Methods and systems for rapid molecule isolation

Abstract

Methods and kits of rapidly isolating molecules from a sample are provided. Methods of treating a subject in need thereof using the rapid isolation kits and / or methods described herein are also provided. In some embodiments, the molecules are selected from ribosomes, DNA, RNA, and protein. In some embodiments, the sample is selected from a tissue sample, a blood sample, a non-blood biological fluid sample, and a whole organism sample.

Description

[0001] METHODS AND SYSTEMS FOR RAPID MOLECULE ISOLATION

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S Provisional Patent Application Nos. 63 / 694,196, filed September 12, 2024 and 63 / 720,824, filed November 15, 2024 the entire contents of which are incorporated herein by reference.

[0004] STATEMENT OF FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with government support under GM136823 and GM1 12824 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] FIELD

[0007] The present disclosure generally relates to rapid isolation of ribosomes and other molecules from biological and non-biological samples.

[0008] BACKGROUND

[0009] Current methods for ribosome isolation (and isolation of associated molecules such as DNA, RNA, protein, etc.) are significantly time consuming and need multiple steps for the clear separation of the ribosomes and associated molecules from other molecules in the cell.

[0010] BRIEF DESCRIPTION OF THE DISCLOSURE

[0011] Among the various aspects of the present disclosure is the provision of molecule isolation from a sample.

[0012] In accordance with an aspect of the present disclosure, a method of isolating a molecule from a sample is provided. The method comprising: lysing cells in the sample to generate a cell lysate; incubating the cell lysate with poly-lysine coated beads; and eluting the incubated beads to recover the isolated molecule.

[0013] In some embodiments, the sample is selected from a non-biological sample and a biological sample. In some embodiments, the biological sample is selected from a single cell sample, a tissue sample, a blood sample, a non-blood biological fluid sample, and a whole organism sample. In some embodiments, the sample is a ribosome. In some embodiments, the lysing is performed in the presence of a translation inhibitor.

[0014] In accordance with another aspect of the present disclosure, a kit for isolating a molecule from a sample is provided. The kit comprising: at least one lysing agent; a plurality of poly-lysine coated beads; and at least one elution agent.

[0015] In accordance with a further aspect of the present disclosure, a method of treating a subject having an infection is provided. The method comprising: isolating ribosomes from a biological sample obtained from the subject, wherein the isolating comprises: lysing cells in the biological sample to generate a cell lysate; incubating the cell lysate with poly-lysine coated beads; and eluting the incubated beads to recover the isolated ribosomes. The method further comprises exposing the isolated ribosomes to at least one therapeutic agent; and treating the subject with the at least one therapeutic agent if the isolated ribosomes were susceptible to the at least one therapeutic agent. In some embodiments, the biological sample is selected from a single cell sample, a tissue sample, a blood sample, a non-blood biological fluid sample, and a whole organism sample. In some embodiments, the at least one therapeutic agent is selected from an antibiotic medication, an antifungal medication, and an antiparasitic medication.

[0016] Another aspect of the present disclosure is the provision of obtaining cytoplasmic ribosome and mitochondrial ribosome translation profiling data from a single sample.

[0017] In accordance with an aspect of the present disclosure, a method of obtaining ribosome translation profiling data from a sample is provided. The method comprising: processing the sample according to a RAPPL-Ribo-Seq protocol; and generating the ribosome translation profiling data from the processed sample.

[0018] In some embodiments, the sample is a biological sample; the sample comprises cytoplasmic ribosomes, mitochondrial ribosomes, or a combination thereof; the biological sample is selected from a tissue sample, a blood sample, a non-blood biological fluid sample, and a whole organism sample; and / or the ribosome translation profiling data comprises cytoplasmic ribosome translation profiling data and mitochondrial ribosome translation profiling data. In some embodiments, the sample is at least one of: a smaller volume than a classical ribosome profiling sample minimum required volume; and a smaller total cell number than a classical ribosome profiling sample minimum required cell number.

[0019] Other objects and features will be in part apparent and in part pointed out hereinafter.

[0020] BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Those of skill in the art will understand that the drawings described herein are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0022] The following figure legends correspond with the figures in Appendix B and the discussion in Appendix A.

[0023] FIG. 1 A shows a schematic describing the advantages of RAPPL over conventional methods.

[0024] FIG. 1 B shows bioanalyzer results of eluates purified from E. coli using RAPPL.

[0025] FIG. 1 C shows Western blot analysis of HEK293 lines uL4-HA and uS4- Flag tagged by CRISPR / Cas9 throughout the RAPPL purification process (lysate, flow-through, elutions).

[0026] FIG. 2A shows RAPPL performed using diluted PureExpress® ribosomes as a control to demonstrate the benefits and limitations of our method, showing the potential to isolate ribosomes in the 1 nM range.

[0027] FIG. 2B shows HEK293 cells in which uL4 was HA-tagged by CRISPR / Cas9 enriched and detected with tagged protein in as few as 5000 cells.

[0028] FIG. 2C shows P. falciparum NF54 cells enriched and detected with tagged protein in as few as one million cells.

[0029] FIG. 3A shows RAPPL is able to purify from several single celled organisms: E. coli, S. cerevisiae, T. gondii, P. falciparum, and C. parvum Multicellular organism tissues such as mouse spleen and liver as well as whole organisms like D. rerio can also be processed using RAPPL.

[0030] FIG. 3B shows the purification of compartment-specific ribosomes is also possible with RAPPL. Fractionation of HEK293 cells was performed. These fractions were then subject to the RAPPL method. Eluates were then visualized by TEM showing purification of cytoplasmic, mitochondrial, and nuclear (ribosome biogenesis) ribosomes.

[0031] FIG. 4A shows HeLA and Wheat germ commercial in vitro lysates can be used with RAPPL, which shows reorganization of ribosomes with the introduction of mRNA upon visualization by TEM, demonstrating that RAPPL can isolate actively translating ribosomes.

[0032] FIG. 4B shows reorganization of ribosomes with the introduction of mRNA upon visualization by TEM, demonstrating that RAPPL can isolate actively translating ribosomes.

[0033] FIG. 4C shows RAPPL is compatible with current methods of studying protein synthesis, such as polysome profiling. HEK293 lysates were fractionated using polysome profiling. Fractions corresponding to ribosome subunit and monosomes, light polysomes, and heavy polysomes were pooled, respectively. These pools were diluted 1 :5 to ensure that sucrose did not interfere with binding. The diluted, pooled samples were subject to RAPPL and the eluates visualized by TEM. The results indicate that ribosome and polysome organizations are maintained throughout the purification process.

[0034] FIG. 4D shows a method to visualize ribosome organization when treated with various translation inhibitors. HEK293 lysates were treated with cycloheximide, anisomycine, and harringtonine with untreated lysate as a control. Ribosomes were purified using RAPPL and the eluates visualized by TEM. Ribosome organization demonstrate treatment-dependent outcomes when compared to each other and untreated control. The untreated control shows relatively uniform dispersion of monosomes, disomes, trisomes, and polysomes. The translation elongation inhibitors cycloheximide and anisomycine show an increase in polysomes, as expected. The translation initiation inhibitor harringtonine eluate harbors less polysomes than the translation elongation inhibitors and ostensibly more monosome or initiating ribosomes than untreated control.

[0035] FIG. 5A shows ribosomes purified by RAPPL from E. coli DH5a cells grown to exponential phase, eluting in 30 pL of buffer. Eluates were then used in the PURExpress® in vitro translation system instead of kit ribosomes. Kit ribosomes were diluted 1 :100 (133 pM) and used as controls. A PCR product encoding for eGFP harboring the T7 promoter and a polyA tail was used in the reaction. Reactions were incubated for four hours. Ribosomes purified using RAPPL are active and able to translation mRNA. Additionally, ribosomes were purified as above, however were not eluted from beads, instead beads were resuspended in 30 pL of wash buffer. Beads were incubated in increasing concentrations of eGFP plasmid. RAPPL beadbound ribosomes are also translationally active and can be used in vitro.

[0036] FIG. 5B shows ribosome-associated mechanisms of resistance using RAPPL, ribosomes were purified from E. coli strains harboring rRNA mutations known to provide antibiotic resistance (SQ110 ATC 16S - A1408G and SQ110 ATC 23S - A2058G) as well as uropathogenic E. coli (UPEC) strains (EC13 and EC24). Strains 10B and DH5a were used as controls. In the presence of the macrolide erythromycin UPEC strain EC24 and SQ110 ATC 23S - A2058G are able to synthesize eGFP and grow on plates with the antibiotic. When the aminoglycoside kanamycin is used, UPEC strain EC13 is unable to synthesize eGFP, but able to grow on plates with the antibiotic, demonstrating a nontranslation associated mechanism of resistance versus the SQ110 ATC 16S - A1408G strain, which is able to both translate and grow in its presence. Application of the bacteriostatic peptidyl transferase translational inhibitor chloramphenicol shows that once again EC24 and SQ110 ATC 23S - A2058G strains are resistant in both in vitro plate assays. Finally, both EC24 and SQ110 ATC 23S - A2058G strains show resistance to the lincosamide clindamycin in in vitro plate assays.

[0037] FIG. 5C shows plate bacterial growth assays performed using erythromycin, kanamycin, chloramphenicol, and clindamycin to demonstrate strain resistance. Ribosomes were purified as above and again used in the PURExpress® in vitro translation kit with 5 pM erythromycin, 1.5 pM or 2 pM kanamycin, 50 pM chloramphenicol, or 50 pM clindamycin. The UPEC strain EC24 ribosomes are resistant to the macrolide erythromycin, as is SQ110 ATC 23S - A2058G.

[0038] FIG. 6A shows the compatibility of RAPPL with structural biology applications we used purified products to generate a cryoEM map of the opportunistic intracellular pathogen Cryptococcus neoformans ribosome. C. neoformans cells (~108) in exponential phase were lysed and the clarified lysate used in RAPPL. The ribosomes were eluted in 30 pL of elution buffer. The Eluate was first screened using TEM. Subsequently, grids were prepared using 5 pL of eluate. Movies were captured on FEI Titan Krios G3 300kV Cryo-TEM with Gatan K2 Summit Direct Electron Detection camera and GIF energy filter. Data was processed using Cryosparc resulting in ~ 2.7 A global resolution. The generated C. neoformans map (blue solid) was then aligned with a S. cerevisiae map (gold mesh, PDB: 6TB3) and displayed in multiple orientations for comparison.

[0039] FIG. 7 shows E. coli ribosome mini prep for 1 mL, 2 mL, and 5 mL cultures.

[0040] FIG. 8 shows RAPPL-Ribo Seq has better yield and coverage than classical ribosome profiling techniques from 15-20 times less material (e.g., 15-20 times less volume than a classical ribosome profiling sample minimum required volume and / or 15-20 times less than a classical ribosome profiling sample minimum required cell number).

[0041] FIG. 9 shows mitochondrial gene data showing RAPPL-Ribo-Seq additionally captures mitochondrial RNA (chr M).

[0042] DETAILED DESCRIPTION OF THE DISCLOSURE

[0043] Aspects of the present disclosure provide a rapid, facile, and economical method enabling the purification of high-quality ribosomes, both translating and nontranslating, as well as associated translation factors that are then compatible with functional and structural studies in about 30-60 mins. This method is compatible with many cells, organs, and whole organisms including, for example, Toxoplasma gondii, Plasmodium falciparum, and Cryptosporidium parvum parasites. Significant data was obtained from ribosomes and other molecules purified from limited quantities, as low as ~1 ,000 cells.

[0044] The method uses poly-lysine, or any other poly-cationic polymer for rapid isolation of the ribosomes and associated proteins, mRNAs and tRNAs from biological or non-biological samples using magnetic or other inert beads coated with poly-cationic polymer of different length. Elution of the purified ribosomes is achieved by increased salt concentration, poly-anionic salts, or polymers or directly by chemicals for downstream use of mRNA, proteins, tRNA or ribosomes. The reduced volumes used in the method enable single cell approaches. Molecule isolation according to the present disclosure includes but is not limited to ribosomes, mRNAs associated with ribosomes from biopsies of patients with different diseases, ribosomes from hard to obtain organisms, cells, or tissue samples. Ribosome isolation and structural determination is further included in the present disclosure, as is development of CryoEM grids for determination of ribosome structure and function (translation) from lysed cells, from complex biological material, and from non-biological material.

[0045] In an aspect of the present disclosure, an application of the disclosed method includes E.coli experiments with Urinary Tract Infection (UTI) causing strains and their sensibility to antibiotics. Using the disclosed method of isolating ribosomes from small samples and in vitro translation kit without ribosomes it was shown whether these bacterial ribosomes were resistant to antibiotics (such as erythromycin, kanamycine, etc.). Consequently, the disclosed method can be used to get very fast data on ribosome-associated antibiotic resistance and therefore used for determining which treatment(s) to administer to subjects / patients (e.g., which therapeutic agent(s) to administer).

[0046] The disclosed method rapidly selects and purifies ribosomes and associated mRNAs from biological samples including small and hard-to-get samples (tissues, biopsies, samples having small number of cells), including development of single cell ribosome isolation. This method has clinical, industrial, and scientific use.

[0047] This disclosure also focuses on the use of RAPPL in ribosome profiling for small cell numbers and clinical samples. Ribosome profiling is a method used to access changes in mRNA translation efficiency as well as in gene expression to get insight into dysregulations associated with multiple diseases.

[0048] The RAPPL-Ribo-Seq protocol disclosed herein can get ribosome translation profiling data for both cytoplasmic and mitochondrial ribosomes from the same sample. This is unique for this type of protocol and is further beneficial as it only requires low input amounts of the cell and clinical material.

[0049] Current ribosome profiling methods cannot be used with small cell numbers and have reduced coverage of the genes due to loss of ribosomal material and associated mRNAs. Current methods are also long and require tedious amount of time and materials. The methods as disclosed herein are simple and fast and enable increased depth of sequencing from 20-50 times over current / typical protocols.

[0050] Aspects of the present disclosure provide ribosome profiling using RAPPL method from small cell numbers and clinical samples. MOLECULAR ENGINEERING

[0051] The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

[0052] The term “transfection,” as used herein, refers to the process of introducing nucleic acids into cells by non-viral methods. The term “transduction,” as used herein, refers to the process whereby foreign DNA is introduced into another cell via a viral vector.

[0053] The terms "heterologous DNA sequence", "exogenous DNA segment", or "heterologous nucleic acid”, “transgene”, “exogenous polynucleotide” as used herein, each refers to a sequence that originates from a source foreign (e.g., nonnative) to the particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling or cloning. The terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides. A "homologous" DNA sequence is a DNA sequence that is naturally associated with a host cell into which it is introduced.

[0054] Sequences described herein can also be the reverse, the complement, or the reverse complement of the nucleotide sequences described herein. The RNA goes in the reverse direction compared to the DNA, but its base pairs still match (e.g., G to C). The reverse complementary RNA for a positive strand DNA sequence will be identical to the corresponding negative strand DNA sequence. Reverse complement converts a DNA sequence into its reverse, complement, or reverse-complement counterpart.

[0055] Complementarity is a property shared between two nucleic acid sequences (e.g., RNA, DNA), such that when they are aligned antiparallel to each other, the nucleotide bases at each position will be complementary. Two bases are complementary if they form Watson-Crick base pairs.

[0056] Expression vector, expression construct, plasmid, or recombinant DNA construct is generally understood to refer to a nucleic acid that has been generated via human intervention, including by recombinant means or direct chemical synthesis, with a series of specified nucleic acid elements that permit transcription or translation of a particular nucleic acid in, for example, a host cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, the expression vector can include a nucleic acid to be transcribed operably linked to a promoter.

[0057] An “expression vector”, otherwise known as an “expression construct”, is generally a plasmid or virus designed for gene expression in cells. The vector is used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for protein synthesis to produce the protein encoded by the gene. Expression vectors are the basic tools in biotechnology for the production of proteins. The vector is engineered to contain regulatory sequences that act as enhancer and / or promoter regions and lead to efficient transcription of the gene carried on the expression vector. The goal of a well-designed expression vector is the efficient production of protein, and this may be achieved by the production of significant amount of stable messenger RNA, which can then be translated into protein. The expression of a protein may be tightly controlled, and the protein is only produced in significant quantity when necessary through the use of an inducer, in some systems however the protein may be expressed constitutively. As described herein, Escherichia coli is used as the host for protein production, but other cell types may also be used.

[0058] In molecular biology, an “inducer” is a molecule that regulates gene expression. An inducer can function in two ways, such as:

[0059] (i) By disabling repressors. The gene is expressed because an inducer binds to the repressor. The binding of the inducer to the repressor prevents the repressor from binding to the operator. RNA polymerase can then begin to transcribe operon genes. An operon is a cluster of genes that are transcribed together to give a single messenger RNA (mRNA) molecule, which therefore encodes multiple proteins.

[0060] (ii) By binding to activators. Activators generally bind poorly to activator DNA sequences unless an inducer is present. An activator binds to an inducer and the complex binds to the activation sequence and activates target gene. Removing the inducer stops transcription. Because a small inducer molecule is required, the increased expression of the target gene is called induction.

[0061] Repressor proteins bind to the DNA strand and prevent RNA polymerase from being able to attach to the DNA and synthesize mRNA. Inducers bind to repressors, causing them to change shape and preventing them from binding to DNA. Therefore, they allow transcription, and thus gene expression, to take place.

[0062] For a gene to be expressed, its DNA sequence (or polynucleotide sequence) must be copied (in a process known as transcription) to make a smaller, mobile molecule called messenger RNA (mRNA), which carries the instructions for making a protein to the site where the protein is manufactured (in a process known as translation). Many different types of proteins can affect the level of gene expression by promoting or preventing transcription. In prokaryotes (such as bacteria), these proteins often act on a portion of DNA known as the operator at the beginning of the gene. The promoter is where RNA polymerase, the enzyme that copies the genetic sequence and synthesizes the mRNA, attaches to the DNA strand.

[0063] Some genes are modulated by activators, which have the opposite effect on gene expression as repressors. Inducers can also bind to activator proteins, allowing them to bind to the operator DNA where they promote RNA transcription. Ligands that bind to deactivate activator proteins are not, in the technical sense, classified as inducers, since they have the effect of preventing transcription.

[0064] A “promoter” is generally understood as a nucleic acid control sequence that directs transcription of a nucleic acid. An inducible promoter is generally understood as a promoter that mediates transcription of an operably linked gene in response to a particular stimulus. A promoter can include necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter can optionally include distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.

[0065] A “ribosome binding site”, or “ribosomal binding site (RBS)”, refers to a sequence of nucleotides upstream of the start codon of an mRNA transcript that is responsible for the recruitment of a ribosome during the initiation of translation. Generally, RBS refers to bacterial sequences, although internal ribosome entry sites (IRES) have been described in mRNAs of eukaryotic cells or viruses that infect eukaryotes. Ribosome recruitment in eukaryotes is generally mediated by the 5' cap present on eukaryotic mRNAs.

[0066] A ribosomal skipping sequence (e.g., 2A sequence such as furin-GSG-T2A) can be used in a construct to prevent covalently linking translated amino acid sequences.

[0067] A "transcribable nucleic acid molecule" as used herein refers to any nucleic acid molecule capable of being transcribed into an RNA molecule. Methods are known for introducing constructs into a cell in such a manner that the transcribable nucleic acid molecule is transcribed into a functional mRNA molecule that is translated and therefore expressed as a protein product. Constructs may also be constructed to be capable of expressing antisense RNA molecules, in order to inhibit translation of a specific RNA molecule of interest. For the practice of the present disclosure, conventional compositions and methods for preparing and using constructs and host cells are well known to one skilled in the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001 ) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754).

[0068] The “transcription start site” or "initiation site" is the position surrounding the first nucleotide that is part of the transcribed sequence, which is also defined as position +1. With respect to this site all other sequences of the gene and its controlling regions can be numbered. Downstream sequences (i.e., further protein encoding sequences in the 3' direction) can be denominated positive, while upstream sequences (mostly of the controlling regions in the 5' direction) are denominated negative.

[0069] "Operably-linked" or "functionally linked" refers preferably to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a regulatory DNA sequence is said to be "operably linked to" or "associated with" a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation. The two nucleic acid molecules may be part of a single contiguous nucleic acid molecule and may be adjacent. For example, a promoter is operably linked to a gene of interest if the promoter regulates or mediates transcription of the gene of interest in a cell.

[0070] A "construct" is generally understood as any recombinant nucleic acid molecule such as a plasmid, cosmid, virus, autonomously replicating nucleic acid molecule, phage, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule where one or more nucleic acid molecule has been operably linked.

[0071] A construct of the present disclosure can contain a promoter operably linked to a transcribable nucleic acid molecule operably linked to a 3' transcription termination nucleic acid molecule. In addition, constructs can include but are not limited to additional regulatory nucleic acid molecules from, e.g., the 3'-untranslated region (3' UTR). Constructs can include but are not limited to the 5' untranslated regions (5' UTR) of an mRNA nucleic acid molecule which can play an important role in translation initiation and can also be a genetic component in an expression construct. These additional upstream and downstream regulatory nucleic acid molecules may be derived from a source that is native or heterologous with respect to the other elements present on the promoter construct.

[0072] The term "transformation" refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance. Host cells containing the transformed nucleic acid fragments are referred to as "transgenic" cells, and organisms comprising transgenic cells are referred to as "transgenic organisms".

[0073] "Transformed," "transgenic," and "recombinant" refer to a host cell or organism such as a bacterium, cyanobacterium, animal, or a plant into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome as generally known in the art and disclosed (Sambrook 1989; Innis 1995; Gelfand 1995; Innis & Gelfand 1999). Known methods of PCR include, but are not limited to, methods using self-replicating primers, paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, and the like. The term "untransformed" refers to normal cells that have not been through the transformation process.

[0074] "Wild-type" refers to a virus or organism found in nature without any known mutation.

[0075] Design, generation, and testing of the variant nucleotides, and their encoded polypeptides, having the above-required percent identities and retaining a required activity of the expressed protein is within the skill of the art. For example, directed evolution and rapid isolation of mutants can be according to methods described in references including, but not limited to, Link et al. (2007) Nature Reviews 5(9), 680- 688; Sanger et al. (1991 ) Gene 97(1 ), 119-123; Ghadessy et al. (2001 ) Proc Natl Acad Sci USA 98(8) 4552-4557. Thus, one skilled in the art could generate a large number of nucleotide and / or polypeptide variants having, for example, at least 95- 99% identity to the reference sequence described herein and screen such for desired phenotypes according to methods routine in the art.

[0076] Nucleotide and / or amino acid sequence identity percent (%) is understood as the percentage of nucleotide or amino acid residues that are identical with nucleotide or amino acid residues in a candidate sequence in comparison to a reference sequence when the two sequences are aligned. To determine percent identity, sequences are aligned and if necessary, gaps are introduced to achieve the maximum percent sequence identity. Sequence alignment procedures to determine percent identity are well known to those of skill in the art. Often publicly available computer software such as BLAST, BLAST2, ALIGN2, or Megalign (DNASTAR) software is used to align sequences. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. When sequences are aligned, the percent sequence identity of a given sequence A to, with, or against a given sequence B (which can alternatively be phrased as a given sequence A that has or comprises a certain percent sequence identity to, with, or against a given sequence B) can be calculated as: percent sequence identity = X / Y100, where X is the number of residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and Y is the total number of residues in B. If the length of sequence A is not equal to the length of sequence B, the percent sequence identity of A to B will not equal the percent sequence identity of B to A. For example, the percent identity can be at least 80% or about 80%, about 81 %, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%.

[0077] Substitution refers to the replacement of one amino acid with another amino acid in a protein or the replacement of one nucleotide with another in DNA or RNA. Insertion refers to the insertion of one or more amino acids in a protein or the insertion of one or more nucleotides with another in DNA or RNA. Deletion refers to the deletion of one or more amino acids in a protein or the deletion of one or more nucleotides with another in DNA or RNA. Generally, substitutions, insertions, or deletions can be made at any position so long as the required activity is retained.

[0078] “Point mutation” refers to when a single base pair is altered. A point mutation or substitution is a genetic mutation where a single nucleotide base is changed, inserted, or deleted from a DNA or RNA sequence of an organism's genome. Point mutations have a variety of effects on the downstream protein product — consequences that are moderately predictable based upon the specifics of the mutation. These consequences can range from no effect (e.g., synonymous mutations) to deleterious effects (e.g., frameshift mutations), with regard to protein production, composition, and function. Point mutations can have one of three effects. First, the base substitution can be a silent mutation where the altered codon corresponds to the same amino acid. Second, the base substitution can be a missense mutation where the altered codon corresponds to a different amino acid. Or third, the base substitution can be a nonsense mutation where the altered codon corresponds to a stop signal. Silent mutations result in a new codon (a triplet nucleotide sequence in RNA) that codes for the same amino acid as the wild type codon in that position. In some silent mutations the codon codes for a different amino acid that happens to have the same properties as the amino acid produced by the wild type codon. Missense mutations involve substitutions that result in functionally different amino acids; these can lead to alteration or loss of protein function. Nonsense mutations, which are a severe type of base substitution, result in a stop codon in a position where there was not one before, which causes the premature termination of protein synthesis and can result in a complete loss of function in the finished protein.

[0079] Generally, conservative substitutions can be made at any position so long as the required activity is retained. So-called conservative exchanges can be carried out in which the amino acid which is replaced has a similar property as the original amino acid, for example, the exchange of Glu by Asp, Gin by Asn, Vai by lie, Leu by lie, and Ser by Thr. For example, amino acids with similar properties can be Aliphatic amino acids (e.g., Glycine, Alanine, Valine, Leucine, Isoleucine); hydroxyl or sulfur / selenium-containing amino acids (e.g., Serine, Cysteine, Selenocysteine, Threonine, Methionine); Cyclic amino acids (e.g., Proline); Aromatic amino acids (e.g., Phenylalanine, Tyrosine, Tryptophan); Basic amino acids (e.g., Histidine, Lysine, Arginine); or Acidic and their Amide (e.g., Aspartate, Glutamate, Asparagine, Glutamine). Deletion is the replacement of an amino acid by a direct bond. Positions for deletions include the termini of a polypeptide and linkages between individual protein domains. Insertions are introductions of amino acids into the polypeptide chain, a direct bond formally being replaced by one or more amino acids. An amino acid sequence can be modulated with the help of art-known computer simulation programs that can produce a polypeptide with, for example, improved activity or altered regulation. On the basis of these artificially generated polypeptide sequences, a corresponding nucleic acid molecule coding for such a modulated polypeptide can be synthesized in-vitro using the specific codon-usage of the desired host cell.

[0080] “Highly stringent hybridization conditions” are defined as hybridization at 65 °C in a 6 X SSC buffer (i.e. , 0.9 M sodium chloride and 0.09 M sodium citrate). Given these conditions, a determination can be made as to whether a given set of sequences will hybridize by calculating the melting temperature (Tm) of a DNA duplex between the two sequences. If a particular duplex has a melting temperature lower than 65°C in the salt conditions of a 6 X SSC, then the two sequences will not hybridize. On the other hand, if the melting temperature is above 65 °C in the same salt conditions, then the sequences will hybridize. In general, the melting temperature for any hybridized DNA: DNA sequence can be determined using the following formula: Tm = 81.5 °C + 16.6(log [Na+]) + 0.41 (fraction G / C content) - 0.63(% formamide) - (600 / I). Furthermore, the Tm of a DNA:DNA hybrid is decreased by 1 -1.5°C for every 1 % decrease in nucleotide identity (see e.g., Sambrook and Russel, 2006).

[0081] Host cells can be transformed using a variety of standard techniques known to the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001 ) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754). Such techniques include, but are not limited to, viral infection, calcium phosphate transfection, liposome-mediated transfection, microprojectile-mediated delivery, receptor-mediated uptake, cell fusion, electroporation, and the like. The transformed cells can be selected and propagated to provide recombinant host cells that comprise the expression vector stably integrated in the host cell genome.

[0082] Conservative Substitutions Negatively Charged (Acidic): D E

[0083] Exemplary nucleic acids that may be introduced to a host cell include, for example, DNA sequences or genes from another species, or even genes or sequences which originate with or are present in the same species, but are incorporated into recipient cells by genetic engineering methods. The term “exogenous” is also intended to refer to genes that are not normally present in the cell being transformed, or perhaps simply not present in the form, structure, etc., as found in the transforming DNA segment or gene, or genes which are normally present and that one desires to express in a manner that differs from the natural expression pattern, e.g., to over-express. Thus, the term “exogenous” gene or DNA is intended to refer to any gene or DNA segment that is introduced into a recipient cell, regardless of whether a similar gene may already be present in such a cell. The type of DNA included in the exogenous DNA can include DNA that is already present in the cell, DNA from another individual of the same type of organism, DNA from a different organism, or a DNA generated externally, such as a DNA sequence containing an antisense message of a gene, or a DNA sequence encoding a synthetic or modified version of a gene.

[0084] Host strains developed according to the approaches described herein can be evaluated by a number of means known in the art (see e.g., Studier (2005) Protein Expr Purif. 41 (1 ), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN- 10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).

[0085] Methods of down-regulation or silencing genes are known in the art. For example, expressed protein activity can be down-regulated or eliminated using antisense oligonucleotides (ASOs), protein aptamers, nucleotide aptamers, and RNA interference (RNAi) (e.g., small interfering RNAs (siRNA), short hairpin RNA (shRNA), single guide RNA (sgRNA), and micro RNAs (miRNA) (see e.g., Rinaldi and Wood (2017) Nature Reviews Neurology 14, describing ASO therapies; Fanning and Symonds (2006) Handb Exp Pharmacol. 173, 289-303G, describing hammerhead ribozymes and small hairpin RNA; Helene, et al. (1992) Ann. N.Y. Acad. Sci. 660, 27-36; Maher (1992) Bioassays 14(12): 807-15, describing targeting deoxyribonucleotide sequences; Lee et al. (2006) Curr Opin Chem Biol. 10, 1 -8, describing aptamers; Reynolds et al. (2004) Nature Biotechnology 22(3), 326 - 330, describing RNAi; Pushparaj and Melendez (2006) Clinical and Experimental Pharmacology and Physiology 33(5-6), 504-510, describing RNAi; Dillon et al. (2005) Annual Review of Physiology 67, 147-173, describing RNAi; Dykxhoorn and Lieberman (2005) Annual Review of Medicine 56, 401 -423, describing RNAi). RNAi molecules are commercially available from a variety of sources (e.g., Ambion, TX; Sigma Aldrich, MO; Invitrogen). Several siRNA molecule design programs using a variety of algorithms are known to the art (see e.g., Cenix algorithm, Ambion; BLOCK-iT™ RNAi Designer, Invitrogen; siRNA Whitehead Institute Design Tools, Bioinformatics & Research Computing). Traits influential in defining optimal siRNA sequences include G / C content at the termini of the siRNAs, Tm of specific internal domains of the siRNA, siRNA length, position of the target sequence within the CDS (coding region), and nucleotide content of the 3' overhangs.

[0086] FORMULATION

[0087] The agents and compositions described herein can be formulated by any conventional manner using one or more pharmaceutically acceptable carriers or excipients as described in, for example, Remington’s Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21 st edition, ISBN: 0781746736 (2005), incorporated herein by reference in its entirety. Such formulations will contain a therapeutically effective amount of a biologically active agent described herein, which can be in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.

[0088] The term "formulation" refers to preparing a drug in a form suitable for administration to a subject, such as a human. Thus, a "formulation" can include pharmaceutically acceptable excipients, including diluents or carriers.

[0089] The term "pharmaceutically acceptable" as used herein can describe substances or components that do not cause unacceptable losses of pharmacological activity or unacceptable adverse side effects. Examples of pharmaceutically acceptable ingredients can be those having monographs in United States Pharmacopeia (USP 29) and National Formulary (NF 24), United States Pharmacopeial Convention, Inc, Rockville, Maryland, 2005 ("USP / NF"), or a more recent edition, and the components listed in the continuously updated Inactive Ingredient Search online database of the FDA. Other useful components that are not described in the USP / NF, etc., may also be used.

[0090] The term “pharmaceutically acceptable excipient,” as used herein, can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic, or absorption delaying agents. The use of such media and agents for pharmaceutically active substances is well known in the art (see generally Remington’s Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21 st edition, ISBN: 0781746736 (2005)). Except insofar as any conventional media or agent is incompatible with an active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[0091] A "stable" formulation or composition can refer to a composition having sufficient stability to allow storage at a convenient temperature, such as between about 0 °C and about 60 °C, for a commercially reasonable period of time, such as at least about one day, at least about one week, at least about one month, at least about three months, at least about six months, at least about one year, or at least about two years.

[0092] The formulation should suit the mode of administration. The agents of use with the current disclosure can be formulated by known methods for administration to a subject using several routes which include, but are not limited to, parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhalation (e.g., in an aerosol), implanted, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, intrathecal, ophthalmic, transdermal, buccal, and rectal. The individual agents may also be administered in combination with one or more additional agents or together with other biologically active or biologically inert agents. Such biologically active or inert agents may be in fluid or mechanical communication with the agent(s) or attached to the agent(s) by ionic, covalent, Van der Waals, hydrophobic, hydrophilic, or other physical forces.

[0093] Controlled-release (or sustained-release) preparations may be formulated to extend the activity of the agent(s) and reduce dosage frequency. Controlled-release preparations can also be used to affect the time of onset of action or other characteristics, such as blood levels of the agent, and consequently, affect the occurrence of side effects. Controlled-release preparations may be designed to initially release an amount of an agent(s) that produces the desired therapeutic effect, and gradually and continually release other amounts of the agent to maintain the level of therapeutic effect over an extended period of time. In order to maintain a near-constant level of an agent in the body, the agent can be released from the dosage form at a rate that will replace the amount of agent being metabolized or excreted from the body. The controlled-release of an agent may be stimulated by various inducers, e.g., change in pH, change in temperature, enzymes, water, or other physiological conditions or molecules.

[0094] Agents or compositions described herein can also be used in combination with other therapeutic modalities, as described further below. Thus, in addition to the therapies described herein, one may also provide to the subject other therapies known to be efficacious for treatment of the disease, disorder, or condition.

[0095] THERAPEUTIC METHODS

[0096] Also provided is a process of treating, preventing, or reversing a disease, disorder, or condition in a subject in need thereof (e.g., a bacterial, fungal, or parasitic infectious disease) via administration of a therapeutically effective amount of a therapeutic agent (e.g., antibiotic medication or treatment, antifungal medication or treatment, antiparasitic medication or treatment, or other drug or treatment), so as to efficiently treat patients more timely than currently available diagnostic or analytical testing.

[0097] Methods described herein are generally performed on a subject in need thereof. A subject in need of the therapeutic methods described herein can be a subject having, diagnosed with, suspected of having, or at risk for developing a disease, disorder, or condition described herein. A determination of the need for treatment will typically be assessed by a history, physical exam, or diagnostic tests consistent with the disease or condition at issue. Diagnosis of the various conditions treatable by the methods described herein is within the skill of the art. The subject can be an animal subject, including a mammal, such as horses, cows, dogs, cats, sheep, pigs, mice, rats, monkeys, hamsters, guinea pigs, and humans or chickens. For example, the subject can be a human subject.

[0098] Generally, a safe and effective amount of a therapeutic agent is, for example, an amount that would cause the desired therapeutic effect in a subject while minimizing undesired side effects. In various embodiments, an effective amount of a therapeutic agent described herein can substantially inhibit, slow the progress of, or limit the development of a disease, disorder, or condition described herein.

[0099] According to the methods described herein, administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, intratumoral, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration. When used in the treatments described herein, a therapeutically effective amount of a therapeutic agent can be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt form and with or without a pharmaceutically acceptable excipient. For example, the compounds of the present disclosure can be administered, at a reasonable benefit / risk ratio applicable to any medical treatment, in a sufficient amount to treat, prevent, or reverse a disease, disorder, or condition described herein (e.g., a bacterial, fungal, or parasitic infectious disease).

[0100] The amount of a composition described herein that can be combined with a pharmaceutically acceptable carrier to produce a single dosage form will vary depending upon the subject or host treated and the particular mode of administration. It will be appreciated by those skilled in the art that the unit content of agent contained in an individual dose of each dosage form need not in itself constitute a therapeutically effective amount, as the necessary therapeutically effective amount could be reached by administration of a number of individual doses.

[0101] Toxicity and therapeutic efficacy of compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals for determining the LDso (the dose lethal to 50% of the population) and the EDso, (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index that can be expressed as the ratio LD50 / ED50, where larger therapeutic indices are generally understood in the art to be optimal.

[0102] The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the seventy of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration; the route of administration; the rate of excretion of the composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts (see e.g., Koda-Kimble et al. (2004) Applied Therapeutics: The Clinical Use of Drugs, Lippincott Williams & Wilkins, ISBN 0781748453; Winter (2003) Basic Clinical Pharmacokinetics, 4thed., Lippincott Williams & Wilkins, ISBN 0781741475; Sharqel (2004) Applied Biopharmaceutics & Pharmacokinetics, McGraw-Hill / Appleton & Lange, ISBN 0071375503). For example, it is well within the skill of the art to start doses of the composition at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose may be divided into multiple doses for purposes of administration. Consequently, single dose compositions may contain such amounts or submultiples thereof to make up the daily dose. It will be understood, however, that the total daily usage of the compounds and compositions of the present disclosure will be decided by an attending physician within the scope of sound medical judgment.

[0103] Again, each of the states, diseases, disorders, and conditions, described herein, as well as others, can benefit from compositions and methods described herein. Generally, treating a state, disease, disorder, or condition includes reversing or delaying the appearance of clinical symptoms in a mammal that may be afflicted with or predisposed to the state, disease, disorder, or condition but does not yet experience or display clinical or subclinical symptoms thereof. Treating can also include inhibiting the state, disease, disorder, or condition, e.g., arresting or reducing the development of the disease or at least one clinical or subclinical symptom thereof. Furthermore, treating can include relieving the disease, e.g., causing regression of the state, disease, disorder, or condition or at least one of its clinical or subclinical symptoms. A benefit to a subject to be treated can be either statistically significant or at least perceptible to the subject or a physician.

[0104] Administration of a therapeutic agent can occur as a single event or over a time course of treatment. For example, a therapeutic agent can be administered daily, weekly, bi-weekly, or monthly. For treatment of acute conditions, the time course of treatment will usually be at least several days. Certain conditions could extend treatment from several days to several weeks. For example, treatment could extend over one week, two weeks, or three weeks. For more chronic conditions, treatment could extend from several weeks to several months or even a year or more.

[0105] Treatment in accord with the methods described herein can be performed prior to or before, concurrent with, or after conventional treatment modalities for diseases, disorder, and conditions disclosed herein (e.g., a bacterial, fungal, or parasitic infectious disease) A therapeutic agent can be administered simultaneously or sequentially with another agent, such as an antibiotic, an anti-inflammatory, or another agent. For example, a therapeutic agent can be administered simultaneously with another agent, such as an antibiotic or an anti-inflammatory. Simultaneous administration can occur through administration of separate compositions, each containing one or more of a therapeutic agent, an antibiotic, an anti-inflammatory, or another agent. Simultaneous administration can occur through administration of one composition containing two or more of a therapeutic agent, an antibiotic, an anti-inflammatory, or another agent. A therapeutic agent can be administered sequentially with an antibiotic, an anti-inflammatory, or another agent. For example, a therapeutic agent can be administered before or after administration of an antibiotic, an antiinflammatory, or another agent.

[0106] Active compounds are administered at a therapeutically effective dosage sufficient to treat a condition associated with a condition in a patient. For example, the efficacy of a compound can be evaluated in an animal model system that may be predictive of efficacy in treating the disease in a human or another animal, such as the model systems shown in the examples and drawings.

[0107] An effective dose range of a therapeutic can be extrapolated from effective doses determined in animal studies for a variety of different animals. In general, a human equivalent dose (HED) in mg / kg can be calculated in accordance with the following formula (see e.g., Reagan-Shaw et al., FASEB J., 22(3):659-661 , 2008, which is incorporated herein by reference):

[0108] HED (mg / kg) = Animal dose (mg / kg) x (Animal Km / Human Km)

[0109] Use of the Km factors in conversion results in more accurate HED values, which are based on body surface area (BSA) rather than only on body mass. Km values for humans and various animals are well known. For example, the Km for an average 60 kg human (with a BSA of 1 .6 m2) is 37, whereas a 20 kg child (BSA 0.8 m2) would have a Km of 25. Km for some relevant animal models are also well known, including: mice Km of 3 (given a weight of 0.02 kg and BSA of 0.007); hamster Km of 5 (given a weight of 0.08 kg and BSA of 0.02); rat Km of 6 (given a weight of 0.15 kg and BSA of 0.025) and monkey Km of 12 (given a weight of 3 kg and BSA of 0.24).

[0110] Precise amounts of the therapeutic composition depend on the judgment of the practitioner and are peculiar to each individual. Nonetheless, a calculated HED dose provides a general guide. Other factors affecting the dose include the physical and clinical state of the patient, the route of administration, the intended goal of treatment, and the potency, stability, and toxicity of the particular therapeutic formulation.

[0111] The actual dosage amount of a compound of the present disclosure or composition comprising a compound of the present disclosure administered to a subject may be determined by physical and physiological factors such as type of animal treated, age, sex, body weight, seventy of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the subject and on the route of administration. These factors may be determined by a skilled artisan. The practitioner responsible for administration will typically determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject. The dosage may be adjusted by the individual physician in the event of any complication.

[0112] In some embodiments, the therapeutic agent may be administered in an amount from about 1 mg / kg to about 100 mg / kg, or about 1 mg / kg to about 50 mg / kg, or about 1 mg / kg to about 25 mg / kg, or about 1 mg / kg to about 15 mg / kg, or about 1 mg / kg to about 10 mg / kg, or about 1 mg / kg to about 5 mg / kg, or about 3 mg / kg. In some embodiments, a therapeutic agent may be administered in a range of about 1 mg / kg to about 200 mg / kg, or about 50 mg / kg to about 200 mg / kg, or about 50 mg / kg to about 100 mg / kg, or about 75 mg / kg to about 100 mg / kg, or about 100 mg / kg.

[0113] The effective amount may be less than 1 mg / kg / day, less than 500 mg / kg / day, less than 250 mg / kg / day, less than 100 mg / kg / day, less than 50 mg / kg / day, less than 25 mg / kg / day or less than 10 mg / kg / day. It may alternatively be in the range of 1 mg / kg / day to 200 mg / kg / day.

[0114] In other non-limiting examples, a dose may also comprise from about 1 micro- gram / kg / body weight, about 5 microgram / kg / body weight, about 10 microgram / kg / body weight, about 50 microgram / kg / body weight, about 100 microgram / kg / body weight, about 200 microgram / kg / body weight, about 350 microgram / kg / body weight, about 500 microgram / kg / body weight, about 1 milligram / kg / body weight, about 5 milligram / kg / body weight, about 10 milligram / kg / body weight, about 50 milligram / kg / body weight, about 100 milligram / kg / body weight, about 200 milligram / kg / body weight, about 350 milligram / kg / body weight, about 500 milligram / kg / body weight, to about 1000 mg / kg / body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg / kg / body weight to about 100 mg / kg / body weight, about 5 microgram / kg / body weight to about 500 milligram / kg / body weight, etc., can be administered, based on the numbers described above.

[0115] ADMINISTRATION

[0116] Agents and compositions described herein can be administered according to methods described herein in a variety of means known to the art. The agents and composition can be used therapeutically either as exogenous materials or as endogenous materials. Exogenous agents are those produced or manufactured outside of the body and administered to the body. Endogenous agents are those produced or manufactured inside the body by some type of device (biologic or other) for delivery within or to other organs in the body.

[0117] As discussed above, administration can be parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhalation (e.g., in an aerosol), implanted, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, intrathecal, ophthalmic, transdermal, buccal, and rectal.

[0118] Agents and compositions described herein can be administered in a variety of methods well known in the arts. Administration can include, for example, methods involving oral ingestion, direct injection (e.g., systemic or stereotactic), implantation of cells engineered to secrete the factor of interest, drug-releasing biomaterials, polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, implantable matrix devices, mini-osmotic pumps, implantable pumps, injectable gels and hydrogels, liposomes, micelles (e.g., up to 30 pm), nanospheres (e.g., less than 1 pm), microspheres (e.g., 1 -100 pm), reservoir devices, a combination of any of the above, or other suitable delivery vehicles to provide the desired release profile in varying proportions. Other methods of controlled-release delivery of agents or compositions will be known to the skilled artisan and are within the scope of the present disclosure.

[0119] Delivery systems may include, for example, an infusion pump which may be used to administer the agent or composition in a manner similar to that used for delivering insulin or chemotherapy to specific organs or tumors. Typically, using such a system, an agent or composition can be administered in combination with a biodegradable, biocompatible polymeric implant that releases the agent over a controlled period of time at a selected site. Examples of polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, and copolymers and combinations thereof. In addition, a controlled release system can be placed in proximity of a therapeutic target, thus requiring only a fraction of a systemic dosage.

[0120] Agents can be encapsulated and administered in a variety of carrier delivery systems. Examples of carrier delivery systems include microspheres, hydrogels, polymeric implants, smart polymeric carriers, and liposomes (see generally, Uchegbu and Schatzlein, eds. (2006) Polymers in Drug Delivery, CRC, ISBN-10: 0849325331 ). Carrier-based systems for molecular or biomolecular agent delivery can: provide for intracellular delivery; tailor biomolecule / agent release rates; increase the proportion of biomolecule that reaches its site of action; improve the transport of the drug to its site of action; allow colocalized deposition with other agents or excipients; improve the stability of the agent in vivo prolong the residence time of the agent at its site of action by reducing clearance; decrease the nonspecific delivery of the agent to nontarget tissues; decrease irritation caused by the agent; decrease toxicity due to high initial doses of the agent; alter the immunogenicity of the agent; decrease dosage frequency; improve taste of the product; or improve shelf life of the product.

[0121] SCREENING

[0122] Also provided are screening methods.

[0123] The subject methods find use in the screening of a variety of different candidate molecules (e.g., potentially therapeutic candidate molecules). Candidate substances for screening according to the methods described herein include, but are not limited to, fractions of tissues or cells, nucleic acids, polypeptides, siRNAs, antisense molecules, aptamers, ribozymes, triple helix compounds, antibodies, and small (e.g., less than about 2000 MW, or less than about 1000 MW, or less than about 800 MW) organic molecules or inorganic molecules including but not limited to salts or metals.

[0124] Candidate molecules encompass numerous chemical classes, for example, organic molecules, such as small organic compounds having a molecular weight of more than 50 and less than about 2,500 Daltons. Candidate molecules can comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl, or carboxyl group, and usually at least two of the functional chemical groups. The candidate molecules can comprise cyclical carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups.

[0125] A candidate molecule can be a compound in a library database of compounds. One of skill in the art will be generally familiar with, for example, numerous databases for commercially available compounds for screening (see e.g., ZINC database, LICSF, with 2.7 million compounds over 12 distinct subsets of molecules; Irwin and Shoichet (2005) J Chem Inf Model 45, 177-182). One of skill in the art will also be familiar with a variety of search engines to identify commercial sources or desirable compounds and classes of compounds for further testing (see e.g., ZINC database; eMolecules.com; and electronic libraries of commercial compounds provided by vendors, for example, ChemBridge, Princeton BioMolecular, Ambinter SARL, Enamine, ASDI, Life Chemicals, etc.).

[0126] Candidate molecules for screening according to the methods described herein include both lead-like compounds and drug-like compounds. A lead-like compound is generally understood to have a relatively smaller scaffold-like structure (e.g., molecular weight of about 150 to about 350 kD) with relatively fewer features (e.g., less than about 3 hydrogen donors and / or less than about 6 hydrogen acceptors; hydrophobicity character xlogP of about -2 to about 4). In contrast, a drug-like compound is generally understood to have a relatively larger scaffold (e.g., molecular weight of about 150 to about 500 kD) with relatively more numerous features (e.g., less than about 10 hydrogen acceptors and / or less than about 8 rotatable bonds; hydrophobicity character xlogP of less than about 5) (see e.g., Lipinski (2000) J. Pharm. Tox. Methods 44, 235-249). Initial screening can be performed with lead-like compounds.

[0127] When designing a lead from spatial orientation data, it can be useful to understand that certain molecular structures are characterized as being “drug-like”. Such characterization can be based on a set of empirically recognized qualities derived by comparing similarities across the breadth of known drugs within the pharmacopoeia. While it is not required for drugs to meet all, or even any, of these characterizations, it is far more likely for a drug candidate to meet with clinical success if it is drug-like.

[0128] Several of these “drug-like” characteristics have been summarized into the four rules of Lipinski (generally known as the “rules of fives” because of the prevalence of the number 5 among them). While these rules generally relate to oral absorption and are used to predict the bioavailability of a compound during lead optimization, they can serve as effective guidelines for constructing a lead molecule during rational drug design efforts such as may be accomplished by using the methods of the present disclosure.

[0129] The four “rules of five” state that a candidate drug-like compound should have at least three of the following characteristics: (i) a weight less than 500 Daltons; (ii) a log of P less than 5; (iii) no more than 5 hydrogen bond donors (expressed as the sum of OH and NH groups); and (iv) no more than 10 hydrogen bond acceptors (the sum of N and O atoms). Also, drug-like molecules typically have a span (breadth) of between about 8A to about 15A.

[0130] KITS

[0131] Also provided are kits. Such kits can include an agent or composition described herein and, in certain embodiments, instructions for administration. Such kits can facilitate performance of the methods described herein. When supplied as a kit, the different components of the composition can be packaged in separate containers and admixed immediately before use. Components include, but are not limited to reagents and materials disclosed herein. Such packaging of the components separately can, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition. The pack may, for example, comprise metal or plastic foil such as a blister pack. Such packaging of the components separately can also, in certain instances, permit longterm storage without losing activity of the components.

[0132] Kits may also include reagents in separate containers such as, for example, sterile water or saline to be added to a lyophilized active component packaged separately. For example, sealed glass ampules may contain a lyophilized component and in a separate ampule, sterile water, sterile saline each of which has been packaged under a neutral non-reacting gas, such as nitrogen. Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramic, metal, or any other material typically employed to hold reagents. Other examples of suitable containers include bottles that may be fabricated from similar substances as ampules and envelopes that may consist of foil-lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, and the like. Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle. Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix. Removable membranes may be glass, plastic, rubber, and the like.

[0133] In certain embodiments, kits can be supplied with instructional materials. Instructions may be printed on paper or another substrate, and / or may be supplied as an electronic-readable medium or video. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an Internet web site specified by the manufacturer or distributor of the kit.

[0134] A control sample or a reference sample as described herein can be a sample from a healthy subject or sample, a wild-type subject or sample, or from populations thereof. A reference value can be used in place of a control or reference sample, which was previously obtained from a healthy subject or a group of healthy subjects or a wild-type subject or sample. A control sample or a reference sample can also be a sample with a known amount of a detectable compound or a spiked sample.

[0135] The methods and procedures of the invention may be fully or partially automated, and thus fully or partially executed by a controller or processor. Furthermore, methods and algorithms of the present invention, can be embodied as a computer-implemented method or methods for performing such computer- implemented method or methods, and can also be embodied in the form of a tangible or non-transitory computer-readable storage medium containing a computer program or other machine-readable instructions (herein “computer program”), wherein when the computer program is loaded into a computer or other processor (herein “computer”) and / or is executed by the computer, the computer becomes an apparatus for practicing the method or methods. Storage media for containing such computer program include, for example, floppy disks and diskettes, compact disk (CD)-ROMs (whether or not writeable), DVD digital disks, RAM and ROM memories, computer hard drives and back-up drives, external hard drives, “thumb” drives, and any other storage medium readable by a computer. The method or methods can also be embodied in the form of a computer program, for example, whether stored in a storage medium or transmitted over a transmission medium such as electrical conductors, fiber optics or other light conductors, or by electromagnetic radiation, wherein when the computer program is loaded into a computer and / or is executed by the computer, the computer becomes an apparatus for practicing the method or methods. The method or methods may be implemented on a general-purpose microprocessor or on a digital processor specifically configured to practice the process or processes. When a general-purpose microprocessor is employed, the computer program code configures the circuitry of the microprocessor to create specific logic circuit arrangements. Storage medium readable by a computer includes medium being readable by a computer per se or by another machine that reads the computer instructions for providing those instructions to a computer for controlling its operation. Such machines may include, for example, machines for reading the storage media mentioned above.

[0136] Compositions and methods described herein utilizing molecular biology protocols can be according to a variety of standard techniques known to the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001 ) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754; Studier (2005) Protein Expr Purif. 41 (1 ), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).

[0137] Definitions and methods described herein are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. In some embodiments, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term “about.” In some embodiments, the term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. In some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. The recitation of discrete values is understood to include ranges between each value.

[0138] In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise. In some embodiments, the term “or” as used herein, including the claims, is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

[0139] The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition or device that “comprises,” “has” or “includes” one or more features is not limited to possessing only those one or more features and can cover other unlisted features.

[0140] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

[0141] Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0142] All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.

[0143] Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing the scope of the present disclosure defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.

[0144] EXAMPLES

[0145] The following non-limiting examples are provided to further illustrate the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the present disclosure, and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

[0146] Example 1 RAPPL enriches ribosomes and associated factors

[0147] RAPPL is an anion exchange and affinity-based purification method that exploits the negatively charged backbone of RNA and positively charged s-amino groups of poly-lysines at the physiological relevant pH 7.5 (FIG. 1 A). For purification of ribosomes and associated factors cells or full organisms were grown and treated per application requirements. Lysis was performed in a typical ribosome isolation low-salt buffer containing DNase I, RNase inhibitors, and protease inhibitors. Crude lysate was clarified from cell membranes and nonsoluble particles by a short centrifugation, and then bound to magnetic poly-lysine beads for an average of 15-30 minutes at 4°C. The beads were then washed in buffer without detergent and elution was carried by incubating the beads in wash buffer containing poly-D-glutamic acid for 15 minutes at either room temperature or 4°C. The overall procedure is as such on the average time frame of 45 min to 1 hour and the eluted material can be then either stored or applied to downstream applications. The workflow, compared with more traditional methods, is displayed in FIG. 1A

[0148] To determine capacity and possibility of poly-lysine beads to bind ribosomes and translation associated material ribosomes from the PURExpress NEB System were incubated with commercially obtained poly-lysine beads. Control and ribosome bound beads were visualized via electron microscopy. The ribosomes were clearly visible associated with magnetic particles coated with poly-lysine when compared to beads without ribosome incubation. The beads incubated with ribosomes also appeared darker, with increased negative staining, and have less feathering at their periphery suggesting dense ribosome binding over the surface of the beads. In support to this observation, an addition of the elution buffer with poly-D-glutamic acid to the beads increased number of ribosomes in imaging fields, arguing for strong surface binding of the ribosomes to the poly-lysine beads.

[0149] To get an assessment of the possible use of poly-lysine beads for purification of ribosomes from complex lysates a cell lysate obtained from E. coli strain DH5a was used. After RAPPL procedure (FIG. 1 A) the quality of the material purified by bioanalyzer was determined (FIG. 1B). The analyzed sample indicated a significant enrichment of both 16S and 23S rRNAs, as well as other shorter RNA species. To further demonstrate RAPPL enrichment of ribosomes and translation- associated factors, such as ribosomal proteins and initiation factors, over those not associated with protein synthesis CRISPR / Cas9 engineered Hek293 cell lines were used. RAPPL was performed using lysates from HEK 293 cell lines in which RPS-9 (uS4) and RPL4 (uL4) have been Flag- and HA-tagged, respectively, by insertion of tag sequences in endogenous loci of appropriate gene. Western blot analyses of RAPPL purified HEK 293 samples indicated complete binding of tagged ribosomal or translation-associated proteins to poly-lysine beads without a visible band in either flowthrough or wash fractions. In addition to the tagged proteins, the binding of native RPS16 (uS9), elF3a and elF4A1 initiation factors, and GAPDH protein was tested. While RPS16 (uS9) and elF3a were readily detectable in poly-lysine bound fractions, heavily abundant elF4A1 was detectable in all fractions and GAPDH remained only in the flow-through fraction during RAPPL method (FIG. 1C).

[0150] Finally, to fully detail the repertoire of proteins enriched by RAPPL, HEK 293 cells and E. coli strain DH5a cells were purified in triplicate on poly-lysine beads followed by quantitative mass spectrometry analysis. Mass spectrometry analysis was performed in triplicate and further demonstrates the binding of ribosome- and translation-associated factors, whereby in E. coli 33 / 34 and 22 / 24 large and small subunit ribosomal proteins are among the first 138 hits by ppm. 13 / 15 ribosomal methyltransferase and all pseudouridine synthases were detected. All initiation factors, all elongation factors and release factors were also detected along with many other known and suspected ribosome-associated proteins. In the case of HEK293 cells, 36 / 44 and 48 / 60 proteins of small and large cytosolic ribosome were found. 31 / 32 and 52 / 56 proteins of small and large mitochondrial ribosomes were also detected. All 13 elF3 complex members, 13 / 15, and 15 / 17 members of 43S and 48S preinitiation complex were also isolated. All translation elongation factors for both cytosolic and mitochondrial ribosomes, including seleno-cysteine elongation factor, were detected. Lastly, release and ribosome biogenesis factors and translation or mRNA associated proteins were detected. The results also indicate that the presence of multiple novel proteins.

[0151] By exploiting the negatively charged RNA backbone and the positively charged s-amino groups of poly-lysines, ribosomes of good quality can be isolated with a relatively fast protocol from cell lysate. Using RAPPL, not only ribosomes, but most of the expected translation-associated factors were isolated. In addition, many factors that have been suspected to be associated with translation were isolated as well as some new candidates. Isolation of these translation-associated components exhibits up to an 80-fold enrichment.

[0152] Example 2 RAPPL overcomes material scarcity limitations

[0153] Limited sample material is a significant hurdle to overcome for many purifications including one associated with ribosomes and translation-associated material. This is often the case for clinically relevant samples, specific cell types or organs and often exaggerated in parasitology - with intercellular parasites being present in small numbers and at certain stages of various parasite life cycles. Therefore, the lower limits of RAPPL was tested by performing purifications on decreasing cell numbers (FIGS. 2A-2C). Ribosomes from the PurExpress® System were used first which provided purified and highly concentrated E. coli ribosomes in a known quantity (13.3 mM). The ribosomes were serially diluted starting at 13.3 pM down to 1 .3 nM by a factor of ten. The ribosomes were then purified using RAPPL and the eluates examined by transmission electron microscopy (TEM). The results show ribosome isolation from the lowest concentration at 1 .3 nM (FIG. 2A).

[0154] To examine method limitations in the context of cell lysates, components of which could easily affect ribosome binding, RAPPL on HEK 293 cells where RPL4 (uL4) has been HA-tagged by CRISPR / Cas9 engineering was performed. A series of cell dilutions was performed and the lysates were used in the method. The eluates were then analyzed by western blot using aHA-HRP antibody to detect the tagged RPL4 (uL4). The results indicate a lower limit of detection by western blot at 5000 cells (FIG. 2B). Mammalian cell lines can be easily cultured in high abundance with larger cells containing significantly more ribosomes than other clinically significant organism such as P. falciparum. Growing and maintaining synchrony of the large parasite cultures in replicates necessary for some studies is difficult and costly. Additionally, growing parasites to high parasitemia to reduce flask numbers and materials generates stress conditions that confound results. Therefore, the cell number limitations of RAPPL to purify from P. falciparum NF54 was tested. Parasites were synchronized at ring stage, grown to ~5% parasitemia in 3% hematocrit. Latestage parasites were isolated via MACS magnet purification. Parasite were counted using the countess cell counter. Cells were then diluted to 5x107, 1 x107, 5x106, 1 x106, lysed, and the clarified lysate used in the RAPPL method. The products were analyzed by western blot analysis, blotting with aRPS14 (uS11 ) antibody. The results indicate detection down to one million cells, although faintly so.

[0155] Taken together the results indicate that RAPPL can purify ribosomes and translation-associated factors from relatively small quantities of starting material with current detection levels associated with 1 nM E. coli ribosome concentration and western blot analysis of 5000 RAPPL purified Hek293 or 1x106of P. falciparum cells.

[0156] Example 3 RAPPL is amenable to a wide variety of material and organism types

[0157] To determine what, if any, were the material limitations of RAPPL, a range of sample types, from single cell organisms and cultured cells to tissues and whole organisms was applied (FIGS. 3A). In each case, translation-associated materials (i.e. , ribosomes) was isolated that were then visualized by TEM (FIG. 3A). RAPPL could isolate ribosomes regardless of starting material type and quantity. However, slight modifications to the lysis step were necessary, but amenable, to ensure success for each sample. For mammalian cell cultures, only detergent is necessary for lysis. To ensure lysis of other cell types (i.e. S. cerevisiae, T. gondii, P. falciparum, and C. parvum), bead-beating was used. For samples containing high tissue organization (i.e. perfused mouse Organs and C. elegans), they were flash frozen, resuspended in buffer, and bead-beating was used to lyse. The D. rerio was further processed by first finely scoring and segmenting the specimen using a scalpel, followed by flash-freezing in liquid nitrogen, and pulverization using a mixer miller. Lysis of all samples was performed in similar buffers with one exception. In case of P. falciparum cells, the well documented specific lysis buffer conditions necessary for ribosome isolation and stripping of ribosomes from endoplasmic reticulum was used. Additionally, high levels of heme present in red blood cells were avoided by magnet purification of the parasite. RAPPL is therefore highly adaptable to various starting materials.

[0158] In comparison to bacterial cells, in eukaryotic cells, one can find organelle specific ribosomes. The mass-spectrometry analyses indicated that RAPPL enriches for both cytoplasmic and mitochondrial ribosomes. Besides, subcellular compartment-localized ribosomes (e.g. mitochondria, and in plants also chloroplasts) ribosome biogenesis is compartmentalized in nucleus. Mass spectrometry analyses of Hek293 lysate also indicated enrichment of multiple ribosome biogenesis factors using RAPPL. As such, to further indicate versatility of the method the compartment-localized ribosomes were separated from the cytoplasmic ones. Using previously published method, the mitochondrial and cytoplasmic cell fractions were isolated, followed by RAPPL procedure. The RAPPL eluates were visualized again by TEM. The results indicate that RAPPL can be used to isolate cytoplasmic and mitochondrial ribosome types (FIG 3B). Furthermore, cellular fractionation on HEK 293 cells was performed using commercial kits and the cytoplasmic and nuclear fractions were subjected to RAPPL purification. The lysates were analyzed using western blots to ensure enrichment of the corresponding fractions while RAPPL eluates were analyzed by TEM (FIGS. 3A and 3B). RAPPL isolated ribosomes undergoing biogenesis from nuclear fractions, making the study of ribosome biogenesis via the method possible (FIG. 3B).

[0159] Taken together the results indicate that RAPPL can robustly isolate translation-associated material from a wide range of substances - single cells, tissues, or whole organisms. The method is further adaptable to the ribosome isolate requirements of different organisms or cellular compartments (cytoplasmic, mitochondrial, nuclear, among others) making it a versatile new tool. Example 4 RAPPL is compatible with current technologies and methodologies for the study of protein synthesis

[0160] To further test the applicability of RAPPL method, the ribosomes and associated translation factors generated by RAPPL were used to look into protein synthesis, effects of known translation associated drugs or for purification of translation associated complexes. Commercial eukaryotic in vitro translation kits were used as proof of concept. Wheat germ and HeLa cell lysates with and without commercially obtained eGFP mRNA were used (FIG. 4A). The lysates were mixed with and without mRNAs, and after protein synthesis was carried on for 2 hours samples were subjected to RAPPL. The eluates were then visualized by TEM (FIG. 4A). The micrographs show that the translation architecture is maintained throughout the purification, whereby ribosomes remain bound to mRNA (FIG. 4A). To determine if RAPPL can be used to assay effects of different drugs on cultured cells RAPPL was performed using HEK cells treated and lysed in the presence of various translation inhibitors. Visualization by TEM showed inhibitor-dependent variation in ribosome organization versus untreated controls (FIG. 4B). The organization of ribosomes treated with translation elongation inhibitors cycloheximide and anisomycine tend to show more polysomes (i.e. ‘beads on a string’), while the translation initiation inhibitor harringtonine reduced this effect having more monosomes (i.e. individual ribosomes). Control samples (without inhibitors) showed combination of monosomes and separated subunits (FIG. 4B). Naturally, cryo-EM at high-resolution can confirm the above-mentioned observations beyond any doubt, but the idea behind the presented panels of FIGS. 4A-4D is to show that the incubation with polyLys beads and the subsequent elution do not seem to interfere with the global translation landscape.

[0161] Polysome profiling is demonstrably an invaluable tool for studying many aspects of protein synthesis. However, isolation of RNA and protein from the generated fractions is cumbersome, with significant loss of sample. To determine if RAPPL was compatible with these sucrose-containing fractions, polysome profiling using HEK cells was performed. The fractions for the subunits / monosomes, light polysomes, and heavy polysomes were pooled, respectively, and subject to RAPPL. The eluates were then visualized by TEM. The results show that not only is RAPPL compatible with polysome profiling, but that ribosome organization is once again maintained (FIG. 4C) allowing the method to be used to enriched from these fractions and remove the contaminating sucrose for further analysis.

[0162] Tandem affinity purifications are often employed to isolate specific translation complexes or improve the final sample purity. Therefore, a tandem RAPPL-Flag purification was performed using both E.coli and HEK293 cells in which eGFP construct was N-term inally tagged with double HA tag followed by engineered TEV cleavage site. In this case translational complexes associated with nascent polypeptide chain were isolated. Cells were lysed in the presence or absence of translation elongation inhibitors for E.coli and human tissue culture cells, chloramphenicol or cycloheximide, respectively. The clarified lysates were incubated with poly-lysine beads, and eluted. The flow-through and eluates from the poly-lysine bead incubations were then incubated with aHA magnetic beads. The aHA beads were then washed and eluted with addition of His-TEV Protease in washing buffer. The aHA flow-through, eluates, and beads were then analyzed by western blot and TEM (FIG. 4D). Immunoblotting shows that RAPPL can be used tandemly to purify specific translation complexes of interest (FIG. 4D). It was also demonstrated how addition of elongation inhibitors prevents run-off of elongating ribosomes and release of nascent polypeptide chain by simply looking at the number of ribosomes associated with HA-beads and growing polypeptide of eGFP. Finally, RAPPL in this case also serves as an enrichment step that would separate complete and release eGFP polypeptide from growing eGFP polypeptide still associated with ribosomes (FIG. 4D).

[0163] RAPPL can therefore be used in combination with current technologies and methodologies to study protein synthesis. This allows for sample enrichment from, and removal of, sucrose, which is often incompatible with downstream techniques. Ribosome binding and organization is maintained throughout the purification process, suggesting efficacy in structural applications as well. Translation inhibitors may be used to perturb ribosomes and translation factor equilibrium in such studies while using tandem purification methods to obtain the desired fractions or purity.

[0164] Example 5 RAPPL eluates are compatible with functional and downstream clinical applications While the products of RAPPL appear to have the visual hallmarks of functional translation, it was determined if they could translate mRNA into protein. To test activity of isolated ribosomes E. coli cells were subjected to the widely used PurExpress in vitro translation system, substituting the kit-supplied ribosomes with increasing amount of RAPPL eluates and adding PCR template that encode eGFP protein. The reactions products were then analyzed by western blot together with control without added PCR template (FIG. 5A). The results indicate that performing RAPPL on E. coli DH5a lysates results in the isolation of functional ribosomes, which can be used for in vitro translation systems. In addition to RAPPL eluted ribosomes incubation of RAPPL beads with purified ribosomes resulted also in active translation of eGFP template by poly-lysine bound ribosomes (FIG. 5A). As such, RAPPL ribosome eluates or an on-bead isolated ribosomes were able to perform in vitro translation reactions and translate reporter genes from DNA or RNA template reporter into protein (FIGS. 5A-5B).

[0165] The further test whether RAPPL could be used for clinical applications, the method was applied to two patient clinical isolates of uropathogenic E. coli (UPEC) EC13 and EC24. These E.coli strains are trimethoprim-sulfamethoxazole and ciprofloxacin resistant and have a broad-spectrum secondary multidrug transporter (MdfA+) providing additional resistance. The EC24 strain has been confirmed to contain active rRNA methylase (ermB), which results in rRNA methylation reducing erythromycin binding to ribosomes. E. coli lab strains DH5a and 10B were used as controls for no resistances to the selected antibiotics. Additional E. coli strains with plasmid encoded rRNAs and engineered rRNA mutations were also used as controls. The SQ110 ATC 16S - A1408G strain carries mutation which provides resistance to spectinomycin, kanamycin, and gentamicin, while the SQ110 ATC 23S

[0166] - A2058G provides resistance to spectinomycin, and erythromycin, and SQ110 plasmid carries selection cassette that encodes aminoglycoside 3'- phosphotransferase II enzyme that inactivates kanamycin by phosphorylation. As such, although both strains have kanamycin antibiotic resistance and can grow on kanamycin containing bacterial agar plates, only small subunit rRNA mutation (A1408G) provides ribosome resistance to kanamycin. However, SQ1 10 ATC 23S

[0167] - A2058G is the only one with mutation in large subunit rRNA (A2058G) that provides resistance to erythromycin. All E.coli cells were grown to exponential phase, harvested and RAPPL was performed. The RAPPL eluates from each of these strains were then used in the PurExpress in vitro translation system as above, varying the concentration of antibiotics. All reactions were analyzed by western blot (FIGS. 5B-5C). As in the original assay (FIG. 5A) all E. coli strain eluates were able to use the eGFP template to synthesize protein (FIGS. 5B-5C) in the absence of antibiotic. The control DH5a eluate was not able to synthesize protein in the presence of the macrolide antibiotic erythromycin (FIG. 5B). However, EC24 and SQ110 ATC 23S - A2058G were able to generate protein in the presence of erythromycin, with EC24 also able to grow on bacterial agar plates containing erythromycin (FIG. 5B). When the aminoglycoside kanamycin is used, neither SQ110 ATC 23S - A2058G, EC13, nor EC24 RAPPL isolated ribosomes could synthesize protein like the SQ110 ATC 16S - A1408G RAPPL isolated ribosomes (FIG. 5C). Although resistant to kanamycin because it harbors the NPTII gene, which inactivates kanamycin by phosphorylation, the SQ1 10 ATC 23S - A2058G does not endow the ribosome itself with resistance to the drug and is unable to synthesize eGFP (FIG. 5C). EC13 is also kanamycin resistant and able to grow on bacterial agar plates supplemented with kanamycin, ostensibly due to its secondary multidrug transporter but not due to ribosome resistance (FIG. 5C). The EC24 was able to grow on chloramphenicol supplemented plates, it was tested whether ribosomes from this strain are chloramphenicol resistant (FIG. 5C). Chloramphenicol is a bacteriostatic antibiotic that inhibits the peptidyl transferase step of translation by binding to large subunit. RAPPL isolated EC24 ribosomes showed resistance to chloramphenicol in in vitro translation assay confirming results observed by growth of this strain on plates (FIG. 5C). The lincosamide clindamycin reversibly binds to the 50S ribosomal subunit, inhibiting peptide bond formation. The A2058G point mutation of the 23S rRNA has previously been shown to confer resistance to clindamycin. Therefore, the ability of the E. coli strains to grow on plates with clindamycin and their ribosomes to synthesize protein in vitro was tested. As expected, the SQ110 ATC 23S - A2058G strain was able to grow and synthesize protein in the presence of clindamycin (FIG. 5C). The UPEC strain EC24 was also able grow and synthesize protein when challenged with clindamycin (FIG. 5C). Taken together the results indicate that E. coli RAPPL-isolated ribosomes are translationally competent and allow the flexibility of on-or off-bead reaction setup. RAPPL can therefore be used to rapidly screen for ribosome or translation factor- associated mechanisms of resistance, with plated-based assays confirming alternative mechanisms. Bacterial strains - lab, clinical, or otherwise - that can be cultured or isolated even in relatively small quantities are accessible for study. These data show the ability of RAPPL to isolate and study translation-associated antibiotic mechanisms of resistances from a clinical setting.

[0168] Example 6 RAPPL generates high-quality materials for structural determination

[0169] The compatibility of RAPPL with structural applications and whether the ribosomes isolated would be of sufficient quality for structural determination using cryo-electron microscopy was tested. RAPPL was applied to Cryptococcus neoformans cells grown in exponential phase isolated in the presence of cycloheximide. C. neoformans was selected for two reasons: 1 ) an 80S structure had yet to be determined and 2) to determine if the anionic polysaccharides heavily present in the cell wall that are released into the lysate during bead-beading would interfere with RAPPL purification. The eluate was first examined by TEM (FIG. 6A). Being of adequate concentration, the sample was applied to cryo-EM grids and images were acquired. A 2.7 A map was generated with the material eluted from RAPPL (FIG. 6A) straightforwardly. The C. neoformans map (FIG. 6A, solid blue) was aligned with a S. cerevisiae map (FIG. 6A, gold mesh, PDB: 6TB3) and ostensibly aligns well (Buschauer R., Matsuo Y, et al. Science 2020).

[0170] Discussion

[0171] The isolation of ribosomes from different types, organs, and organisms imposes several challenges, primarily due to the unique biochemical environment and cellular compositions. The most common problem associated with ribosome isolation is contaminant presence, which can affect RNA quality and protein content during isolation. Different cell / tissue types require different adjustments based on the cell type. Further, the cellular structure of the tissue can affect ribosome isolation; tissues with dense cellular arrangements, such as brain or heart tissue, can be more challenging to homogenize, leading to inefficient lysis and ribosome extraction. Further, different cell types / tissues require specific pH levels and ionic conditions for optimal ribosome extraction. Maintaining such conditions during isolation is critical, as deviations can reduce ribosomal activity and purity. Traditional procedures are time-consuming, complex, and often not suitable for all tissue types, especially when rapid isolation is needed for downstream application. Maintaining RNA integrity or achieving high yield and purity simultaneously can be challenging. Methods that obtain purity may reduce yield, making it difficult to obtain ribosomes that are both functional and free of contaminants. Further, the activity of isolated ribosomes can vary depending on the tissue and isolation method used. In summary, the isolation of ribosomes from different cells / tissues is fraught with challenges that stem from tissue-specific characteristics.

[0172] To overcome these challenges, a rapid, facile, and economical method to purify ribosomes and associated factors was developed. RAPPL is a robust method, capable of enriching ribosome-associated materials from a wide range of sample types, even those in low abundance, and cellular compartments. The downstream application of isolated ribosomes can go in many directions, like translational studies, drug development, functional analysis, ribosome profiling, structural biology, and posttranslational modification. The compatibility of RAPPL with several of these is demonstrated. The method is compatible with currently available technologies and methodologies (FIGS. 4A and 4B). RAPPL can also be used to study the effects of translational inhibitors on eukaryotic ribosomes (FIG. 4C), which could be coupled with factor-specific tandem purifications for further analysis (FIG. 4D). Using various E. coli strains, including patient isolated strains, ribosomes can be isolated via RAPPL and subsequently translation-associated mechanisms of antibiotic resistance can be determined (FIGS. 5A-5C).

[0173] The ability to isolate translational machinery from limited starting materials would prove advantageous for those fields in which this is a major limiting factor (e.g. patient samples, clinical isolates, and several parasites). Here, the ability of RAPPL to isolate ribosomes from as little as 5000 mammalian cells is demonstrated (FIG. 2B). However, mammalian cells harbor significantly more ribosomes than other organisms. The method can also isolate and detect ribosomes from one million P. falciparum NF54 cells (FIG. 2C). These are significantly less than necessary for other methods used to study ribosomes and protein synthesis. Often ~108P. falciparum cells are used in other methodologies such as polysome profiling, which is dependent on gradient volume, with larger gradient sizes requiring up to five times more material. This limitation prevents the use of many clinically relevant organisms as well as patient biopsies in such studies. While RAPPL will not replace current methods used to study ribosomes and translation, the lower cellular threshold enables researchers to gain access to this data for those organisms and clinical samples that cannot be isolated in sufficient quantities for said traditional methods.

[0174] RAPPL purified ribosomes from a wide range of organisms with minor modifications to cell lysis when necessary. This method worked with single celled organisms - intracellular and extracellular - and cultured cells as well as tissues and whole organisms (FIG. 3A). Processing single-celled organisms and cultured cells is done similarly. Lysis of these organisms is done using detergent (triton- X100). For those that have cells walls (bacteria, yeast) or multiple membranes (P. falciparum), bead-beating is introduced to ensure membrane rupture. In the case of the intracellular parasite T. gondii, the host cells are lysed by shearing prior to parasite lysis. P. falciparum requires lysis in potassium acetate to ensure ribosome release from the endoplasmic reticulum (REF), which must then be diluted (1 :8) to enable ribosome binding to the beads. This dilution did not prevent enrichment by RAPPL. However, these lysis methods are known and currently used in their respective fields. Tissues, such as the perfused mouse organs used here, and whole organisms require breakdown of the tissue structure and cell wall by flashfreezing and subsequent bead-beading or milling to ensure release of the cytoplasmic contents. Whole organisms with complex tissue organization like the D. rerio used here require scoring of the sample prior to flash-freezing and milling. These methods, again, are those already currently employed, demonstrating the adaptability of RAPPL for a multitude of sample specimen types. Of import are the conditions under which RAPPL lysis and binding are performed in regards to those necessary to maintain ribosome subunit association with mRNA, should this be desired, as well as those needed for other translation-associated factors. The study of mitoribosome dysfunction is of clinical import with a host of lifethreatening outcomes. RAPPL allows for the rapid purification of mitoribosomes (FIG. 3B). The method could therefore be combined with current laboratory or clinical studies to examine mitoribosomes for functional, composition, and structural analysis. Although the study prioritized the use of RAPPL for the study of protein synthesis, the method is capable of purify other pertinent ribosome- associated activities, such as biogenesis. Ribosome biogenesis is essential, vital to the cell cycle (proliferation, differentiation, apoptosis, et cetera), cell and organismal development, as well as playing roles in malignant cell transformation and therapeutic resistance. The study of ribosome biogenesis also provides insights into microbial diversity through ribosome evolution, function, and the development of therapeutic resistance. The ability to quickly and easily harvest this material enables study in these areas, which is demonstrated herein (FIG. 3B). Thus, RAPPL enables the purification and study of ribosomes from various cellular compartments, not only cytosolic ribosomes.

[0175] Functional analysis of purified ribosomes can provide insight into from the effects of drug treatments on the ribosome translation cycles to the outcomes of different cell stressors. Using in vitro protein synthesis kits, it was visually demonstrated that ribosome organization is maintained by RAPPL (FIG. 4A). In addition, the effects of translation inhibitors on this organization can be visualized using the method (FIG. 5B), suggestion that further study of such drug treatments or other stress factors is possible. However, it should be noted, as previously mentioned, that adaptations may be necessary for more nuanced investigation, such as any specific conditions to ensure accessory protein binding, and high concentrations of anionic compounds that disrupt polylysine-RNA interactions will reduce, if not inhibit, purification by RAPPL.

[0176] The compatibility of RAPPL with current technologies like in vitro kits and methodologies like polysome profiling provides further flexibility. Enriching from polysome profiling fractions via magnetic bead isolation (FIG. 5C) without the necessity of genetic manipulation to introduce affinity tags enables researchers to quickly and freely pursue various avenues of study. It also reduces the time to use of isolated products, thereby reducing degradation or complex dissociation that may occur during long centrifugations. Product enrichment using RAPPL over loss often seen with centrifugation is also a benefit, requiring less starting sample. Purifying and enriching ribosomes is a useful tool for their study. Purifying and enriching ribosomes is a useful tool for their study in various conditions. However, enriching functioning ribosomes can provide significantly more information through in vitro studies. The current results indicate that RAPPL products are functional and can be used on (FIG. 5A) or off bead (FIG. 5B) for protein synthesis. Furthermore, the clinical applications of RAPPL to study ribosome-associated mechanisms of resistance by using clinical UPEC isolates, with mutagenized and lab strains as controls was demonstrated (FIG. 5C). These methods can then be further adapted to plate-based assays, which lends to the possibility of high throughput assays using ribosomes isolated from various pathogenic organisms on compound libraries. Furthermore, with the right supplementation (i.e. S100 fraction), in vitro protein synthesis studies may be possible with eukaryotic organisms.

[0177] The purification of ribosomes and ribosomal complexes for structural determination can be quite time and labor intensive. Herein, the ability to obtain cryoEM-ready sample using RAPPL in approximately one hour that produces high- quality maps is demonstrated (FIG. 6A). This processes typically requires a significant amount of cells, as is the case with many clinically relevant organisms like the parasites P. falciparum or C. parvum. To isolate certain ribosomal complexes, such as the pre-initiation complex, even more material be needed, followed by polysome profiling, and finally isolation from the desired sucrose fraction(s) by lengthy ultracentrifugation. Throughout this process, sample loss to handling, degradation, and complex dissociation inevitably occurs. RAPPL provides a means of rapid sample enrichment, which can be performed instead of, prior to, or following polysome profiling depending on what ribosomal complexes are sought. These options can decrease the required starting sample and / or sample loss at key bottlenecks in the process reducing the time from lysis to grid preparation, and ultimately structural determination.

[0178] The development of methods like polysome profiling has been instrumental in furthering our understanding of ribosomes, protein synthesis, and gene regulation. However, there are some limitations to this method such as meeting cell material requirements, lengthy centrifugation times, costly equipment, and sucrose contamination, by necessity, of the fractionated samples. To reduce, and in some cases circumvent, these limitations the presented RAPPL, is a method define by its ease of use, wide range of applications, and adaptability. Using RAPPL, ribosomes as well as ribosome- and translation-associated factors from a wide variety of specimens and in limited ribosome or cell numbers can be isolated. RAPPL significantly enriches ribosome- and translation-associated factors as shown by mass spectrometry. Ribosome organization is also maintained during purification and can be visualized by TEM, demonstrating the effects of the addition of mRNA on in vitro protein synthesis kits or mRNA translation inhibitors on mammalian cell lysates. Ribosomes isolate by RAPPL can be use in functional studies, as disclosed herein, showing ribosome-associated mechanisms of resistance using in vitro protein synthesis kits. This suggests the ability of RAPPL to generate ribosomes for in vitro protein synthesis from virtually any organism given the right additional factors are supplied, such as those in the S100 fraction. RAPPL is compatible with current methods, enable ribosome and ribosome-bound protein enrichment from polysome profiling fractions while removing sucrose. RAPPL eluates are also of sufficient quality for structural studies, capable of producing high-resolution maps by cryoEM for structural determination of ribosomes and ribosome-associated complexes.

[0179] Methods

[0180] Escherichia coli

[0181] E. coli DH5a cells, uropathogenic patient isolate E. coli strains Ec13 and Ec24, and E. coli rRNA mutagenized lines SQ110 ATC 16S - A1408G and SQ110 ATC 23S - A2058G were cultured overnight in Luria-Bertani (LB) medium. From this overnight culture, 2 mL was used to inoculate 50 mL of LB medium. For mass spectrometry, E. coli DH5a cells were grown for 1.5-2 hours. Otherwise, all cells were incubated for 3 hours at 37°C while shaking at 200 rpm. In the case of rRNA mutagenized lines, double the culture was used as they grew at approximately half the rate of the other lines.

[0182] Plasmodium falciparum Parasites were cultured as previously described (Trager W and Jense JB, J. parasitol. 1976). Briefly, P falciparum Dd2 or NF54 were maintained by continuous culture at 2-5% hematocrit in human erythrocytes with malaria culture medium (RPMI 1640 supplemented with 5 g / L Albumax II (Gibco), 0.12 mM hypoxanthine (1.2 ml 0.1 M hypoxanthine in 1 M NaOH), and 10 pg / ml gentamicin). Cultures were grown statically under hypoxic conditions in candle jar atmosphere. Synchronization was done by 5% sorbitol treatment as well as magnetic purification using MACS cell separation magnets over LD columns.

[0183] Toxoplasma gondii

[0184] T. gondii ME49 parasites were continuously cultured in human foreskin fibroblast (HFF) cell monolayers as previously described (Olias P et al. Cell Host & Microbe 2016). HFF cells were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% HyClone fetal bovine serum (GE Healthcare Life Sciences), 10 pg / mL gentamicin (ThermoFisher Scientific) and 10 mM glutamine (ThermoFisher Scientific) (D10). T. gondii parasites were isolated from host cells as previous (Brow KM et al Bio Protoc. 2018). Briefly, parasites were cultured to high parasitemia (~75%) in two T25 flasks. The monolayers were scraped and combined in 10 mL of D10 medium. The cell suspension was passed through 22G blunt-end syringe 3 times to disrupt host cells. Host cell debris was filtered out by passing through a pre-wet 3 pm polycarbonate membrane, which was then washed with and additional 5 mL of D10 medium. The freed T. gondii cells were pelleted by centrifugation (400 x g for 10 mins). The parasites were washed with PBS prior to lysis.

[0185] Cryptosporidium parvum

[0186] Purified C. parum oocysts were graciously provided by the Sibley Lab per the lab protocol (REF). Oocysts (107) were bleached by treating with 40% bleach and incubating on ice for 10 mins. The oocysts were removed by centrifugation (900 x g for 3 mins at 4°C). The supernatant was removed, and the oocysts washed three times with 1X DPBS + 1 % BSA. Excystation was performed by combining equal volumes of resuspended oocysts (100 pL) and 1X DPBS + 1 .5% sodium taurocholate. The oocysts were then incubated for 60-75 minutes at 37°C. Excystation was confirmed by brightfield microscopy (~80%). The parasites were centrifuged for 3 mins at 1400 x g and washed with 1X DPBS twice prior to lysis. Saccharomyces cerevisiae

[0187] An overnight culture of S. cerevisiae was grown by inoculating 10 mL of yeast- peptone-dextrose (YPD) growth medium with 200 pL of glycerol stock. The culture was harvested by centrifugation at 3500 x g for 5 mins at 4°C. The culture was washed with PBS prior to lysis.

[0188] Cryptococcus neoformans

[0189] C. neoformans KN99a were grown and generously provided by the Doer lab (Washington University in St. Louis). Briefly, cultures were grown on yeast extract- peptone-dextrose (YPD) plates for two days at 30°C. YPD liquid medium was inoculated with single colonies and grown overnight at 30°C while shaking at 230 RPM. Overnight cultures were diluted to an OD600 of 0.2 and grown to 0.6 (exponential phase). Cultures were pelleted and washed with PBS prior to lysis. Danio rerio

[0190] Zebra fish (D. rerio wild-type AB) were provided by the Stratman lab and experimental procedures done per approved guidelines by the Washington University in St. Louis School of Medicine Institutional Animal Care and Use Committee (IACUC). Fish were euthanized by ice water bath (5 parts ice / 1 part water, 0-4°C) separated from ice chips by a fine mesh strainer for a minimum of 10 minutes after cessation of opercular movement. Following euthanasia, the sample was finely scored and segmented using a scalpel then flashed frozen by plunging into a liquid nitrogen bath. The frozen sample was then pulverized into a fine powder using a RETSCH mixer miller MM 400 (). This powder was then resuspended into 1 mL of RAPPL lysis buffer.

[0191] Sample and Lysate Preparation

[0192] Cultured cells were maintained as mentioned above. Cells were centrifuged, their growth mediums removed, washed with PBS, and transferred to 2.0 mL microcentrifuge tubes. Cells were then resuspended in lysis buffer (100 mM HEPES KOH solution, pH 7.5, 50 mM KCI, 10 mM Mg(OAc)2, 1 % Triton-X, 1 mM DTT, Protease Inhibitors, 40 U / mL RNaseOUT, 20 U / mL Superas IN™ RNase Inhibitor, 4 U / mL DNase I) Transmission Electron Microscopy

[0193] For analyses of ribosome preparations, samples were allowed to absorb onto freshly glow discharged formvar / carbon-coated copper grids (200 mesh, Ted Pella Inc., Redding, CA)) for 10 min. Grids were then washed two times in dH20 and stained with 1 % aqueous uranyl acetate (Ted Pella Inc.) for 1 min. Excess liquid was gently wicked off and grids were allowed to air dry. Samples were viewed on a JEOL 1200EX transmission electron microscope (JEOL USA, Peabody, MA) equipped with an AMT 8 megapixel digital camera (Advanced Microscopy Techniques, Woburn, MA).

Claims

CLAIMSWhat is claimed is:1 . A method of isolating a molecule from a sample, the method comprising: lysing cells in the sample to generate a cell lysate; incubating the cell lysate with poly-lysine coated beads; and eluting the incubated beads to recover the isolated molecule.

2. The method of claim 1 , wherein the molecule is a ribosome.

3. The method of claim 1 , wherein the sample is a biological sample.

4. The method of claim 3, wherein the biological sample is selected from a tissue sample, a blood sample, a non-blood biological fluid sample, and a whole organism sample.

5. The method of claim 1 , wherein the sample is a non-biological sample.

6. The method of claim 1 , wherein the lysing is performed in the presence of a translation inhibitor.

7. A kit for isolating a molecule from a sample matrix, the kit comprising: at least one lysing agent; a plurality of poly-lysine coated beads; and at least one elution agent.

8. A method of treating a subject having an infection, the method comprising: isolating ribosomes from a biological sample obtained from the subject, wherein the isolating comprises: lysing cells in the biological sample to generate a cell lysate; incubating the cell lysate with poly-lysine coated beads; and eluting the incubated beads to recover the isolated ribosomes;exposing the isolated ribosomes to at least one therapeutic agent; and treating the subject with the at least one therapeutic agent if the isolated ribosomes were susceptible to the at least one therapeutic agent.

9. The method of claim 8, wherein the biological sample is selected from a tissue sample, a blood sample, a non-blood biological fluid sample, and a whole organism sample.

10. The method of claim 8, wherein the at least one therapeutic agent is selected from an antibiotic medication, an antifungal medication, and an antiparasitic medication.11 . A method of obtaining ribosome translation profiling data from a sample, the method comprising: processing the sample according to a RAPPL-Ribo-Seq protocol; and generating the ribosome translation profiling data from the processed sample.

12. The method of claim 11 , wherein the sample is a biological sample.

13. The method of claim 12, wherein the sample comprises cytoplasmic ribosomes, mitochondrial ribosomes, or a combination thereof.

14. The method of claim 12, wherein the biological sample is selected from a tissue sample, a blood sample, a non-blood biological fluid sample, and a whole organism sample.

15. The method of claim 11 , wherein the ribosome translation profiling data comprises cytoplasmic ribosome translation profiling data and mitochondrial ribosome translation profiling data.

16. The method of claim 11 , wherein the sample is at least one of: a smaller volume than a classical ribosome profiling sample minimum required volume; anda smaller total cell number than a classical ribosome profiling sample minimum required cell number.

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