WO2022162363A1

WO2022162363A1 - Method of preparing a biological sample for electron microscopy

Info

Publication number: WO2022162363A1
Application number: PCT/GB2022/050209
Authority: WO
Inventors: Dr Aditi BORKAR
Original assignee: Cambridge Enterprise Limited
Priority date: 2021-01-28
Filing date: 2022-01-27
Publication date: 2022-08-04
Also published as: GB202101188D0

Abstract

The present invention relates to a method of preparing a biological sample for electron microscopy, wherein the sample comprises a target nucleic acid, the method comprising (i) incubating an electron permeable substrate with an aqueous solution comprising an oligonucleotide probe; (ii) incubating the biological sample with the primed substrate; and (iii) washing the hybridised primed substrate. The invention also relates to a primed electron permeable substrate, and to the use of such a substrate.

Description

METHOD OF PREPARING A BIOLOGICAL SAMPLE FOR ELECTRON MICROSCOPY

Field of the Invention

The invention relates to a method of preparing a biological sample for electron microscopy. In addition, the invention relates to a method of determining one or more biological properties of a biological sample. The invention also relates to a primed electron permeable substrate, and to the use of such a substrate for the in situ analysis of a native nucleic acid-protein complex, such as a ribonucleoprotein (RNP), in a biological sample.

Background to the Invention

The preparation of samples for structure determination by electron microscopy (EM) traditionally requires a biochemical pipeline involving cloning/vector generation, transfection/transformation, recombinant expression, purification, in vitro reconstitution, etc. of the target molecule, which is both time and resource intensive and sometimes unpredictable. Moreover, such methods are unsuitable for the preparation of short-lived systems, such as native RNA complexes, that are difficult to clone and express in heterologous systems or unstable when constituted in vitro outside their biological context.

There remains a need for a sample preparation method which is capable of providing a sample which is suitable for downstream analysis using EM whilst also bypassing the burdensome biochemical pipeline. Not only will such methods be useful in the analysis of biological molecules that are difficult to overexpress via recombinant means and/or purify, but the methods can also allow for analysing biological molecules in situ. As will be appreciated in the art, many biological macromolecules (such as ribosomes) undergo transient forms that only visible in situ, which are otherwise lost when purified and reconstituted.

Summary of the Invention

The present invention relates to a novel technique, known as RHyTEM (pronounced “rhythm”), which enables the enrichment of native nucleic acids and nucleic acid-protein complexes. RHyTEM stands for Ribonucleic acids (RNA) and their complexes (RNP) through Hybridisation on a solid phase for biochemical, biophysical and structural characterisation using, for example, Transmission Electron Microscopy (TEM), although the technique can readily be applied to DNA and DNA-protein complexes too. RHyTEM involves first priming the solid phase substrate with an oligonucleotide probe that is designed to be complementary to the exposed regions of the nucleic acid (such as RNA) molecule which is part of the target molecule or complex of interest (such as an RNP complex). The primed substrate surface is then incubated with the biological sample (e.g. a whole cell lysate) containing the molecule or complex of interest. Following incubation, the substrate surface then undergoes washing steps.

Following sample preparation using the RHyTEM technique, the solid phase can be directly analysed using multiple biophysical techniques, such as UV Spectroscopy or TEM, for biochemical or structural characterisation of the molecules of interest. The RHyTEM technique allows one to completely bypass the typical biochemistry pipeline for sample preparation and can be completed within about 2 hours using only a few microliters (less than 20 microlitres, such as less than 15 microlitres) of cell lysate.

Advantageously, the invention does not require the production of recombinant macromolecules (e.g. His-tagged proteins) using heterologous protein expression systems. This concurrently avoids the need for expensive and unpredictable purification and/or reconstitution procedures. It thus provides a more efficient method of sample preparation for the purposes of electron microscopy, which is less resource- and labour-intensive than prior art methods. A further advantage of RHyTEM is that it can enrich native, wild-type complexes from cell lysates without the need for source modification. As such, it is suitable for sample preparations of highly challenging systems, such as low-abundance, short-lived and dynamic native RNP complexes.

The present invention provides a method of preparing a biological sample for electron microscopy, wherein the sample comprises a target nucleic acid and/or its complex and the method comprises: (i) incubating an electron permeable substrate with an aqueous solution comprising an oligonucleotide probe for a period sufficient for the oligonucleotide probe to adsorb onto the surface of the substrate to produce a primed substrate, wherein the oligonucleotide probe comprises a region that is complementary to the target nucleic acid; (ii) incubating the biological sample with the primed substrate for a period sufficient for the target nucleic acid to hybridise to the oligonucleotide probe to produce a hybridised primed substrate; and (iii) washing the hybridised primed substrate with a buffer.

The present invention also provides a method of determining one or more biological properties of a biological sample, wherein the sample comprises a target nucleic acid and the method comprises: (i) carrying out steps (i) to (iii) as defined in the method of preparing a biological sample according to the present invention; and (ii) performing biophysical analysis on the washed, hybridised primed substrate; wherein the biophysical analysis is selected from UV spectroscopy, TEM, or cryo-EM.

The present invention further provides a primed electron permeable substrate comprising: (a) an electron-conducting mesh support having one or more apertures; (b) a biocompatible layer coating the mesh support; and (c) one or more oligonucleotide probes adsorbed onto the biocompatible layer.

The present invention additionally provides use of a substrate of the present invention for the in situ analysis of a native ribonucleoprotein (RNP) in a biological sample, wherein the analysis is carried out using UV spectroscopy, TEM, or cryo-EM.

For the avoidance of doubt, the embodiments described herein can be combined unless context clearly dictates otherwise.

Figure 1 - RHyTEM method for solid phase enrichment of RNA protein complexes suitable for downstream TEM applications, (a) An electron permeable substrate having a hydrophobic surface (b) is charged/primed with oligomeric nucleic acid probes, (c) incubated with a clarified whole cell lysate to allow hybridisation with native RNP complexes and (d) washed several times with compatible buffer solutions to remove the unbound and non-specifically bound cellular material from the primed surface, leading to specific enrichment of the RNP complex of interest. The solid phase can be used directly, for instance, in negative stain TEM imaging. Figure 2 - Specificity and efficiency of different oligonucleotide probes in enriching E. coli ribosomes. The figure shows that multiple specific oligonucleotide probes designed for 16S and 23 S rRNA are able to hybridise with and enrich E. coli ribosomes on a solid phase. The average particle numbers for these specific probes are also significantly higher than the control experiments where no probes (0) or a completely non-specific (NS) probes where used. Among the specific probes tested, the 23S-1 probe led to the highest number of single, well-dispersed ribosome particles per micrograph. Error bars represent the minimum and maximum number of particles identified on the electron micrographs.

Figure 3 - Representative negative stain TEM micrographs for (a) RHyTEM enriched E.coli ribosomes through 23S-1 probe along with (b) purified E. coli ribosomes and solid phase negative controls with (c) 0 nM probe concentration and (d) non-specific probe. The scale bars represent 100 nm.

Figure 4 - Representative control micrographs obtained by adsorbing E. coli whole cell lysate on (a) hydrophobic and (b) negatively charged hydrophilic surface without 23 S rRNA specific probes.

Figure 5 - Reversibility of the RHyTEM enrichment. The figure shows RNP content estimated after RHyTEM enriched surface was washed with the standard buffer supplemented with high concentrations of KC1. Compared to the baseline value for each buffer supplemented with 0 M, 1.5 M and 3 M KC1, RNPs are only eluted from the surface primed with 100 nM 23 S RNA specific probe at 1.5 M KC1 concentration. Moreover, 1.5 M KC1 is sufficient to completely elute the RNPs from the surface without the need for proceeding to higher concentrations.

Figure 6 - Optimisation of time of incubation of E. coli whole cell lysate with oligonucleotide probe specific to the 23 S rRNA. After 30 mins of incubation at 4°C, the negative stain Electron Micrographs show the highest number of single, well-dispersed 70S particles per micrograph. Error bars represent the minimum and maximum number of particles identified on the electron micrographs. Figure 7 - Optimisation of oligonucleotide probe concentration and specificity of the RHyTEM enrichment. A concentration series (0 to 50 micromolar) of an oligonucleotide probe specific to the 23 S rRNA demonstrates that at a concentration of 100 nM, the negative stain Electron Micrographs show the highest number of single, well- dispersed 70S particles per micrograph. This number is significantly enriched compared to a 0 nM control experiment or over the range of the concentrations for a completely nonspecific oligonucleotide probe that is not able to hybridise to the E. coli ribosomes. Error bars represent the minimum and maximum number of particles identified on the electron micrographs.

Figure 8 - Effect of Ribosome modifying factors on RHyTEM enriched (magenta) and purified (black) E. coli 70S ribosomes. The graph shows that compared to control experiments, the samples treated with Ribosome Recycling Factor (RRF) had nearly half the number of polysome particles and those treated with Hibernation Promoting Factor (HBF) had more than twice the number of polysomes in the negative stain Electron Micrographs.

Figure 9 - Representative 2D classes obtained after data processing in RELION for (a) purified and (b) RHyTEM enriched E. coh 70S particles, (c) The latter also shows presence of ribosome subunit dimers enriched directly from the lysate. The two rows in (d and e) show 3D reconstructions of the (d) purified and (e) RHyTEM enriched 70S at two different orientations. Both these reconstructions were obtained at 36A resolution, (f) PDB ID 5WDT was used as reference for the 3D reconstruction in (d) and (e). (g) Comparison between the purified (green mesh) and enriched 70S (solid grey) reconstructions shows a total volume overlap of 76% and a correlation coefficient of 0.85.

Detailed Description of the Invention

It is to be understood that different applications of the disclosed methods may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting. In addition, as used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “an oligonucleotide” includes two or more such oligonucleotides, and the like. All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

Biological sample

As used herein, a biological sample refers to a sample derived from a biological specimen, such as an organ, tissue or cell. A biological sample comprises a target nucleic acid, such as a target DNA or RNA, optionally wherein the nucleic acid is part of a nucleic acid-protein complex (such as a ribonucleoprotein (RNP) or DNA-protein (DNP) complex). Any biological sample can be suitable for use with the methods of the present invention as long as it comprises a target nucleic acid. A biological sample for use with the methods of the present invention may be a cell lysate. The cell lysate may be derived from prokaryotic, eukaryotic or archaeal cells. The cell lysate may be obtained by the lysis of a population of cells through any known method in the art, such as enzymatic treatment, sonication, freeze-thaw, high pressure, etc.

As used herein, a cell lysate may also be a clarified cell lysate. Clarified cell lysates are known in the art as a cell lysate from which insoluble cellular material, such as cells walls, has been removed, e.g. by centrifugation.

Thus, a biological sample for use with the methods of the present invention may be derived from a cell lysate. It is understood in the art that a cell lysate can be further processed through various purification techniques known in the art in order to isolate a specific molecule(s) from the sample. Examples of purification techniques include chromatographic techniques, such as affinity chromatography, ion exchange chromatography and hydrophobic interaction chromatography. As such, a biological sample for use with the methods of the present invention may also be a purified aqueous solution comprising a target nucleic acid which is derived from a cell lysate.

In some embodiments, the volume of biological sample incubated with the primed substrate (i.e. in step (ii) of the method of the invention) is from about 10 pl to about 15 pl. Target nucleic acid

The target nucleic acid in the biological sample may comprise or consist of DNA or RNA. In a preferred embodiment, the target nucleic acid in the sample is RNA. The target nucleic acid may also be associated with one or more additional molecules to form a nucleic acid complex. For example, the target nucleic acid may form a complex with one or more additional nucleic acids. The target nucleic acid may form a complex with one or more additional polypeptides to form a nucleic acid-protein complex.

In some embodiments, the target nucleic acid is associated with a polypeptide. The polypeptide may be any polypeptide which binds or associates with nucleic acids, such as ribosomal proteins, histones, transcription factors, restriction enzymes, polymerases, etc.

In one embodiment, the target nucleic acid is a DNA, and the target nucleic acid is part of a DNA-protein complex (DNP). Optionally, the target nucleic acid is an RNA and is part of a ribonucleoprotein (RNP) complex. In a preferred embodiment, the target nucleic acid is an RNA and the RNA is associated with one or more proteins to form an RNP complex. Preferably, the target nucleic acid is part of a native RNP complex.

The target nucleic acid may comprise regions which are exposed to the solution. Regions which are exposed to the solution may comprise loop regions of the nucleic acid when present in a nucleic acid-protein complex such as an RNP. Loop regions for known nucleic-acid protein complexes may be known from structural or biochemical studies into the complex. In other words, the regions which are exposed to the solution are regions of the target nucleic acid which is capable of hybridising to a complementary sequence (e.g. on an oligonucleotide probe) when in an aqueous medium.

Specific examples of target nucleic acids which are part of the E. coli ribosome RNP are provided herein. For example, the target nucleic acid may be the 23 S rRNA or the 16S rRNA.

Electron permeable substrate

Solid phase substrates for use with methods of the present invention include electron permeable substrates, i.e. substrates that allow electrons to pass through. Electron permeable substrates which can be used in sample preparation for electron microscopy are also known as electron microscopy grids (EM grids), and the terms can be used interchangeably herein. The electron microscopy may be transmission electron microscopy (TEM) or cryo-electron microscopy (cryo-EM). Thus, the EM grid may be a TEM grid and/or a cryo-EM grid. When used in a cryo-electron microscope, a vitreous ice layer may be present on the EM grid to fix the configuration of the molecules (such as the target nucleic acid) within the biological sample.

Electron permeable substrates are known in the art and generally comprise an electron-conducting mesh support layer having one or more apertures (or perforations). Electron permeable substrates may additionally comprise a layer coating the mesh support layer, such as a biocompatible layer. The mesh support layer can be made of a number of materials or a combination or alloy of such materials provided they are electron conducting. In embodiments, the mesh support layer may comprise (or consist of) a metal. In embodiments, the metal may be selected from the list of Cu, Ni, Ti, Si, Au, CuRh, Mo, Al and W. In some embodiments, the mesh support may comprise carbon, copper, nickel, molybdenum, beryllium, gold, silicon, GaAs, or oxide (e.g., SiCh, TiCh, ITO, or AI2O3). In some embodiments, the electron microscopy grid comprises copper (Cu).

The layer coating the mesh support layer may be a biocompatible layer. A biocompatible layer may comprise carbon, and preferably be in the form of a carbon film disposed on at least one surface of the mesh support layer. In some embodiments, the carbon film is hydrophilic. In preferred embodiments, the carbon film is hydrophobic. In some embodiments, the carbon film has a thickness of about 10 nm to 25 nm, or about 12 nm.

In some embodiments, electron permeable substrate is an about 3 mm diameter disc (e.g., 3.05 mm diameter). In some embodiments, the electron permeable substrate has a thickness and mesh size ranging from about 3 microns to 100 microns. In some embodiments, an electron microscopy grid has a thickness of about 30 microns and a mesh (aperture) size of about 100 microns.

The present invention also provides for a primed electron permeable substrate, wherein the electron permeable substrate comprises: (a) an electron-conducting mesh support having one or more apertures; (b) a biocompatible layer coating the mesh support; and (c) one or more oligonucleotide probes adsorbed onto the biocompatible layer.

Oligonucleotide probe

As used herein, an oligonucleotide probe is an oligonucleotide that: (a) is capable of adsorbing onto the surface of the substrate; and (b) comprises a region that is complementary to the target nucleic acid. Suitable oligonucleotide probes are selected based on the target nucleic acid of interest, and having regard to the regions of the target nucleic acid which are exposed to the solution and/or are capable of hybridising to a complementary sequence (e.g. on an oligonucleotide probe) when in an aqueous medium.

The oligonucleotide probe comprises a region (having a sequence) that is (partly or fully) complementary to the target. Alternatively, the oligonucleotide probe may consist entirely of a sequence which is (partly or fully) complementary sequence to the target nucleic acid. The length of the probe sequence that is complementary to the target is sufficient to provide specific hybridisation to the target nucleic acid. The length of the complementary sequence in the oligonucleotide probe is typically at least 10 nucleotides, more preferably at least 15, 16, 17 or 18 nucleotides. The length of the complementary sequence in the oligonucleotide probe is typically at most 40 nucleotides, more preferably at most 30, at most 25, or at least 21 nucleotides. The length of the complementary sequence in the oligonucleotide probe may be 10-25, 15-25, 10-40, 15-40 or 15-30 nucleotides.

Mismatches may be present between the oligonucleotide probe and the target nucleic acid sequence at particular positions while still allowing for specific hybridisation to the target sequence. For example, there may be 1, 2, 3, 4 or 5 mismatches between the complementary region of the probe and the corresponding region of the target sequence.

Preferably, the oligonucleotide probe is designed to allow for specific hybridisation and binding to the target nucleic acid. Thus, the oligonucleotide probe typically specifically or selectively hybridises to a complementary sequence found only in target nucleic acid of interest. As will be appreciated in the art, specific or selective hybridisation refers to the binding of a probe only to a particular target nucleic acid under given conditions, when that sequence is present in a nucleic acid in a sample, such as a biological sample which can be a biological mixture including total cellular and foreign DNA or RNA (such as is the case in a cell lysate).

Typically, the total length of the oligonucleotide probe will be 15 - 40 nucleotides, more preferably at most 30 nucleotides, such as 15 to 25, or 18 to 21 nucleotides in length.

Specific examples of suitable oligonucleotide probes for binding to a target nucleic acid (sequence) from the E. coli ribosome RNP are provided herein. Preferred oligonucleotide probes for targeting the E. coli 23 S rRNA are the oligonucleotide probes of SEQ ID NO: 1 and 2 (or variants thereof). Preferred oligonucleotide probes for targeting the E. coli 16S rRNA are the oligonucleotide probes of SEQ ID NO: 3 and 4 (or variants thereof). Most preferably, the oligonucleotide probe for targeting the E. coli 23 S rRNA is SEQ ID NO: 1.

Variants of SEQ ID NOs: 1 to 4 may be oligonucleotides of up to 40 nucleotides in length comprising a region which is partly or fully complementary or identical to at least 10 contiguous nucleotides of the corresponding original probe sequence of SEQ ID NOs: 1 to 4 respectively. Preferably, said variants of SEQ ID NOs: 1 to 4 will comprise a region which is partly or fully complementary or identical to at least 11, 12, 13, 14 or 15 contiguous nucleotides of the corresponding original probe sequence of SEQ ID NOs: 1 to 4 respectively.

The above variants may comprise a region which has 1, 2, 3, 4, or 5 mismatches (substitutions) with respect to the corresponding region of the original probe sequence (and thus the target sequence) and thus is partly complementary or identical thereto. Thus, for instance, the variants may comprise a region of at least 10 nucleotides in length which has 1, 2, or 3 mismatches, such as 1 or 2 mismatches to a corresponding region of at least ten contiguous nucleotides of the corresponding original probe sequence. The variants may comprise a region of at least 13, 14 or 15 nucleotides in length which has 1, 2, 3, 4 or 5 mismatches, such as 1-3 mismatches to a corresponding region of an equivalent length in the corresponding original probe sequence.

Variants of SEQ ID NOs: 1-4 may also be oligonucleotides of up to 30 nucleotides in length which have at least 70% sequence identity to the sequence of the corresponding original probe sequence, preferably at least 75%, at least 80%, more preferably at least 85%, at least 90%, at least 95% sequence identity.

Any oligonucleotide probe used in the invention may comprise one or more modified nucleotides and/or a detectable label, for example a fluorescent dye

As used herein, the oligonucleotide probe is typically present in an aqueous solution. In the aqueous solution, the oligonucleotide probe in the aqueous solution may have a concentration from about 50 nM to about 50 pM. Preferably, the concentration of the oligonucleotide probe in the aqueous solution may be from about 50 nM to about 5 pM, from about 50 nM to about 500 nM, from about 50 nM to about 300 nM, from about 50 nM to about 200 nM, or from about 50 nM to about 150 nM. Preferably, the concentration of the oligonucleotide probe in the aqueous solution is from about 50 to about 150 nM or about 100 nM.

Incubation of the substrate with the oligonucleotide probe - priming

In accordance with the methods of the present invention, the electron permeable substrate is incubated with an aqueous solution comprising an oligonucleotide probe in order to produce a primed substrate. This is known as priming the substrate (in particular priming the surface of the substrate).

A substrate (or surface of the substrate) becomes primed when the oligonucleotide probe has adsorbed onto the surface of the substrate. As such, the period for the incubation of the substrate with the oligonucleotide probe should be sufficient for the oligonucleotide probe to adsorb onto the surface of the substrate.

In some embodiments, the period sufficient for the oligonucleotide probe to adsorb onto the surface of the substrate is from about 10 minutes to about 120 minutes. For example the period sufficient for the oligonucleotide probe to adsorb onto the surface of the substrate may be about 20 minutes, about 40 minutes, about 60 minutes, about 80 minutes, or about 100 minutes. In some embodiments, the period sufficient for the oligonucleotide probe to adsorb onto the surface of the substrate is from about 30 minutes to about 90 minutes. Preferably, the period sufficient for the oligonucleotide probe to adsorb onto the surface of the substrate is about 60 minutes. In some embodiments, excess liquid from the aqueous probe solution is removed from the surface of the electron permeable substrate following contact between the aqueous probe solution and substrate. In some embodiments, the excess liquid is removed in no more than 20 seconds, no more than 15 seconds, or no more than 10 seconds following the initial contact between the aqueous probe solution and the substrate. In some embodiments, the excess liquid is removed by blotting with an absorbent material, preferably with filter paper.

In some embodiments, the electron permeable substrate is incubated with at most about 15 pl of an aqueous solution comprising an oligonucleotide probe. Preferably, the electron permeable substrate is incubated with at most about 10 pl of an aqueous solution comprising an oligonucleotide probe. More preferably, the electron permeable substrate is incubated with at most about 5 pl of an aqueous solution comprising an oligonucleotide probe.

In some embodiments, the electron permeable substrate is incubated with the aqueous solution comprising an oligonucleotide probe in a humidified chamber. Any enclosed chamber that is humidified can be suitable for the incubation. For example, the humidified chamber may be an enclosed chamber comprising a reservoir of water, wherein the reservoir is not in contact with the electron permeable substrate. In some embodiments, the humidified chamber is set at a temperature of about 2 to about 10 degrees Celsius, preferably wherein the humidified chamber is set at a temperature of about 4 degrees Celsius.

Incubation of the primed substrate with the biological sample - hybridising

In accordance with the methods of the present invention, the primed electron permeable substrate is incubated with the biological sample comprising a target nucleic acid (which may, for example, be part of an RNP). This leads to hybridisation between the oligonucleotide probe and the complementary sequence on the target nucleic acid and is the step that leads to enrichment of the target nucleic acid (which may, for example, be part of an RNP) on the electron permeable substrate. As such, the period for the incubation of the substrate with the biological sample should be sufficient for the target nucleic acid to hybridise to the oligonucleotide probe on the primed substrate.

In some embodiments, the period sufficient for the target nucleic acid to hybridise to the oligonucleotide probe is from about 15 minutes to about 60 minutes. For example, the period sufficient for the oligonucleotide probe to adsorb onto the surface of the substrate may be about 15 minutes, about 30 minutes, or about 60 minutes. In some embodiments, the period sufficient for the target nucleic acid to hybridise to the oligonucleotide probe is from about 15 minutes to about 45 minutes. Preferably, the period sufficient for the target nucleic acid to hybridise to the oligonucleotide probe is about 30 minutes.

The period of time for which the primed electron permeable substrate is incubated with the biological sample comprising a target nucleic acid should preferably not exceed a period sufficient for the target nucleic acid to hybridise to the oligonucleotide probe. In particular, exceeding a period sufficient for the target nucleic acid to hybridise to the oligonucleotide probe may lead to crowding of the substrate surface which decrease particle resolution in subsequent analysis via e.g. TEM. As such, in preferred embodiments, the period for the incubation of the substrate with the biological sample should be no more than about 30 minutes.

In some embodiments, excess liquid from the biological sample is removed from the surface of the electron permeable substrate following the incubation period sufficient for the target nucleic acid to hybridise to the oligonucleotide probe. In some embodiments, the excess liquid is removed by blotting with an absorbent material, e.g. filter paper.

In some embodiments, the electron permeable substrate is incubated with at most about 20 pl of the biological sample. Preferably, the electron permeable substrate is incubated with at most about 15 pl of the biological sample. More preferably, the electron permeable substrate is incubated with at most about 10 pl of the biological sample.

In some embodiments, one or more modifying factors may be added to the biological sample during the incubation the electron permeable substrate with at the biological sample. As used herein, the modifying factor may be any molecule (such as a polypeptide, a small molecule, a polysaccharide, a nucleic acid, etc.) which is capable of altering the three-dimensional structure of the target nucleic acid (such as when present in a nucleic acid-protein complex such as an RNP) in the sample. Preferably, the target nucleic acid is part of a ribonucleoprotein (RNP) complex and the modifying factor is a ligand which is capable of binding to the RNP complex or an enzyme which acts on the RNP complex. In a preferred embodiment, the modifying factor is a polypeptide which is capable of altering the three-dimensional structure of a RNP in the sample. When the target nucleic acid is the 16S rRNA or 23 S rRNA and it is part of a ribosome RNP, the modifying factor may be Ribosome Recycling Factor (RRF) or Hibernation Promoting Factor (HPF).

In some embodiments, the primed electron permeable substrate is incubated with the biological sample in a humidified chamber. In some embodiments, the humidified chamber is set at a temperature of about 2 to about 10 degrees Celsius, preferably wherein the humidified chamber is set at a temperature of about 4 degrees Celsius.

Washing

Following incubation of the primed electron permeable substrate with the biological sample, the primed and hybridised substrate (which is now enriched with the target nucleic acid present in the biological sample) undergoes a wash step. Preferably, the washing step is carried out with a buffer, such as a compatible buffer solution or a biological buffer.

In some embodiments, the buffer may be a Tris buffer. The buffer may have a pH from about pH 6 to about pH 8, preferably from about pH 6.5 to about pH 7.5, preferably about pH 7. In some embodiments, the buffer may comprise 0.2-1 mM EDTA, 0.3 to 0.9 mM EDTA or about 0.5 mM EDTA. In some embodiments, the buffer may comprise from 50 to 500 mM NH4CI, such as from 50 to 250 mM NH4CI, or from 50 to 100 mM NH4CI. In some embodiments, the buffer may contain monovalent and/or divalent ions (such as Mg²⁺) sufficient to maintain stability of the target nucleic acid and/or nucleic acid-protein complex. In some embodiments, the buffer may contain chelating and/or reducing agents. In some embodiments, the buffer may comprise protease inhibitors and/or RNAse inhibitors. In a particularly preferred embodiment, the buffer comprises 20 mM Tris, pH 7, 100 mM NH4CI, 10.5 mM Magnesium Acetate, 0.5 mM EDTA and 5 mM P- mercaptoethanol.

Washing may comprise incubating the (enriched) primed and hybridised substrate surface with about 20 pl (or about 15 pl or about 25 pl) of wash buffer for at most about 20 minutes, or at most about 15 minutes. Preferably, washing comprises incubating the (enriched) primed and hybridised substrate surface with wash buffer for at about 10 minutes.

In some embodiments, the (enriched) primed and hybridised substrate surface is incubated with the wash in a humidified chamber. In some embodiments, the humidified chamber is set at a temperature of about 2 to about 10 degrees Celsius, preferably wherein the humidified chamber is set at a temperature of about 4 degrees Celsius.

In some embodiments, excess liquid from the wash buffer is removed from the surface of the (enriched) primed and hybridised substrate following the incubation. In some embodiments, the excess liquid is removed by blotting with an absorbent material, preferably with filter paper.

In some embodiments, the incubation with the wash buffer is repeated at least once (for example, at least twice or at least three times). In a preferred embodiment, the incubation with the wash buffer is repeated three times.

Overall period of time taken for the method of the invention

Preferably, steps (i) to (iii) of the above-described method of preparing a biological sample for EM are performed within a period of no more than 180 minutes, preferably a period of no more than 120 minutes.

Methods of analysis and uses

The present invention further provides a method of determining one or more biological properties of a biological sample, wherein the sample comprises a target nucleic acid and the method comprises preparing a biological sample according to the present invention performing biophysical analysis on the washed, hybridised primed substrate. The biophysical analysis may be selected from UV spectroscopy, TEM, or cryo-EM.

In some embodiments, the electron permeable substrate comprises: (a) an electronconducting mesh support having one or more apertures and (b) a biocompatible layer coating the mesh support.

Preferably, the electron permeable substrate comprises a hydrophobic surface. In embodiments, the mesh support layer may comprise (or consist of) a metal. In embodiments, the metal may be selected from the list of Cu, Ni, Ti, Si, Au, CuRh, Mo, Al and W. In some embodiments, the mesh support may comprise carbon, copper, nickel, molybdenum, beryllium, gold, silicon, GaAs, or oxide (e.g., SiCh, TiCh, ITO, or AI2O3). Preferably, the biocompatible layer comprises carbon. Preferably, the oligonucleotide probe has a length of 15 to 45 nucleotides.

The present invention additionally provides use of a substrate of the present invention for in situ analysis of a native ribonucleoprotein (RNP) in a biological sample, wherein the analysis is carried out using UV spectroscopy, TEM, or cryo-EM.

Kits

The invention also provides compositions and kits comprising: (a) an electron permeable substrate, (b) one or more oligonucleotide probes or one or more aqueous compositions, each comprising an oligonucleotide probe. The compositions and kits may also comprise a wash buffer and/or instructions for use.

The following Examples are provided to illustrate, but not limit the invention. Example 1 - RHyTEM method

RHyTEM (Figure 1) is a novel technique to enrich native Ribonucleic acids (RNA) and their complexes (RNP) through Hybridisation on a solid phase for biochemical, biophysical and structural characterisation using, for example, Transmission Electron Microscopy (TEM). RHyTEM comprises of three critical steps - surface/substrate priming, sample hybridisation and wash - which directly affect the spatial dispersion of the enriched particles on the solid phase for TEM applications.

First, a biocompatible and electron permeable solid phase surface (for direct TEM visualisation) such as a carbon layer coated onto a standard copper mesh TEM grid (Figure la), is primed by incubating it with oligonucleotide probes (Figure lb) which are specific (i.e. complementary) to an exposed regions of the RNA in the target molecule.

Next, the primed surface is incubated with 10-15 pl of clarified whole cell lysate (Figure 1c) in a humidified chamber to allow hybridisation of the probes with the target molecule and pull it down on the solid phase from solution.

Finally, the solid phase is washed several times with compatible buffer solutions (Figure Id) to remove the unbound and non-specifically bound cellular material from the primed surface. The washed surface can be used directly, for instance, in negative stain TEM imaging (Figure 3 a)

The RHyTEM processing pipeline can be summarised as follows.

Standard Buffer: 20 mM Tris, pH 7, 100 mM NH4CI, 10.5 mM Magnesium Acetate, 0.5 mM EDTA and 5 mM f-mercaptoethanol supplemented with protease inhibitors and RNAse inhibitors.

1. Apply ~5 pl of the solution containing the oligonucleotide probe to the electron permeable substrate surface to prime the substrate surface. Ensure that the droplet covers the whole surface of the grid.

2. Incubate for ~10s at room temperature.

3. Removing the excess liquid (for example, by pipetting the liquid away or blotting the liquid away with a piece of Whatmann paper), taking care that the surface of the substrate is undisturbed (by e.g. the blotting paper or pipette) and does not dry completely. 4. Further incubate the priming substrate for ~1 hr in a humidified chamber at 4°C to allow the probes to be adsorbed on to the surface.

5. Apply ~10 pl of whole cell lysate to the primed surface and further incubate for 30 mins at 4°C in a humidified chamber.

6. Blot excess liquid away as described in Step 3.

7. Wash the surface by incubating the surface with ~20 pl droplet of wash buffer for 10 mins at 4°C in a humidified chamber.

8. Blot excess liquid away as described in Step 3.

9. Repeat the wash steps 3 times.

The RHyTEM primed surface which is enriched for the target RNP complex can be directly used in further negative stain TEM analysis by, for example, following the standard staining protocol with 0.75% Uranyl Acetate solution at room temperature.

Example 2 - Probe design for RHyTEM

To test the RHyTEM method, oligonucleotide probes were designed for enrichment of ribosomal ribonucleoprotein (RNP) complexes. The oligonucleotide probes were chosen to be between 15 - 40 nucleotides in length and complementary to the exposed RNA loops and turns in the RNP complex of interest. This length was optimised for ease of chemical synthesis and for minimising the chance of random pairing to non-specific RNA or DNA fragments in the whole cell lysate. For RHyTEM enrichment of the E. coli 70S ribosome, two oligonucleotide probes complementary to different regions of the 16S and 23 S rRNA each (

Table 1) were chosen from the ProbeBASE database (Greuter et al., 2016; Loy et al., 2007). The probes specific to the 23 S rRNA were already shown to hybridise specifically to the E. coli ribosome in fluorescent in situ hybridisation (FISH) experiments and thus were already validated for recognising and binding to the ribosomes under in vivo and in vitro conditions. Table 1 : Sequence of the oligonucleotide probes used for RHyTEM enrichment of E. coli ribosomes.

Example 3 - Enrichment of ribosomes using RHyTEM

To test the efficiency of individual probes in enriching the ribosomes, we conducted a series of experiments (Figure 2) where a standard TEM grid comprising a carbon-covered copper mesh was primed (incubated) with the four oligonucleotide probes listed in Table 1 above along with two control conditions - one in which the grid was just primed with buffer (Figure 3c) and second where it was primed with a oligonucleotide probe with a random 20 nucleotide sequence non-specific to any E. coli RNP components (Figure 3d). Here we observed that most of the specific probes led to significant enrichment of the E. coli ribosomes (compared to control conditions) and amongst them, the 23S-1 probe led to the highest number of single, well-dispersed ribosome particles per micrograph (Figure 3 a). The 23S-1 probe was therefore selected to be used in later optimisation experiments. We also observed that compared to a separate grid incubated with a purified ribosome sample (Figure 3b). This clearly shows that the RHyTEM method is capable of enriching ribosomes within polysome particles (multiple ribosomes bound to an mRNA molecule formed during translation) in the micrograph.

E. coli whole cell lysate was incubated with either a hydrophobic (Figure 4a) or hydrophilic (Figure 4b) solid phase and did not observe any non-specific enrichment of the ribosomes on either of these surfaces. This shows that priming of a surface using oligonucleotide probes as per the RHyTEM method is required to enrich for ribosome particles, and an unprimed surface is not suitable for sample enrichment with the RHyTEM method as described herein.

Example 4- Reversible hybridisation to RHyTEM oligonucleotides

Since RHyTEM enrichment works on the principle of nucleic acid hybridisation, where interactions can be disrupted by buffers containing high ionic concentrations, a series of experiments were performed in which a primed RHyTEM substrate/surface, which has been subsequently incubated with a sample and enriched with ribosome particles, was washed with buffer solutions. The buffer solutions tested contained increasing concentrations of KC1 and the RNP content in the wash buffer eluent was estimated using absorbance at 280 nm (Figure 5). This confirmed that the oligonucleotide probe enriches for target ribosome particles, and further that a high ionic strength buffer reverses the hybridisation to the primed RHyTEM substrate surface, as expected.

Example 5 - Optimisation of sample incubation

Specific parameters of incubation can affect the quality of the sample resulting from the RHyTEM method. A high quality sample which is particularly suitable for micrographs and preventing overcrowding or under saturation of the surface.

Typically, in accordance with the RHyTEM method described in Example 1, 10- 15 pl of clarified whole cell lysate can be accommodated on a standard 3mm TEM grid such that the whole of the grid surface is covered by the lysate without causing any overruns to the opposing side (which has typically not been primed with oligonucleotide probes). Further, to maximize sample stability, we chose a standard ribosome compatible buffer (20 mM Tris, pH 7, 100 mM NH4CI, 10.5 mM Magnesium Acetate, 0.5 mM EDTA and 5 mM P-mercaptoethanol) supplemented with protease inhibitors and RNAse inhibitors for cell lysis and grid wash. Similarly, all incubation steps were performed at 4°C to maintain structural integrity of the RNP complex of interest in the cell lysate and during RHyTEM enrichment. To optimise the time of incubation of the cell lysate with the probe-primed substrate surface, a time series experiment was performed (Figure 6) for 10, 30, 60, 90 and 120 mins at 4°C in a humidified chamber before blotting the excess cell lysate away from the surface using Whatmann filter paper and washing the grids as detailed in the RHyTEM method in Example 1. Here, it was observed that an incubation period of the cell lysate (containing the RNP) with the probe-primed substrate surface for 30 mins at 4°C leads to the best spatial distribution of ribosomes in the TEM micrographs. At 10 mins, the grids were sparsely populated. At 60 mins or longer incubation times, the grids became increasingly crowded, which inhibited single particle image analysis.

Example 6 - Optimisation of oligonucleotide robe concentration

It is known that the optimal spatial distribution and number of particles in a cryo- TEM micrograph is related to the concentration of the target molecules (Vinothkumar & Henderson, 2016). For the 2700 kDa E. coli ribosome RNP complex, this number lies between 25 and 100 particles and corresponds to from about 0.5 mg/ml to about 2 mg/ml (100 to 750 nM) concentration of the ribosome complex in the sample. Thus, in the present study, probe concentrations in the nanomolar to micromolar range were tested to obtain approximately 100 RHyTEM enriched particles per negative stain TEM micrograph (Figure 7). It was observed that the 23S-1 probe at 100 nM concentration gave the highest number of resolved single, well-dispersed ribosomes particles per micrograph in the negative stain TEM experiment. This number is significantly enriched compared to a 0 nM control experiment or over the range of the concentrations for a completely nonspecific oligonucleotide probe that is not able to hybridise to E. coli ribosomes.

To test the utility of RHyTEM enrichment in biochemical and structural analysis of native RNP complexes, the effect of supplementing the cell lysate with ribosome modifying factors on the structural properties of the enriched ribosomes was analysed. Purified Ribosome Recycling Factor (RRF) and Hibernation Promoting Factor (HPF) were added to the cell lysate at a final concentration of 1 pM while the lysate was incubating with the probe primed surface in accordance with the RHyTEM method as described in Example 1 (Figure 8). RRF promotes splitting of the polysome complexes (as discussed above) into individual ribosome subunits and thus leads to release of the bound mRNA after completion of translation. HPF, on the other hand, stabilizes dimerization of the 70S ribosomes leading to formation of inactive 100S polysome particles. Thus, as expected, the number of RHyTEM enriched polysomes (2 or more ribosomes) per micrograph in the RRF treated sample was nearly half the number of polysome particles in control sample and those treated with HBF had more than twice the number of polysomes (Figure 8). For purified ribosomes, since the conventional protocol exclusively selects 70S particles, the number of polysomes in the control experiment of purified ribosomes is fewer than that of RHyTEM enrichment and is comparable to RRF treated samples. Here, the effect of ribosome modification is markedly seen in HBF treatment where the number of polysomes are significantly increased compared to control and RRF treatment.

Example 8 - Structural analysis

To demonstrate the utility of the RHyTEM method for structural analysis of RNP complexes via TEM, E. coli ribosomes from whole cell lysate were enriched directly on copper mesh TEM grids (Figure 3a) in accordance with the RHyTEM method as described in Example 1, using the 23S-1 oligonucleotide and according to the optimised incubation time (30 minutes) and oligonucleotide concentration values (100 nM) calculated above. For comparison, we also incubated copper mesh TEM grids (which had not been primed in accordance with the RHyTEM method) with purified ribosome sample at approximately lOOnM concentration (Figure 3b). The grids were then immediately stained with uranyl formate and imaged using a Tecnai G2 80-200keV Microscope. Between 10 to 70 micrographs (typically 20 to 30 micrographs) were collected from each sample, each containing approximately 100 well-dispersed particles per micrograph and analysed the images using EMAN2 (Tang et al., 2007) and RELION 3.0 (Scheres, 2016; Scheres, 2012; Zivanov et al., 2018) software. The analysis first distributed the particles into well resolved 2D classes (Figure 9a- c). As expected, the purified 70S sample yielded classes of ribosome monomers (Figure 9a), whereas the micrographs from the RHyTEM enriched sample distinguished 70S and 100S (polysome) particles classes (Figure 9). These classes were next used for reconstructing a 3D model of the ribosome (Figure 9d, e, g) and yielded a coarse model at 36 A resolution for both the RHyTEM enriched and purified ribosomes using only tens of hundred particles (typically 1500-3500 particles) and within few minutes of GPU processing time. These models have 76% volume overlap with correlation coefficient of 0.85 indicating stark similarities between the structures constructed for ribosomes obtained by the two methods. Further, any differences in the structures are also anticipated as the RHyTEM enriched ribosomes are expected to co-enrich with various cellular factors while these factors are selectively removed during conventional ribosome purification protocols. This comparison also indicates that the RHyTEM enriched particles do not have any significant orientation preference, probably due to the intrinsic flexibility of the probes and are suitable for 3D model reconstructions.

References

Blaha, G., Stanley, R. E., & Steitz, T. A. (2009). Formation of the first peptide bond: The structure of EF-P bound to the 70S ribosome. Science. https://doi.org/10.1126/science.l 175800

Greuter, D., Loy, A., Horn, M., & Rattei, T. (2016). ProbeBase-an online resource for rRNA-targeted oligonucleotide probes and primers: New features 2016. Nucleic Acids Research, https://doi.org/10.1093/nar/gkvl232

Loy, A., Maixner, F., Wagner, M., & Hom, M. (2007). probeBase - An online resource for rRNA-targeted oligonucleotide probes: New features 2007. Nucleic Acids Research. https://doi.org/10.1093/nar/gkl856

Scheres, S. H.W. (2016). Processing of Structurally Heterogeneous Cryo-EM Data in

RELION. In Methods in Enzymology, https://doi.org/10.1016/bs.mie.2016.04.012 Scheres, Sjors H.W. (2012). RELION: Implementation of a Bayesian approach to cryo-EM structure determination. Journal of Structural Biology, 180(3), 519-530. https://doi.Org/10.1016/j.jsb.2012.09.006

Tang, G., Peng, L., Baldwin, P. R., Mann, D. S., Jiang, W., Rees, I., & Ludtke, S. J. (2007). EMAN2: An extensible image processing suite for electron microscopy. Journal of

Structural Biology, https://doi.org/10.1016/j jsb.2006.05.009

Vinothkumar, K. R., & Henderson, R. (2016). Single particle electron cryomicroscopy: trends, issues and future perspective. Quarterly Reviews of Biophysics, 49, el3. https://doi.org/10.1017/S0033583516000068 Zivanov, J., Nakane, T., Forsberg, B. O., Kimanius, D., Hagen, W. J. H., Lindahl, E., & Scheres, S. H. W. (2018). New tools for automated high-resolution cryo-EM structure determination in RELION-3. ELife, 7. https://doi.org/10.7554/eLife.42166

Claims

1. A method of preparing a biological sample for electron microscopy, wherein the sample comprises a target nucleic acid and the method comprises:

(i) incubating an electron permeable substrate with an aqueous solution comprising an oligonucleotide probe for a period sufficient for the oligonucleotide probe to adsorb onto the surface of the substrate to produce a primed substrate, wherein the oligonucleotide probe comprises a region that is complementary to the target nucleic acid;

(ii) incubating the biological sample with the primed substrate for a period sufficient for the target nucleic acid to hybridise to the oligonucleotide probe to produce a hybridised primed substrate; and

(iii) washing the hybridised primed substrate with a buffer.

2. The method of claim 1, wherein the biological sample is a cell lysate, preferably a clarified cell lysate.

3. The method of claim 2, wherein the cell is a prokaryotic cell or a eukaryotic cell.

4. The method of claims 1 to 3, wherein the volume of biological sample incubated in step (ii) is from about 10 pl to about 15 pl.

5. The method of any one of claims 1 to 4, wherein the target nucleic acid comprises or consists of DNA.

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6. The method of any one of claims 1 to 4, wherein the target nucleic acid comprises or consists of RNA.

7. The method of any one of claims 1 to 6, wherein the target nucleic acid is associated with a polypeptide.

8. The method of any one of claims 1 to 7, wherein the target nucleic acid is part of a ribonucleoprotein (RNP) complex.

9. The method of any one of claims 1 to 8, wherein the target nucleic acid is part of a native RNP complex.

10. The method of any one of claims 1 to 9, wherein the concentration of the oligonucleotide probe in the aqueous solution is from about 50 to about 150 nM.

11. The method of claim 10, wherein the concentration of the oligonucleotide probe in the aqueous solution is about 100 nM.

12. The method of any one of claims 1 to 11, wherein the period sufficient for the oligonucleotide probe to adsorb onto the surface of the substrate is from about 10 minutes to about 120 minutes.

13. The method of claim 12, wherein the period sufficient for the oligonucleotide probe to adsorb onto the surface of the substrate is from about 30 minutes to about 90 minutes, optionally wherein the period sufficient for the oligonucleotide probe to adsorb onto the surface of the substrate is about 60 minutes.

14. The method of any one of claims 1 to 13, wherein the period sufficient for the target nucleic acid to hybridise to the oligonucleotide probe on the primed substrate is from about 15 minutes to about 45 minutes.

15. The method of claim 14, wherein the period sufficient for the target nucleic acid to hybridise to the oligonucleotide probe on the primed substrate is about 30 minutes.

16. The method of any one of claims 1 to 15, wherein the incubation in step (i) and/or step (ii) is carried out in a humidified chamber.

17. The method of claim 16, wherein the humidified chamber is set at a temperature of about 2 to about 10 degrees Celsius, optionally wherein the humidified chamber is set at a temperature of 4 degrees Celsius.

18. The method of any one of claims 1 to 17, wherein one or more modifying factors are added to the biological sample during the incubation of step (ii).

19. The method of claim 18, wherein the target nucleic acid is part of a nucleic acidprotein complex and wherein:

(a) the modifying factor is a polypeptide which is capable of altering the three- dimensional structure of the nucleic acid-protein complex in the sample, and preferably the nucleic acid-protein complex is a ribonucleoprotein (RNP) complex; or

(b) the modifying factor is a ligand which is capable of binding to the nucleic acidprotein complex or an enzyme which acts on the nucleic acid-protein complex, and preferably the nucleic acid-protein complex is a ribonucleoprotein (RNP) complex.

20. The method of any one of claims 1 to 19, wherein steps (i) to (iii) are performed within a period of no more than 180 minutes, preferably a period of no more than 120 minutes.

21. A method of determining one or more biological properties of a biological sample, wherein the sample comprises a target nucleic acid and the method comprises carrying out steps (i) to (iii) as defined in any one of claims 1 to 20 and performing biophysical analysis on the washed, hybridised primed substrate; wherein the biophysical analysis is selected from UV spectroscopy, TEM, or cryo-EM.

22. The method of any one of claims 1 to 21, wherein the electron permeable substrate comprises:

(a) an electron-conducting mesh support having one or more apertures; and

(b) a biocompatible layer coating the mesh support.

23. A primed electron permeable substrate comprising:

(a) an electron-conducting mesh support having one or more apertures;

(b) a biocompatible layer coating the mesh support; and

28 (c) one or more oligonucleotide probes adsorbed onto the biocompatible layer.

24. The method of claim 22 or substrate of claim 23, wherein the electron permeable substrate comprises a hydrophobic surface.

25. The method or substrate of any one of claims 22 to 24, wherein the support comprises carbon, copper, nickel, molybdenum, beryllium, gold, silicon, GaAs, or oxide (e g., SiCh, TiC>2, ITO, or AI2O3).

26. The method or substrate of any one of claims 22 to 25, wherein the biocompatible layer comprises carbon.

27. The method or substrate of any one of claims 1 to 26, wherein the oligonucleotide probe has a length of 15 to 45 nucleotides.

28. Use of a substrate as defined in any one of claims 23 to 27 for the in situ analysis of a native nucleic acid-protein complex in a biological sample, wherein the analysis is carried out using UV spectroscopy, TEM, or cryo-EM, preferably wherein the nucleic acid-protein complex is a ribonucleoprotein (RNP) complex.

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