRs and 3′UTRs are known in the art, which are used in various embodiments. Some specific embodiments of rationally designed RNAs select the 5′UTR from natural or modified 5′UTR elements, including SEQ ID NOs: 1-55. And, certain specific embodiments select the 3′UTR from SEQ ID NOs: 56-80. Tables 1 and 2 list various 5′UTRs and 3′UTRs, respectively, with their respective SEQ ID NOs.

TABLE 1 5′UTR Sequences Name: SEQ ID NO: SynJ 1 hHBB30 2 CYP2E1 3 CYBA 4 mRpl18a 5 RpS25 6 scrUTR 7 TEV 8 hHBB 9 APOA2 10 TOP_hHBB 11 C3 12 hHBB_pA 13 TCV 14 PoV_pA_scrUTR 15 TMV 16 PoV_pA_hHBB 17 RpL38 18 CoV-2-TTG-dSL4-5 19 hACTB 20 RpL31 21 mRpl18a_hHBB 22 DEN2 23 RBCS3B 24 mActb 25 mActb_inv 26 TEV_CERT_hHBB 27 Tubb2b 28 P4_hACTB 29 hCOL1A2 30 BYDV 31 CoV-2-TTG-dSL5 32 P4_mActb 33 P4_mActb_inv 34 CoV-2-TTG-dSL5A-C 35 CoV-2-TTG-dSL1-3 36 mRpl18a_P4_mActb 37 TEV_P4_mActb 38 P4_TEV_mActb 39 CoV-2-TTG-dSL4-full 40 CoV-2-TTG-dSL4-1 41 CoV-2-TTG-dSL5A 42 CoV-2-TTG-TTGfull-dSL1-3 43 CoV-2-TTG-dSL5B, C 44 CoV-2-TTG-dSL1 45 CoV-2-TTG-dSL4-2 46 CoV-2-TTG-dSL2 47 CoV-2-TTG-dSL3 48 CoV2 49 CoV2_TTG 50 CoV2_P4 51 CoV-2-TTG-TTGfull 52 mHoxa9_IRES 53 HCV_IRES 54 RBCS1A 55

TABLE 2 3′UTR Sequences Name: SEQ ID NO: SINV_URE 56 CYBA 57 PV 58 hHBA1 59 CYBA_1.5x 60 BMV 61 hHBB 62 AMV 63 ENE_Wilusz 64 ENE_Weissman 65 BYDV 66 TSV 67 hHBB_F30Pepper 68 P4P6 69 TCV 70 hHBBx2 71 CrPV 72 SINV 73 CoV2 74 DV 75 RV 76 WPRE 77 hActb 78 mActb 79 hCOL1A2 80

FIG. 1B illustrates a method 100 for iteratively selecting elements to increase RNA translatability and/or stability. Such embodiments identify sequences or segments of an expression-affecting region (e.g., 5′UTR, 3′UTR, and/or coding region) that increase translatability, stability, and/or other property of the RNA molecule.

At 102, various embodiments obtain a library of RNA molecules. In certain embodiments, the library comprises RNA molecules with degenerate sequences in regions that affect RNA expression. In certain embodiments, the degenerate expression-affecting region are truncated at its 5′- and/or 3′-end. In some embodiments, the degenerate expression-affecting region contains internal deletions, such that the 5′- and/or 3′-end remain intact, but the overall region is smaller. In certain embodiments, the degenerate sequences are random, ambiguous, and/or mutated sequences to identify specific bases that may allow for an outsized role in translatability and/or stability.

At 104, many embodiments assess stability and/or translatability of the molecules. Various methods to assess translatability and/or stability are described herein. At 106, certain embodiments select for molecules having a minimum level of translatability and/or stability, such as through selection of a specific fraction of stability and/or translatability. For example, many embodiments select for fractions having high levels of stability (e.g., at longer time points) and/or translatability (e.g., higher polysome fractions).

Upon assessing stability and/or translatability, certain embodiments sequence the selected for molecules at 108. Sequencing the selected molecules identifies the specific sequences that correlate to the tested characteristic (e.g., translatability and/or stability).

It should be noted that many embodiments may perform several features multiple times, such as the assessing 104 and selecting 106 features, in order to identify the sequences having the highest rates of translatability and/or stability. For example, numerous embodiments take the selected for molecules (e.g., ones having high levels of translatability and/or stability) and reassess the translatability and/or stability of these molecules, selecting for high levels of high levels of translatability and/or stability. Various embodiments repeat the assessing 104 and selecting 106 features 2 times, 3 times, 4 times 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, or more times to identify molecules having the highest levels of translatability and/or stability. Additionally, various embodiments repeat sequencing 108, such as after each selection 106, or just after every second selection 106.

Methods, such as method 100, allow for iterative selection of expression-affecting regions to further increase translatability, stability, and/or other property. Some exemplary embodiments utilize a pool 5′-UTR selected from SEQ ID NOs: 81-111. Table 3 identifies specific pools of 5′UTR sequences for iterative selection.

TABLE 3 5′UTR Sequence Pools Pool 1 SEQ ID NOs: 81-90 Pool 2 SEQ ID NOs: 91-101 Pool 3 SEQ ID NOs: 102-106 Pool 4 SEQ ID NOs: 107-111

Returning to FIG. 1A, many embodiments of an RNA molecule possess a coding sequence, or CDS, located 3′ from the 5′UTR, and 5′ of the 3′UTR. In many embodiments, the CDS begins (e.g., at its 5′-end) with a start codon (e.g., the canonical AUG and/or any other codon known to begin translation). In many embodiments, the CDS terminates (e.g., at its 3′-end) with a stop codon. In various embodiments the stop codon is a canonical stop codon (e.g., UAG, UAA, UGA), while further embodiments comprise a noncanonical stop codon or another sequence shown to terminate translation. Certain embodiments comprise more than one stop codon in the CDS.

The coding sequence is a designed sequence of interest to encode a protein or peptide of interest. In certain embodiments, the coding sequence encodes an epitope or other antigen to induce an immune response, thus allowing creation of a vaccine. In various embodiments, the protein or peptide of interest is used as a therapeutic directly, such that the protein or peptide of interest replaces or supplements a dysfunctional protein or peptide. In some embodiments, the protein or peptide of interest corrects for dysfunction of another protein or peptide. While protein coding sequences are described in the context of this exemplary embodiment, additional embodiments possess sequences for non-coding RNAs, such as RNAs that guide genome editing and/or coat chromatin. Various embodiments possess a CDS encoding a reporter gene; for example, nanoluciferase (“Nluc”, SEQ ID NO: 112), green fluorescence protein (“GFP”, SEQ ID NO: 113), and/or any other reporter gene of interest. Various embodiments encode a therapeutic, such as a multi-epitope vaccine (“MEV”, SEQ ID NO: 114).

Additional embodiments of an RNA molecule include a barcode to identify particular molecules based on unique sequences. Many barcode schemes are known in the art and range from 2 to 12 or more nucleotides. In many embodiments, the barcodes are 6-9 nucleotides in length. Certain embodiments select one or more barcodes from SEQ ID NOs: 115-1380.

To read barcodes, an RNA molecule can include one or more profiling sequences that can be used by PCR primers or sequencing primers to amplify and/or sequence the barcode region. In some embodiments profiling sequences are located at the 5′ and/or 3′ end of a barcode. In many embodiments, profiling sequences flank the barcode. In various embodiments profiling sequences are selected from profiling sequence 1 (SEQ ID NO: 1381) and profiling sequence 2 (SEQ ID NO: 1382).

As noted above, some embodiments of an RNA molecule possess a tailing sequence located at the 3′ end of a molecule. In various embodiments the tailing sequence is used to add a poly-A tail or other structural sequence to an RNA molecule. In some embodiments, the tailing sequence is selected as SEQ ID NO: 1383.

Structures, such as those described above in regard to FIG. 1 allow for modular and combinatorial testing of various 5′UTRs, CDSs, and 3′UTRs.

Methods of Assessing RNA Translatability

Certain embodiments assess translatability of RNA molecules, such as those described elsewhere herein. An exemplary embodiment of a method 200 to assess translatability is illustrated in FIG. 2. In method 200, an RNA molecule is obtained at 202 of many embodiments. In certain embodiments, the RNA molecule is generated via in vitro transcription. Additionally, certain embodiments generate an RNA transcript and/or further modify RNA transcripts to be used for translation (e.g., including a 5′ cap and/or a 3′ polyA tail). Some embodiments obtain DNA templates from a commercial vendor. In various embodiments, polymerase chain reaction (PCR) is used to amplify a full-length DNA template for the RNA molecule. Additional embodiments assess amplicon quality via electrophoresis, including gel (agarose and/or polyacrylamide) and/or capillary electrophoresis (e.g., ABI 3700 and/or Agilent Bioanalyzer). Further embodiments transcribe DNA amplicons to RNA using a DNA-dependent RNA polymerase. Certain embodiments perform the in vitro transcription using commercial kits, including Thermo's T7 MEGAScript kit. Various embodiments modify the RNA transcripts with a 5′ cap and/or polyA tail. These modifications can be accomplished using kits, such as the Cellscript kit and/or any other applicable and commercially available kit. Additional cleanups can be accomplished at various stages (e.g., after PCR, after transcription, and/or after modification), using columns or reagents, such as Thermo's MEGAClear columns. And, quality of the transcribed and/or modified RNAs can be accomplished via electrophoresis, including gel and capillary electrophoresis. Further embodiments quantify the RNA pool via various known means, such as spectrophotometry, fluorometry, or and/or any other known method for quantifying nucleic acids.

In various embodiments, the RNA molecule is obtained as a pool of RNA molecules, where each unique RNA sequence in the pool comprises a unique barcode, such as described herein. In certain embodiments, the RNA molecules within the pool are approximately the same length. In certain embodiments, the RNA molecules within the pool vary in length.

Various embodiments transfect RNA transcripts into cells or add the transcripts to a cellular lysate at 204. In certain embodiments, transfection occurs on cultured cells or tissue, including mammalian cells, while other embodiments use yeast, bacteria, or plant cells. Some specific embodiments transfect HEK293T cells. Various embodiments incubate the transfected cells to allow for translation of the RNAs. Incubation can last between 1 hour and several days (e.g., 7-10 days) at temperatures and/or conditions to encourage cellular growth and translation. Culture media can include antibiotics or other selective reagents to prevent growth of non-transfected cells and/or contamination. Certain embodiments utilize a cellular lysate as a proxy of in vivo stress on RNA. In such embodiments, cultured cells are lysed via a known method, such as sonication, hydrodynamic stress, or any other method to generate cellular lysate. In various embodiments, the RNA molecule(s) are added to the lysate and allowed to react for a period of time, such as between 1 hour and several days (e.g., 7-10 days) and at temperatures commensurate with the operating temperature for the RNA (e.g., average body temperature, 37° C.).

At 206, certain embodiments perform polysome profiling. In various embodiments, the polysome profiling separates RNA molecules or transcripts based on the number of ribosomes located on, or bound to, a transcript or RNA molecule. As ribosomes are the machinery for translation, the number of ribosomes located on a transcript is indicative of the translatability of a particular transcript.

In certain embodiments, polysome profiling uses a sucrose gradient (e.g., a continuous sucrose gradient) to fractionate RNA molecules based on the number of ribosomes (e.g., polysomes) located on the transcript. Various embodiments perform polysome profiling by lysing transfected cells and applying the lysate to a column containing a sucrose gradient. In embodiments, where RNA transcripts are applied to a cellular lysate, the lysate is directly added to a sucrose gradient column. Centrifugation is applied to the column to separate transcripts based on the number of ribosomes attached to a transcript.

At 208, many embodiments isolate or extract one or more fractions of RNA molecules from the polysome profile. In certain embodiments, the fractions or isolated from the sucrose gradient. In various embodiments, the fractions are isolated as slices, drops, and/or other method of obtaining a fraction from a sucrose gradient. Actively translating RNA molecules have a higher number of ribosomes associated with them and are found in polysomal fractions (e.g., more ribosomes bound to the RNA molecule) whereas non-translating/poorly-translating RNA molecules are present in a free RNA fraction or associated with ribosomal subunits (e.g., 40S ribosomal subunit). In certain embodiments, fractions representing higher amounts of ribosomes bound to RNA are isolated, while some embodiments isolate fractions representing a range of ribosomes bound to RNA in order to identify a distribution of ribosomes present for a particular transcript sequence. RNA molecules from an isolated fraction can be cleaned up via known procedures or kits, including columns.

Certain embodiments introduce a known amount of one or more RNA molecules as a spike-in. Spike-ins serve as an internal reference to allow for quantification of molecules within the assessed RNA library. Such spike-ins are unique RNA molecules that are not present in the analyzed RNA library. The spike-ins can be similar in length to the molecules in the library, and/or possess unique sequences or barcodes.

Various embodiments identify the RNA molecules located in the one or more fractions based on their barcodes at 210. As noted above in relation to FIG. 1, many embodiments of RNA molecules contain a barcode sequence (e.g., SEQ ID NOs: 115-1360). The profiling sequences flanking the barcodes (e.g., SEQ ID NOs: 1381-1382) can be used to amplify the barcode or can be used as sequencing primers for barcoding reads of the RNA molecules of certain embodiments. Further embodiments utilize hybridization probes, quantitative PCR (qPCR), or any other known method with or without pooling strategies to identify which RNAs are present in each fraction.

Methods of Assessing In Vivo or In-Cell RNA Stability

Certain embodiments assess the stability of RNA molecules, including stability within in vivo and in vitro environments. An exemplary embodiment of a method 300 to assess stability is illustrated in FIG. 3. In method 300, RNA is obtained at 302. Obtaining RNA at 302 can be accomplished via many methods, including such steps as described in regard to method 200 (FIG. 2), including the obtention of a pool of RNA molecules, where each unique RNA sequence is identifiable by a unique barcode.

Various embodiments transfect RNA transcripts into cells or add the transcripts to a cellular lysate at 304. In certain embodiments, transfection occurs on cultured cells or tissue, including mammalian cells, while other embodiments use yeast, bacteria, or plant cells. Some specific embodiments transfect HEK293T cells. Various embodiments incubate the transfected cells. Incubation can last between 1 hour and several days (e.g., 7-10 days) at temperatures and/or conditions to encourage cellular growth. Culture media can include antibiotics or other selective reagents to prevent growth of non-transfected cells and/or contamination. Certain embodiments utilize a cellular lysate as a proxy of in vivo stress on RNA. In such embodiments, cultured cells are lysed via a known method, such as sonication, hydrodynamic stress, or any other method to generate cellular lysate. Then, the RNAs are added to the lysate and allowed to react for a period of time, such as between 1 hour and several days (e.g., 7-10 days) and at temperatures commensurate with the operating temperature for the RNA (e.g., average body temperature, 37° C.).

At 306, certain embodiments isolate RNAs based on in-cell stability. In various embodiments, RNAs are isolated from transfected cells, while some embodiments isolate the RNAs from a cellular lysate. Certain embodiments isolate RNA from transfected cells at various time points (e.g., after 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, 24 hours, etc.) to create time-based fractions of RNAs. Based on the relative amounts of an RNA at the different timepoints, assessment of RNA stability can be derived, and a RNA half-life can be calculated. Additionally, isolated RNA molecules can be cleaned up via known procedures or kits, including isolation protocols, kits, columns, or any other known method for isolating RNA from cells or a lysate.

Some embodiments select for stable RNAs by performing reverse transcription PCR (RT-PCR) to amplify long, full length RNA regions, for example the full-length mRNA, full-length CDS, 5′ UTR-CDS, 3′ UTR-CDS, or any other length covering functional region, or only the barcode region, into complimentary DNA (cDNA). By creating cDNAs, downstream amplifications can utilize DNA-dependent polymerases to create sequencing libraries or other molecules for analysis. Such embodiments select for full length or any longer functional length of RNAs rather than RNAs that may have been hydrolyzed but may still be of sufficient length that electrophoresis or other methods do not remove them.

Certain embodiments introduce a known amount of one or more RNA molecules as a spike-in. Spike-ins serve as an internal reference to allow for quantification of molecules within the assessed RNA library. Such spike-ins are unique RNA molecules that are not present in the analyzed RNA library. The spike-ins can be similar in length to the molecules in the library, and/or possess unique sequences or barcodes.

Various embodiments identify the RNAs based on their barcodes at 308. As noted above in relation to FIG. 2, many embodiments of RNA molecules contain a barcode sequence (e.g., SEQ ID NOs: 115-1380). The profiling sequences flanking the barcodes (e.g., SEQ ID NOs: 1381-1382) can be used to amplify the barcode or can be used as sequencing primers for barcoding reads of the RNA molecules of certain embodiments. Further embodiments utilize hybridization probes, quantitative PCR (qPCR), or any other known method with or without pooling strategies to identify which RNAs are present in timepoint based fractions.

Determination of In Vitro or in Solution RNA Stability

An additional challenge for RNA therapeutics, including vaccines, include the stability in storage, such as between manufacture and actual treatment or delivery to an individual. Such stability is referred to as in vitro stability, as it emphasizes stability in non-biological environments, such as in vials, syringes, or other method of storage. Various embodiments provide a method to measure in vitro stability of RNAs. Turning to FIG. 4, a method 400 to determine in vitro RNA stability in accordance with various embodiments is illustrated. Within method 400, RNA is obtained at 402. Obtaining RNA at 402 can be accomplished via many methods, including such steps as described in regard to method 200 (FIG. 2), including the obtention of a pool of RNA molecules, where each unique RNA sequence is identifiable by a unique barcode.

At 404 of many embodiments, the RNA pool is treated or subjected to an experimental condition. The experimental conditions include any condition that may cause degradation of an RNA molecule in a storage situation, including (but not limited to) temperature, pH, presence of certain molecules and/or ions, concentration of certain molecules and/or ions, irradiation, time, buffer type, buffer concentration, and/or any other condition that can affect RNA stability. Such conditions are meant to reproduce actual conditions that can induce one or more hydrolytic events within the RNA molecules. A hydrolytic event, in accordance with various embodiments, causes a break within the RNA molecule, resulting in a broken or incomplete RNA molecule. Incomplete or broken RNA molecules may be insufficient for use as a therapeutic, as they may be prone to degradation or ineffective in protein production, thus incomplete or broken RNA molecules may limit the efficacy of the molecule as a therapeutic.

Further embodiments further select for stable RNAs in the pool at 406. In some embodiments, the selection occurs by size selecting for full length RNAs, such as through electrophoresis, including (but not limited to) agarose gel electrophoresis, polyacrylamide electrophoresis, and capillary electrophoresis.

Some embodiments select for stable RNAs by performing reverse transcription PCR (RT-PCR) to amplify long RNA regions, for example the full-length mRNA, full-length CDS, 5′ UTR-CDS, 3′ UTR-CDS, or any other length covering functional region, or only the barcode region, into complimentary DNA (cDNA). By creating cDNAs, downstream amplifications can utilize DNA-dependent polymerases to create sequencing libraries or other molecules for analysis. Such embodiments select for full length or any longer functional length of RNAs rather than RNAs that may have been hydrolyzed but may still be of sufficient length that electrophoresis or other methods do not remove them.

Certain embodiments introduce a known amount of one or more RNA molecules as a spike-in. Spike-ins serve as an internal reference to allow for quantification of molecules within the assessed RNA library. Such spike-ins are unique RNA molecules that are not present in the analyzed RNA library. The spike-ins can be similar in length to the molecules in the library, and/or possess unique sequences or barcodes.

Many embodiments isolate RNAs based on in vitro or in solution stability at 408. Certain embodiments isolate RNA from a solution at various time points (e.g., after 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, 24 hours, etc.) to create time-based fractions of RNAs from a solution. Based on the amount of an RNA at the timepoint 0, relative assessment of RNA stability can be derived, and a RNA half-life can be calculated. Additionally, isolated RNA molecules can be cleaned up via known procedures or kits, including isolation protocols, kits, columns, or any other know method for isolating RNA from cells or a lysate.

At 410, stable RNAs are identified. In various embodiments, the undigested or gel-extracted RNAs are sequenced using the barcode to identify the particular molecules that are stable. In many embodiments, cDNAs created in 406 are utilized as templates to create a sequencing library to avoid the amplification of RNAs that may be near full length.

Identifying RNAs Having Enhanced Translatability, Stability, and/or Other Property

Turning to FIG. 5A, certain embodiments are capable of simultaneously assessing one or more of translatability, stability, and/or any other property. Such embodiments assess one or more of translatability, in vivo (or in cell) stability, in vitro (or in solution) stability, and/or any other property. Within method 500, RNA is obtained at 502. Obtaining RNA at 502 can be accomplished via many methods, including such steps as described in regard to method 200 (FIG. 2), including the obtention of a pool of RNA molecules, where each unique RNA sequence is identifiable by a unique barcode. Many embodiments perform one or more of assessing translatability 504, assessing in vivo (or in cell) stability 506, and/or assessing in vitro (or in solution) stability 508. Assessing translatability 504 can be performed via methods, such as method 200 (FIG. 2), while in vivo stability 506 can be performed via method 300 (FIG. 3), and assessing in vitro stability can be performed via method 400 (FIG. 4). Upon obtaining fractions from the one or more of assessing translatability 504, assessing in vivo stability 506, and/or assessing in vitro stability 508, various embodiments can identify RNAs at 510.

Turning to FIG. 5B, various embodiments identify RNA molecules possessing increased translatability in method 550. At 552, many embodiments obtain identities of RNA molecules present in various fractions of translatability (e.g., RNAs assessed via methods 200, 300, 400, and/or 500). In various embodiments, these identities include the barcode or barcodes that identify each of the RNA molecules in a fraction and a read count of each barcode in each fraction.

At 554, various embodiments determine the translatability of each RNA molecule by identifying prevalence of each barcode in each fraction. Certain embodiments perform statistical analyses to relative prevalence of the barcode in each fraction. The presence of RNAs in fractions correlating to more ribosomes, indicate increased translatability of that particular RNA molecule as compared to other fractions across the whole polysome profile gradient.

Some embodiments filter RNA molecules based on particular characteristics at 556. Particular characteristics may be specific cutoffs, minimum levels of translatability, or a statistical distribution of a particular barcode. For example, certain embodiments may select barcodes that have a narrower distribution with a lower average ribosomal load (e.g., fewer ribosomes on RNA molecules), while other embodiments may select for a higher average with a broader overall distribution.

Various embodiments deconvolve the barcodes at 558, where deconvolution involves matching the specific RNA sequence or sequence name with the barcode sequence comprised within that RNA molecule.

Additional embodiments output results of translatability, stability, and/or other property at 560. Certain embodiments provide lists of each of the sequences providing a specific cutoff or parameter for minimum translatability, stability, and/or other property. Various embodiments produce a graphical display or visualization, such as a dot plot, heat map, or other graph or chart to visualize stability (e.g., in vivo, in vitro, in cell, in solution, etc.), translatability, and/or any other property of a particular RNA molecule.

Additional embodiments output results of predicted protein expression at a given time or total protein expression over time, from experimentally determined stability and translatability. For this, additional embodiments can use modelling of the empirical data to estimate the predicted protein expression in a pool of hundreds of different RNA molecules based on measurements of a selected number of RNA designs.

Enhanced Translatability of RNA Molecules

Turning to FIGS. 6A-6C, exemplary results of embodiments showing translation efficiency are illustrated, where FIG. 6A illustrates a heatmap and FIGS. 6B-6C show enlarged portions of FIG. 6A. FIGS. 6A-6C illustrate the relative prevalence of 64 unique RNA molecules in accordance with various embodiments based on polysome fraction. Darker cells indicate a lower relative prevalence of the molecule in a particular fraction, while lighter colors indicate a higher relative prevalence of the molecule in a particular fraction.

Additionally, FIGS. 7A-7C illustrate exemplary data plotting ribosomal load (FIG. 7A), half-lives for in-cell (or in vivo) stability (FIG. 7B) and in solution (or in vitro) stability (FIG. 7C) of various mRNA molecules, including mRNA molecules having 5′UTR variants, 3′UTR variants, both 5′UTR and 3′UTR variants, and various CDS sequences, including from Nluc, eGFP, and MEV. For ribosomal load in FIG. 7A, ribosomal load is determined by the equation listed in FIG. 7A.

FIGS. 8A-8C illustrate exemplary data showing correlations of in cell mRNA half-life to ribosomal load (FIG. 8A), in cell mRNA half-life to monosome-to-free-subunit ratio (FIG. 8B), and in cell mRNA half-life to polysome-to-monosome ratio (FIG. 8C).

Given the assessment of in cell stability and translatability in accordance with various embodiments, further embodiments determine protein expression levels of proteins encoded in a CDS of the molecule. Certain embodiments determine protein expression via the equation:

$P (t) ~ k_{t} \frac{e^{- k_{p} t} - e^{- k_{m} t}}{k_{m} - k_{p}}$

Where P(t) is protein quantity at time t; k_tis translation rate; and k_mand k_pare rates of mRNA and protein decay, respectively.

An exemplary demonstration of predicted expression is illustrated in FIGS. 9A-9C, where FIG. 9A illustrates in cell half-life of various mRNA constructs, FIG. 9B illustrates ribosomal load of various constructs, and FIG. 9C illustrates the predicted expression.

EXEMPLARY EMBODIMENTS

Although the following embodiments provide details on certain embodiments of the inventions, it should be understood that these are only exemplary in nature, and are not intended to limit the scope of the invention.

Example 1: In Vitro Transcription of Reporter mRNAs

Method: Preparation of mRNAs were based on in vitro transcription from DNA templates. DNA templates were amplified by PCR using AccuPrime Pfx (Life Technologies, 12344024) and purified using the Monarch PCR & DNA Cleanup Kit (NEB, T1030L). The source of the 3×HA-Nluc starting CDS (“Nluc start”) is derived from the pcDNA3.1-5′UTR-3×HA-Nluc plasmid encoding the HA-tagged Nanoluc CDS. Individual template DNA or the 233-mRNA library was amplified from linear DNA synthesized on a BioXP 3200 system (Codex DNA) or by Twist Bioscience, using the fixed forward (T7_F_28nt) and reverse (const3_R) primer. The forward primer binds to the T7 RNA polymerase promoter common in DNA template for all mRNA designs; the reverse primer is complementary to a common “const3” region at the end of all tested mRNA 3′ UTRs. For the IVT template pool, individual DNA templates were pooled for a template pool of hundreds of constructs at an equimolar concentration and are amplified with outer primers in a pooled format. For the pooled template, 1 μL of each construct (˜20 ng/μL stock concentration) was pooled to be used as the PCR template. The Pfx PCR contained the following: 2.5 μL 10×Pfx buffer, 0.25 μL forward primer (100 uM), 0.25 μL reverse primer (100 uM), 0.75 μL DMSO (NEB), 0.25 μL Pfx Polymerase (Thermo), 20.5 water, and 0.5 μL template DNA (˜20-50 ng/ul), in a total 25 μL reaction with the following program: 2 min at 95° C.; 10 sec at 95° C.; 30 sec at 58° C.; 30 s or 1 min at 68° C.; cycled 9×; final extension of 5 min at 68° C. PCR reactions were purified with Monarch PCR & DNA Cleanup Kit (NEB, T1030L). For the hHBB-Fluc control mRNA, the DNA template was amplified from the pGL3-HBB plasmid using the primers KL588/KL589 which yielded a PCR product of 1,750 kb in length. For cloning the MALAT1 ENE 3′ UTR stem-loop, we first amplified the ENE region using primers ENE-1/ENE-2 with flanking constant regions. The resulting amplicon was assembled with a hHBB-Nluc sequence that lacked a 3′ UTR but maintained a unique barcode using a NEBuilder HiFi Assembly Kit (NEB, ES2621).

In vitro transcription was performed with the MEGAscript T7 kit (Ambion, AM1333) according to the manufacturer's instructions. A 20 μL transcription reaction contained max. 5 μg linear DNA template, 4 mM of each NTP (Ambion), 2 μL/200 U MEGAscript T7 RNA polymerase (Ambion) and 1×T7 MEGAscript Transcription Buffer (Ambion). After a total incubation for 3 hours at 37° C., the DNA was digested by addition of 1 μL/2 U Turbo DNase (Ambion, AM2238) for 15 min at 37° C. For pseudouridylated mRNAs, pseudouridine triphosphate (Trilink Biotechnologies, N1019-5) was substituted for uridine triphosphate at an equivalent concentration. mRNA was purified using MegaClear columns (Thermo Scientific, Ambion, AM1908). A 20 μL reaction usually yielded 100-150 μg of RNA.

For mRNA transfection of HEK293T cells, m⁷G-capped and polyadenylated mRNAs were generated as follows. In vitro transcribed mRNA was then m⁷G-capped and polyadenylated using the ScriptCap m7G Capping System (CellScript, C-SCCE0625) and A-Plus Poly(A) Polymerase Tailing Kit (CellScript, C-PAP5104H), respectively, according to the manufacturer's instruction with the following modifications. Aliquots of 30 μg of each RNA were processed in parallel, diluted to 34.25 μL in water and heated for 5 min at 65° C. to denature and placed on ice. The 50 μL capping reaction contained 5 μL 10× ScriptCap buffer (Cellscript), 5 μL 10 mM GTP (Cellscript), 2.5 μL 2 mM S-adenosyl-methionine (SAM, 20 mM stock, Cellscript), 1.25 μL ScriptGuard RNase Inhibitor (Cellscript), and 2 μL Capping enzyme (20 U, Cellscript, 10 U/μL). For the capping step, the 37° C. incubation was performed for 1 hour and the capped RNA was placed on ice. Polyadenylation was performed from the resulting RNAs without purification in between. The polyA reaction contained 30 μg of capped mRNA in 50 μL, 6.6 μL 10× A-Plus polyA tailing buffer (Cellscript), 6.6 μL 10 mM ATP (Cellscript), 0.3 μL ScriptGuard RNase Inhibitor (Cellscript), and 2.5 μL A-Plus PolyA Polymerase (10 U, 4 U/μL, Cellscript) in a total reaction volume of 66 μL. We aimed to add a 150 nt-long polyA-tail for which we incubated the capped mRNA for 30 min at 37° C. with 10 U of polyA enzyme, after which the reaction was placed on ice. The mRNA was again purified using MegaClear columns. mRNA concentration was determined on a Nanodrop 2000 (Thermo Fisher). This usually yields 30-40 μg of capped and polyadenylated mRNA. mRNA quality was determined by 4% urea-PAGE, 1% formaldehyde agarose gel or capillary electrophoresis with an Agilent 2100 Bioanalyzer (Agilent Technologies).

Example 2: Cell Culture and Transfections

Method: HEK293T (ATCC: CRL-3216) cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, 11965-118) containing 2 mM L-glutamine, supplemented with 10% fetal bovine serum (EMD Millipore, TMS-013-B), 100 U/ml penicillin and 0.1 mg/ml streptomycin (EmbryoMax ES Cell Qualified Penicillin-Streptomycin Solution 100×; EMD Millipore, TMS-AB2-C or Gibco, 15140-122) at 37° C. in 5% CO₂-buffered incubators. For transfection of pooled 5‘ m’G-capped and poly(A)-tailed RNAs, 5.0×10⁶HEK293T cells were seeded in a 10 cm plate 24 h before transfection. 10 μg of pooled RNAs were transfected using Lipofectamine MessengerMax as per manufacturer's instructions (Life Technologies). Media was changed 3 h after transfection and replaced with complete DMEM supplemented with 10% FBS and Pen/Strep. For transfections of individual m⁷G-capped RNAs, 3.0×10⁴HEK293T cells were seeded per well 24 h before transfection in a 96-well plate. Subsequently, 10 ng of Nluc RNA was co-transfected with 20 ng of m⁷G-capped HBB-Fluc control RNA using Lipofectamine MessengerMax as per manufacturer's instructions (Life Technologies).

Example 3: Sucrose Gradient Fractionation Analysis

Method: Cell culture media was replaced with cycloheximide (MilliporeSigma, C7698-1G) containing media at 100 ug/mL. After 2 minutes, cells were washed, trypsinized and harvested using PBS, trypsin, and culture media containing 100 g/mL cycloheximide. ˜10×10⁶cells were resuspended in 400 μL of following lysis buffer on ice for 30 min, vortexing every 10 min: 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 15 mM MgCI2, 1 mM DTT, 8% glycerol, 1% Triton X-100, 100 μg/mL cycloheximide, 0.2 U/μL Superase-In RNase inhibitor (ThermoFisher Scientific, AM2694), 1× Halt protease inhibitor cocktail (ThermoFisher Scientific, 78430), 0.02 U/μL TURBO DNase (ThermoFisher Scientific, AM2238). After lysis, nuclei were removed by two step centrifuging, first at 1300 g for 5 min and second at 10000 g for 5 min, taking the supernatants from each. 25%-50% sucrose gradient was prepared in 13.2 mL ultracentrifuge tubes (Beckman Coulter, 331372) using Biocomp Gradient Master with the following recipe: 25 or 50% sucrose (w/v), 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 15 mM MgCI2, 1 mM DTT, 100 μg/mL cycloheximide. The lysate was layered onto the sucrose gradient and ultracentrifuged on Beckman Coulter SW-41Ti rotor at 40000 rpm for 150 min at 4° C. The gradient was density fractionated using Brandel BR-188 into 16×750 μL fractions, and in vitro transcribed spike-in RNA mix (120002B1, 120010B1, 220023B1, 310333T3; 1000, 100, 10, 1-fold dilutions respectively) were added to each fraction. 700 μL of each fraction was mixed with 100 μL 10% SDS, 200 μL 1.5 M sodium acetate, and 900 μL acid phenol-chloroform, pH 4.5 (ThermoFisher Scientific, AM9720), heated at 65° C. for 5 min, and centrifuged at 20.000 g for 15 min at 4° C. for phase separation. 600 μL aqueous phase was mixed with 600 μL 100% ethanol and RNA was purified on silica columns (Zymo, R1013).

Example 4: Polysome Selection and Library Preparation

Method: The variant 5′ UTR is composed of: fixed first 29 nt of hHBB, variable 35 nt (initially degenerate) and 6 nt Kozak consensus. To generate the reporter mRNA pool containing the variant 5′ UTR library, IVT template was first assembled by PCR under the following conditions: 4 μL 10× AccuPrime Pfx Reaction Mix, 0.4 pmol HBB29_N35 amplicon, 0.4 pmol Nluc_HBB_3UTR, 0.4 μL AccuPrime Pfx Polymerase in 40 μL of total reaction volume. Cycling conditions are: 95° C. for 120 sec, and 19 cycles of 95° C. for 15 sec, 66° C. for 30 sec, 68° C. for 75 sec. PCR product was purified on silica columns (NEB T1034) and amplified with under the following conditions: 4 uL 10× AccuPrime Pfx Reaction Mix, 4 μL 10 μM T7_28_HBB_30_F, 4 μL 10 μM Nanoluc_ORF_R, 0.4 μL AccuPrime Pfx Polymerase in 40 μL total reaction volume. Cycling conditions are: 95° C. for 120 sec, and 4 cycles of 95° C. for 15 sec, 66° C. for 30 sec, 68° C. for 75 sec. The mRNA was in vitro transcribed, capped and polyadenylated as described above. This yields an estimated initial starting degenerate pool complexity of ˜2.4×10¹¹.

Transfection of HEK-293 cells and sucrose gradient fractionation were performed as described above. Equal volumes of fractions 10-16 were pooled and RNA was by acidic phenol chloroform extraction followed by column purification (Zymo Research, R1013) as described above. 1/3 lysate volume was kept as input before layering onto the sucrose gradient and RNA was extracted from the input lysate by Trizol extraction followed by column purification. 1.5 μg RNA in 5.5 μL was mixed with 0.5 μL 2 uM RT_Nluc26_UM112_Read1Partial and 0.5 μL 10 mM dNTPs each. The RNA samples were then denatured at 65° C. for 5 min and chilled to 4° C. 3.5 μL reverse transcription mix was added to 10 μL total reaction volume: 2 μL 5× Superscript IV buffer, 0.5 μL 10 mM DTT, 0.5 μL Superase-In (ThermoFisher Scientific, AM2694), 0.5 μL Superscript IV (Thermo 18091050). The reaction was incubated at 55° C. for 45 min and inactivated at 80° C. for 10 min. Variant 5′ UTR amplicon was amplified from the reverse transcription reaction via PCR under the following reaction conditions: 4 μL RT reaction, 40 μL 2× Q5 Hot Start Master Mix (NEB M0494S), 0.8 μL 100×SYBR (Thermo S7563), 4 μL 10 μM T7_28_HBB_29_F, 4 μL 10 μM Nanoluc_ORF_R, in 80 μL total reaction volume. Cycling conditions were as follows: 98° C. for 60 sec, and 15 cycles of 98° C. for 10 sec, 68° C. for 10 sec, 72° C. for 10 sec. PCR product was purified on silica columns (NEB T1034) and assembly with Nluc_HBB_3UTR fragment was performed as described above for initial preparation of IVT template using HBB29_N35 amplicon. The mRNA was in vitro transcribed, capped and polyadenylated as described above. The same process of transfection, fractionation, reverse transcription, PCR amplification, assembly and in vitro transcription was repeated.

For sequencing library preparation, the RT reaction was PCR amplified under the following conditions: 1 μL RT reaction, 10 μL 2× Q5 Hot Start Master Mix (NEB M0494S), 0.2 μL 100×SYBR (Thermo S7563), 1 μL 10 μM Read1, 1 μL 10 μM Read2Partial_HBB29 in 20 μL total reaction volume. Cycling conditions were as follows: 98° C. for 60 sec, and 15 cycles of 98° C. for 10 sec, 68° C. for 10 sec, 72° C. for 10 sec. Sequencing adaptors were added using the following conditions for final round PCR: 1 μL first round PCR reaction, 10 μL 2× Q5 Hot Start Master Mix, 0.2 μL 100×SYBR, 1 μL 10 uM NEBNext Index Primer (NEB E7335, NEB E7500, NEB E7710, NEB E7730, NEB E6609), 1 μL 10 uM NEBNext Universal PCR Primer in 20 μL total volume. Cycling conditions are: 98° C. for 60 sec, and 5 cycles of 98° C. for 10 sec, 72° C. for 10 sec. All barcoded samples were then pooled at equal volumes and purified with 1.1×SPRiselect beads Beckman Coulter B23317). Sequencing was performed at the Stanford Functional Genomics Facility (SFGF) at Stanford University, on the Illumina NextSeq 550 instrument, using a high output kit, 1×81 cycles.

Example 5: In Cell and In-Solution RNA Degradation Time Courses

Method: For in-cell RNA stability, the 233-member in vitro transcribed mRNA pool (m⁷G-capped and polyA) was transfected into HEK293T cells as described above and RNA was harvested at 1, 7, 12, and 24 h in Trizol (ThermoFisher Scientific, 15596026). RNA was extracted from the aqueous phase on silica columns (Zymo, R1013).

For in-solution RNA degradation experiments, 750 ng of the 233-mRNA pool (not m⁷G-capped or polyA) was incubated in 30 μL of Degradation Buffer (50 mM CHES at pH 10 and 10 mM MgCI2) and collected over 10 time points: 0, 0.5, 1, 2, 3, 4, 5, 6, 16 and 24 h. To each sample, 15 μL of 0.5 M Tris-HCl pH 7 and 3 μL of 0.5 M EDTA-Na was added to quench the degradation. The integrity of each sample was checked by loading 5 μL of total RNA alongside a spike-in control (P4P62HP, 50 ng) onto a PAGE-Urea-TBE gel and visualized by SYBR Gold (Thermo Fisher). Subsequently, RNA was purified using Ampure beads +40% polyethylene glycol 8000 (7:3) and checked again by PAGE-Urea-TBE gel and visualized by SYBR Gold.

Example 6: Measurement of In-Solution mRNA Stability by Capillary Electrophoresis

Method: For one-by-one measurement of in-solution mRNA stability, in vitro transcribed mRNA was incubated in a degradation buffer over ten time points (0, 0.5, 1.0, 1.5, 2, 3, 4, 5, 18, and 24 hours), then analyzed by capillary electrophoresis.

For each time point, 1.6 pmol of mRNA brought to 10 μL in a buffer containing 50 mM Na-CHES at pH 10 with 10 mM MgCI2, and the reaction was incubated at 25° C. When the incubation period was reached for each time point, 5 μL of Tris-HCl at pH 7 and 1 μL of 500 mM EDTA in nuclease free water was added to quench the degradation reaction, and frozen for further analysis. After the final time point (24 hours), 4 μL of each mRNA degradation sample (out of a total stored volume of 16 μL) was taken, and mixed with 1 μL of a control RNA at a concentration of 50 ng/μL. For these experiments we opted to use the P4-P6 domain of the Tetrahymena ribozyme with two added hairpins (˜239 nt) as a control. The RNA mixture was then purified using a mixture of AMPure XP beads (Beckman Coulter) with 40% polyethylene glycol (mixed in a 7:3 ratio). The resulting RNA was eluted into 4.5 μL of RNAse-free water for analysis on the 2100 Bioanalyzer (Agilent) using the RNA-Nano Eukaryote protocol.

The data from the Bioanalyzer were analyzed using a custom script that performs the following analysis. We first converted elution times to nucleotides based on a ladder control (25, 200, 500, 1000, 2000, and 4000 nts). Relative mRNA amounts were estimated based on peak areas at expected band lengths (for example, ˜900 nucleotides for the mRNAs of interest and ˜265 nucleotides for the control). When calculating peak areas, background subtraction was performed, where the background was defined as the area under a linear line in the range of nucleotides used for the peak area. Normalization was performed using two different methods used to cross-validate. First, the peak areas of full-length mRNA were normalized to the control P4-P6 domain RNA that was spiked into the samples after degradation was performed. Second, peak areas of full-length mRNAs were also normalized to the total amount of RNA in the lane less the peak area of the bands of interest (between ˜20-1000 nucleotides in our case), assuming that the majority of the other RNA in the lane were degradation products from the mRNA of interest. These distinct approaches to normalizing the data gave the same results within estimated error (see below). After calculations of normalized peak areas, fraction intact values were then calculated for each mRNA by dividing the normalized area across the ten timepoints by the normalized area at the start (0 hours).

$Fraction {Intact}_{i} = \frac{Normalized {Area}_{i}}{Normalized {Area}_{0 hours}}$

For each sample, fraction intact values were fit across the different timepoints to an exponential function:

F_i=Ae^−τ/t

Where F; is an array of fraction intact values across multiple time points, A is the amplitude of the exponential decay function, x is the time constant, and t is an array of time points in hours. The time constant was then used to calculate the in vitro half-life of mRNA:

Half-life=ln(2)τ

Example 7: Library Preparation and Amplicon Sequencinq

Method: Up to 250 ng RNA in 2.75 μL was mixed with 0.25 μL 2 μM RT_Const2_N12_Read1Partial and 0.25 μL 10 mM dNTPs each. The RNA samples were then denatured at 65° C. for 5 min and chilled to 4° C. 1.75 μL reverse transcription mix was added to 5 μL total reaction volume: 1 μL 5× Superscript IV buffer, 0.25 μL 10 mM DTT, 0.25 μL Superase-In (ThermoFisher Scientific, AM2694), 0.25 μL Superscript IV (Thermo 18091050). The reaction was incubated at 55° C. for 45 min and inactivated at 80° C. for 10 min.

First round PCR was performed under following conditions: 1 μL RT reaction, 10 μL 2× Q5 Hot Start Master Mix (NEB M0494S), 0.2 μL 100×SYBR (Thermo S7563), 1 μL 10 uM Read1Partial_F, 1 μL 10 uM 50:50 Hbb_Fwd:Nluc_Fwd mix in 20 μL total volume. Cycling conditions were: 98° C. for 60 sec, and 15 cycles of 98° C. for 10 sec, 68° C. for 10 sec and 72° C. Second round PCR was performed under the following conditions: 1 μL first round PCR, 10 μL 2× Q5 Hot Start Master Mix, 0.2 μL 100×SYBR, 1 μL 10 uM Read1Partial_F, 1 μL 10 uM Read2Partial_Const1_R in 20 μL total volume. Cycling conditions were: 98° C. for 60 sec, and 5 cycles of 98° C. for 10 sec, 72° C. for 5 sec. Sequencing adaptors were added using the following conditions for final round PCR: 1 μL second round PCR, 10 μL 2× Q5 Hot Start Master Mix, 0.2 μL 100×SYBR, 1 μL 10 μM NEBNext Index Primer (NEB E7335, NEB E7500, NEB E7710, NEB E7730, NEB E6609), 1 μL 10 μM NEBNext Universal PCR Primer in 20 μL total volume. Cycling conditions were: 98° C. for 60 sec, and 5 cycles of 98° C. for 10 sec, 72° C. for 5 sec. All barcoded samples were then pooled at equal volumes and purified with 1.1×SPRiselect beads (Beckman Coulter B23317). Sequencing was performed at the Stanford Functional Genomics Facility (SFGF) at Stanford University, on an Illumina NextSeq 550 instrument, using a high output kit, 1×76 cycles. The SEQ ID NOs for the various PCR primers are listed in Table 4.

TABLE 4 Primer Sequences Name: SEQ ID NO: RT_Const2_N12_Read1Partial 1384 Const3_R 1385 Hbb_Fwd 1386 Nluc_Fwd 1387 Read1Partial_F 1388 Read2Partial_Const1_R 1389 T7_F_28nt (forward) 1390

Example 8: Amplicon Sequencing Data Analysis

Method: After bcl conversion and demultiplexing with Illumina bcl2fastq, the constant regions were trimmed using cutadapt. The trimmed reads were aligned to the indexed reference of barcode sequences using Bowtie2 with the following options: -L 11-N 0--nofw. The alignments were deduplicated based on UMIs using UMicollapse with -p 0.05 and counted using samtools idxstats. This pipeline yields a matrix of barcode read counts where rows are the different constructs in the library and columns are the different samples.

The count matrix was log transformed and normalized column-wise using a linear fit on the dilution series of spike-in constructs in each sample. For the calculation of RNA degradation coefficients in cells, we carried out a linear fit to log RNA abundance from the time course data, i.e. we fit an expression of Y=β₀+β₁t where Y is the normalized log RNA abundance and t is the number of hours after transfection; pi is the degradation constant. For the calculation of in solution degradation coefficients, sufficient data points were available to carry out a nonlinear fit directly to an exponential model, i.e. an expression of y=A exp(−τ/t) was fit, where y is the fraction intact (RNA abundance normalized to initial abundance), A is the amplitude, t is the time of incubation in degradation buffer in hours, and T is the degradation time constant. Time courses in which the observed fraction intact exceeded the fitted exponential by more than 0.05 in the last time point signaled RT-PCR amplification of misprimed, non-full-length products and were filtered out of downstream analysis.

For polysome profiles, percent RNA abundances for each fraction were first calculated by scaling per-fraction values by the sum of all fractions. For the heatmap displays in the figures, column medians were also subtracted from each percent RNA value. For the calculation of ribosome load, the matrix of percent RNA abundances in fractions 4-9 (1-3 are free RNP fractions, and >9 have negligible abundance) were first multiplied by a weight vector representing the number of ribosomes in each fraction as determined by the A260 trace from the fractionator, then the weighted abundances were summed across the row. For the calculation of polysome to monosome ratio, the sum of fractions 7-9 (>3 ribosomes) abundances were divided by fraction 4 (80S) abundance. For the calculation of monosome to 40S/60S ratio, fraction 4 (80S) abundance was divided by the sum of fraction 2 (40S/60S) abundance.

To calculate the expected protein levels assuming first order kinetics of mRNA translation and mRNA/protein decay, the following differential equations were used:

$\frac{dM}{dt} = - k_{m} \cdot M (t)$ $\frac{dP}{dt} = k_{t} \cdot M (t) - k_{p} \cdot P (t)$

where dM/dt and dP/dt are rates of change in mRNA and protein levels, respectively; M(t) and P(t) are moles of mRNA and protein at time t, respectively; k_tis the translation rate constant; and k_mand k_pare rate constants of mRNA and protein decay, respectively. The analytical solution for P(t) is proportional to:

$P (t) ~ k_{t} \frac{e^{- k_{p} t} - e^{- k_{m} t}}{k_{m} - k_{p}}$

where m₀is the mass of mRNA present at t=0, and l is the mRNA length in nucleotides. k_pis set to 0 since Nluc protein has negligible degradation as measured by luciferase activity in transiently Nluc-expressing HEK293 cells for at least 6 hours after cycloheximide treatment, which allows assessment of protein degradation in the absence of further translation⁹⁹. k_mis the degradation constant obtained from the linear fit of in-cell time course RNA data (−β₁above). k_tis the ribosome load calculated by summing weighted RNA abundances from polysome profile data.

Example 9: Luciferase Activity Assay after mRNA Transfection

Method: Media from transiently transfected HEK293T cells was aspirated and cells were lysed in 40 μL of 1× passive lysis buffer from the Dual-Luciferase Reporter Assay System (Promega, E1980) and either directly assayed or frozen at −20° C. After thawing, 20 μL of supernatant was transferred to a new plate and assayed for luciferase activity using the Nano-Glo Dual-Luciferase Reporter Assay System (Promega, N1610) to measure Firefly (Fluc) and NanoLuc (Nluc) luciferase activities. In particular, 50 μL of ONE-Glo Ex Reagent was added to each well of lysate and incubated for 3 minutes at room temperature before measuring Fluc activities. Subsequently, 50 μL of NanoDLR Stop & Glo reagent was added to each well, and incubated for 10 min at room temperature before measuring luciferase activities on a GloMax-Multi (Promega) plate reader. Luciferase reporter activity is expressed as a ratio between Nluc and Fluc. Each experiment was performed a minimum of three independent times. Because this assay relies on accumulation of luciferase in the cytosol, any signal peptide sequences were removed from the CDS for templates and mRNA for these transfection and luciferase activity experiments.

Example 10: Polysome Selection Library Sequencing Data Analysis

Method: Following adapter trimming, 670440 sequences with at least 10 summed read count across all libraries combined were set as the reference. Each library was aligned to this indexed reference using Bowtie2. Only uniquely mapping reads with edit distance ≤3 were retained. Alignments were further deduplicated using UMicollapse (-p 0.05, -k 1). This results in the matrix of read count where rows are different sequence variants and columns are the samples.

Normalized counts were obtained by dividing the matrix column-wise by total read counts per sample. For sequence variants with at least 15 reads in any one of the samples, a regression model was fitted on normalized read counts with the sequential selection rounds as ordinal predictors, penalizing differences between coefficients of adjacent groups (R package ordPens). False discovery rate was estimated by Benjamini-Hochberg procedure. For choosing the final set of candidates, the criteria of 15 read counts in the final round polysome selection library and 2 fold enrichment over input in the final round was also required.

DOCTRINE OF EQUIVALENTS

Having described several embodiments, it will be recognized by those skilled in the art that various modifications, alternative constructions, and equivalents may be used without departing from the spirit of the invention. Additionally, a number of well-known processes and elements have not been described in order to avoid unnecessarily obscuring the present invention. Accordingly, the above description should not be taken as limiting the scope of the invention.

Those skilled in the art will appreciate that the foregoing examples and descriptions of various preferred embodiments of the present invention are merely illustrative of the invention as a whole, and that variations in the components or steps of the present invention may be made within the spirit and scope of the invention. Accordingly, the present invention is not limited to the specific embodiments described herein, but, rather, is defined by the scope of the appended claims.

Claims

1-33. (canceled)

34. A method for identifying RNA molecules possessing increased translatability and stability, comprising:

obtaining a pool of RNA molecules, wherein each RNA molecule is uniquely encoded with a barcoding sequence and each barcoding sequence is flanked by at least one profiling sequence;

assessing translatability of the pool of RNA molecules by: transfecting a cell or cell lysate with a first subset of the pool of RNA molecules; performing polysome profiling on the first subset of the pool of RNA molecules to segregate RNA molecules based on the number of ribosomes bound to the RNA molecule; and isolating a fraction from the polysome profile to generate a first set of RNA molecules showing a first level of ribosomes bound to the RNA molecules in the set of RNA molecules; and

assessing stability of the pool of RNA molecules by: treating a second subset of the pool of RNA molecules under an experimental condition; and isolating a fraction from the second subset the pool of RNA molecules at a specified timepoint to generate a second set of RNA molecules showing stability under the experimental condition for the specified timepoint.

35. The method of claim 34, further comprising sequencing the barcode sequence of the first set of RNA molecules and the second set of RNA molecules to identify the presence of each RNA molecule in each fraction of RNA molecules.

36. The method of claim 35, further comprising determining translatability and stability of the RNA molecules associated with each barcode sequence in the first set of RNA molecules and the second set of RNA molecules by identifying the prevalence of each barcode in each fraction of RNA molecules.

37. The method of claim 34, wherein the barcoding sequence is selected from SEQ ID NOs: 115-1380.

38. The method of claim 34, wherein the profiling sequence is selected from SEQ ID NOs: 1381-1382.

39. A method to select for RNA elements, comprising:

obtaining a library of RNA molecules, wherein each RNA molecule comprises a coding sequence, a 5′ untranslated region (5′UTR), and a 3′ untranslated region (3′UTR), wherein one of the coding sequence, the 5′UTR, or the 3′UTR comprises a degenerate region;

assessing a property of the library of RNA molecules, wherein the property is selected from the group consisting of translatability, in vivo stability, and in vitro stability; and

selecting an RNA molecule from the library of RNA molecules showing increase in the property over other RNA molecules in the library of RNA molecules.

40. The method of claim 39, further comprising sequencing the selected RNA molecule.

41. The method of claim 39, wherein the selected RNA molecule is a pool of RNA molecules.

42. The method of claim 41, further comprising:

reassessing the property of the pool of RNA molecules; and

selecting an RNA molecule from the pool of RNA molecules showing increase in the property over other RNA molecules in the pool of RNA molecules.

43. The method of claim 42, further comprising sequencing the selected RNA molecule from the pool of RNA molecules.

44. The method of claim 39, wherein the property is translatability.

45. The method of claim 39, wherein the degenerate region is selected from the group consisting of: a deletion, a random sequence, an ambiguous sequence, and a truncation.

46. The method of claim 34, wherein the RNA molecules are transfected into a collection of cells.

47. The method of claim 46, wherein the collection of cells is selected from mammalian cells, yeast cells, bacteria cells, and plant cells.

48. The method of claim 34, wherein the RNA molecules are added to a cell lysate.

49. The method of claim 34, wherein polysome profiling comprises adding a cell lysate to a sucrose gradient and centrifuging the sucrose gradient to segregate the RNA molecules.

50. The method of claim 34, further comprising isolating a second fraction from the polysome profile to generate a second set of RNA molecules showing a second level of ribosomes bound to the RNA molecules in the set of RNA molecules, wherein the first level and second level represent different amounts of bound ribosomes.

51. The method of claim 50, further comprising sequencing the barcode sequence of each RNA molecule in the first set of RNA molecules and the second set of RNA molecules to identify the presence of each RNA molecule in the first set of RNA molecules and the second set of RNA molecules.

52. The method of claim 34, wherein the treatment condition is selected from temperature, pH, presence of certain molecules, presence of certain ions, concentration of certain molecules, concentration of certain ions, irradiation, buffer type, and buffer concentration.

53. The method of claim 34, further comprising size selecting for full-length RNA molecules.