Kits and methods for detecting mRNA

Abstract

Kits and methods for detecting messenger RNA (mRNA) are generally provided. The kits and methods described herein may make use of probes that, upon exposure to mRNA, bind the mRNA thereto. When the mRNA is present in a liquid with which the probe is contacted, the probe may bind the mRNA thereto in an amount and / or at a rate that depends on the concentration of the mRNA in the liquid. In some embodiments, contacting a probe with a liquid comprising mRNA may result in the generation of a signal that is indicative of the concentration of the mRNA in the liquid.

Description

[0001] KITS AND METHODS FOR DETECTING MRNA

[0002] RELATED APPLICATIONS

[0003] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 748,326, filed January 22, 2025, and entitled “Kits and Methods for Detecting mRNA,” which is incorporated herein by reference in its entirety for all purposes.

[0004] FIELD

[0005] Kits and methods for detecting mRNA are generally provided.

[0006] BACKGROUND

[0007] It can be desirable to detect mRNA in various samples. However, some existing methods of detecting mRNA experience drawbacks, like low signal and / or long detection times. Accordingly, improved methods and kits are needed.

[0008] SUMMARY

[0009] The present disclosure generally describes kits and methods for detecting mRNA. The subject matter described herein involves, in some cases, interrelated products, alternative solutions to a particular problem, and / or a plurality of different uses of one or more systems and / or articles.

[0010] Paragraph 1: In some embodiments, a kit is provided. The kit comprises a probe, a salt comprising a divalent cation, and a salt comprising a monovalent cation. The probe comprises a body portion, a first coating disposed on the body portion, and a second coating disposed on the first coating. The first coating has a composition and an arrangement such that light impinging on the first coating after traveling through the body portion is partially reflected at an interface between the first coating and the body portion and partially transmitted through the first coating. The second coating comprises a molecule. The molecule comprises a plurality of repeat units. The plurality of repeat units comprises deoxy thymidine and / or a thymidine analog.

[0011] Paragraph 2: In some embodiments, a method is provided. The method comprises contacting a probe with a liquid comprising mRNA at an mRNA concentration, after contacting the probe with the liquid comprising mRNA at the mRNA concentration, contacting the probe with a liquid comprising a divalent cation, while and / or after contacting the probe with the liquid comprising the divalent cation, detecting a signal, and 1

[0012] #14820240vldetermining the mRNA concentration based on the signal. The probe comprises a body portion and a coating disposed on the body portion. The coating disposed on the body portion comprises a molecule. The molecule comprises a plurality of repeat units. The plurality of repeat units comprises deoxythymidine and / or a thymidine analog. The signal is indicative of an amount and / or a rate of binding between the mRNA and the molecule.

[0013] Paragraph 3: In some embodiments, a method comprises contacting a probe with a liquid comprising mRNA at an mRNA concentration, while and / or after contacting the probe with the liquid comprising mRNA at the mRNA concentration, detecting a signal, and determining the mRNA concentration based on the signal. The liquid comprising mRNA further comprises a divalent cation and a monovalent cation. The probe comprises a body portion and a coating disposed on the body portion. The coating disposed on the body portion comprises a molecule. The molecule comprises a plurality of repeat units. The plurality of repeat units comprises deoxythymidine and / or a thymidine analog. The signal is indicative of an amount and / or a rate of binding between the mRNA and the molecule.

[0014] Paragraph 4: In some embodiments, in a kit or method of any preceding Paragraph, the divalent cation is Mg2+.

[0015] Paragraph 5: In some embodiments, in a kit or method of any preceding Paragraph, the divalent cation is Ca2+.

[0016] Paragraph 6: In some embodiments, in a kit or method of any preceding Paragraph, the monovalent cation is Na+.

[0017] Paragraph 7: In some embodiments, in a kit or method of any preceding Paragraph, the monovalent cation is K+.

[0018] Paragraph 8: In some embodiments, in a kit or method of any preceding Paragraph, the coating is a second coating, and wherein the probe further comprises a first coating.

[0019] Paragraph 9: In some embodiments, in a kit or method of any preceding Paragraph, the probe further comprises a third coating positioned between the first coating and the second coating.

[0020] Paragraph 10: In some embodiments, in a kit or method of any preceding Paragraph, the third coating comprises streptavidin.

[0021] Paragraph 11: In some embodiments, in a kit or method of any preceding Paragraph, the molecule is chemically bound to biotin.

[0022] Paragraph 12: In some embodiments, in a kit or method of any preceding Paragraph, the plurality of repeat units comprises deoxythymidine.

[0023] 2

[0024] #14820240vlParagraph 13: In some embodiments, in a kit or method of any preceding Paragraph, the plurality of repeat units comprises the thymidine analog.

[0025] Paragraph 14: In some embodiments, in a kit or method of any preceding Paragraph, the thymidine analog is an engineered thymidine analog.

[0026] Paragraph 15: In some embodiments, in a kit or method of any preceding Paragraph, the engineered thymidine analog is LNA thymidine.

[0027] Paragraph 16: In some embodiments, in a kit or method of any preceding Paragraph, the liquid comprising the mRNA comprises a crude sample.

[0028] Paragraph 17: In some embodiments, in a kit or method of any preceding Paragraph, the liquid comprising the mRNA comprises RNA polymerase.

[0029] Paragraph 18: In some embodiments, in a kit or method of any preceding Paragraph, the liquid comprising the mRNA comprises dithiothreitol.

[0030] Paragraph 19: In some embodiments, in a kit or method of any preceding Paragraph, the liquid comprising the mRNA comprises a DNA template.

[0031] Paragraph 20: In some embodiments, in a kit or method of any preceding Paragraph, the liquid comprising the mRNA comprises cellular lysates.

[0032] Paragraph 21: In some embodiments, in a kit or method of any preceding Paragraph, the liquid comprising the mRNA comprises tissue homogenates.

[0033] Paragraph 22: In some embodiments, in a kit or method of any preceding Paragraph, the liquid comprising the mRNA comprises a surfactant.

[0034] Paragraph 23: In some embodiments, in a kit or method of any preceding Paragraph, the surfactant is NP-40 or Triton X-100.

[0035] Paragraph 24: In some embodiments, in a kit or method of any preceding Paragraph, the surfactant makes up greater than or equal to 0.01 wt% and less than or equal to 2 wt% of the liquid.

[0036] Paragraph 25: In some embodiments, in a kit or method of any preceding Paragraph, the liquid comprising the mRNA comprises a DNase inhibitor.

[0037] Paragraph 26: In some embodiments, in a kit or method of any preceding Paragraph, the liquid comprising the mRNA comprises an RNase inhibitor.

[0038] Paragraph 27: In some embodiments, in a kit or method of any preceding Paragraph, the liquid comprising the mRNA comprises a protease inhibitor.

[0039] Paragraph 28: In some embodiments, in a kit or method of any preceding Paragraph, the divalent cation is dissolved in water.

[0040] 3

[0041] #14820240vlParagraph 29: In some embodiments, in a kit or method of any preceding Paragraph, the salt comprising the monovalent cation is dissolved in water.

[0042] Paragraph 30: In some embodiments, in a kit or method of any preceding Paragraph, the kit comprises a buffer in which the salt comprising the divalent cation and the salt comprising the monovalent cation are dissolved.

[0043] Paragraph 31: In some embodiments, in a kit or method of any preceding Paragraph, the kit comprises a first buffer in which the salt comprising the divalent cation is dissolved and a second buffer in which the salt comprising the monovalent cation is dissolved.

[0044] Paragraph 32: In some embodiments, in a kit or method of any preceding Paragraph, the liquid comprising the mRNA is a buffer.

[0045] Paragraph 33: In some embodiments, in a kit or method of any preceding Paragraph, the liquid comprising the mRNA is a buffer comprising the monovalent cation.

[0046] Paragraph 34: In some embodiments, in a kit or method of any preceding Paragraph, the liquid comprising the mRNA is a buffer comprising the divalent cation.

[0047] Paragraph 35: In some embodiments, in a kit or method of any preceding Paragraph, the liquid comprising the mRNA is a Tris buffer.

[0048] Paragraph 36: In some embodiments, in a kit or method of any preceding Paragraph, the liquid comprising the mRNA is a HEPES buffer.

[0049] Paragraph 37: In some embodiments, in a kit or method of any preceding Paragraph, the liquid comprising the mRNA is a citrate buffer.

[0050] Paragraph 38: In some embodiments, in a kit or method of any preceding Paragraph, a concentration of the divalent cation in the buffer is greater than or equal to 1 mM and less than or equal to 100 mM.

[0051] Paragraph 39: In some embodiments, in a kit or method of any preceding Paragraph, a concentration of the monovalent cation in the buffer is greater than or equal to 50 mM and less than or equal to 200 mM.

[0052] Paragraph 40: In some embodiments, a kit or method of any preceding Paragraph further comprises contacting the probe with a liquid comprising the divalent cation after contacting the probe with the liquid comprising the mRNA at the mRNA concentration.

[0053] Paragraph 41: In some embodiments, a kit or method of any preceding Paragraph further comprises contacting the probe with a regeneration liquid after contacting the probe with the liquid comprising the mRNA at the mRNA concentration.

[0054] Paragraph 42: In some embodiments, in a kit or method of any preceding Paragraph, the regeneration liquid is a buffer.

[0055] 4

[0056] #14820240vlParagraph 43: In some embodiments, in a kit or method of any preceding Paragraph, the buffer comprises NaOH.

[0057] Paragraph 44: In some embodiments, a kit or method of any preceding Paragraph further comprises contacting the probe with a neutralization liquid after contacting the probe with the regeneration liquid.

[0058] Paragraph 45: In some embodiments, in a kit or method of any preceding Paragraph, the neutralization liquid is a buffer.

[0059] Paragraph 46: In some embodiments, in a kit or method of any preceding Paragraph, the mRNA comprises a poly(A) tail.

[0060] Paragraph 47: In some embodiments, in a kit or method of any preceding Paragraph, the binding between the mRNA and the molecule occurs via binding between the poly(A) tail and the deoxythymidine and / or between the poly(A) tail and the thymidine analog.

[0061] Paragraph 48: In some embodiments, a kit or method of any preceding Paragraph further comprises, while and / or after contacting the probe with the liquid comprising mRNA at the mRNA concentration, detecting a signal.

[0062] Paragraph 49: In some embodiments, in a kit or method of any preceding Paragraph, the signal is an optical signal.

[0063] Paragraph 50: In some embodiments, in a kit or method of any preceding Paragraph, the optical signal comprises light reflected from two different locations associated with the probe.

[0064] Paragraph 51: In some embodiments, in a kit or method of any preceding Paragraph, the optical signal comprises light reflected from an interface between the body portion and the coating and light reflected from the end of the probe.

[0065] Paragraph 52: In some embodiments, in a kit or method of any preceding Paragraph, the optical signal comprises light interference generated by interferometry.

[0066] Paragraph 53: In some embodiments, in a kit or method of any preceding Paragraph, the interferometry is biolayer interferometry.

[0067] Paragraph 54: In some embodiments, in a kit or method of any preceding Paragraph, the method is performed by an instrument.

[0068] Paragraph 55: In some embodiments, a kit or method of any preceding Paragraph further comprises contacting the probe with a standard.

[0069] Paragraph 56: In some embodiments, in a kit or method of any preceding Paragraph, the liquid comprising mRNA is contained by a container.

[0070] 5

[0071] #14820240vlParagraph 57: In some embodiments, in a kit or method of any preceding Paragraph, the liquid comprising the divalent cation is contained by a container.

[0072] Paragraph 58: In some embodiments, in a kit or method of any preceding Paragraph, the standard is contained by a container.

[0073] Paragraph 59: In some embodiments, in a kit or method of any preceding Paragraph, the regeneration liquid is contained by a container.

[0074] Paragraph 60: In some embodiments, in a kit or method of any preceding Paragraph, the neutralization liquid is contained by a container.

[0075] Paragraph 61: In some embodiments, in a kit or method of any preceding Paragraph, the container is a well in a multiwell plate.

[0076] Paragraph 62: In some embodiments, in a kit or method of any preceding Paragraph, the multiwell plate further comprises a well containing the liquid comprising the divalent cation, a well containing a standard, a well containing a regeneration liquid, and / or a well containing a neutralization liquid.

[0077] Paragraph 63: In some embodiments, a kit or method of any preceding Paragraph further comprises translating the probe to contact the liquids sequentially.

[0078] Paragraph 64: In some embodiments, in a kit or method of any preceding Paragraph, the containers are wells in a multiwell plate.

[0079] Other advantages and novel features of the present invention will become apparent from the following detailed description of various non-limiting embodiments of the invention when considered in conjunction with the accompanying figures. In cases where the present specification and a document incorporated by reference include conflicting and / or inconsistent disclosure, the present specification shall control. If two or more documents incorporated by reference include conflicting and / or inconsistent disclosure with respect to each other, then the document having the later effective date shall control.

[0080] BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the

[0082] 6

[0083] #14820240vlinvention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention. In the figures:

[0084] FIG. 1 shows one non-limiting example of such a method, in accordance with some embodiments;

[0085] FIG. 2 shows one non-limiting example of a kit, in accordance with some embodiments;

[0086] FIG. 3 shows one non-limiting example of a probe comprising a body portion and a coating disposed on the body portion, in accordance with some embodiments;

[0087] FIG. 4 shows one non-limiting example of a probe comprising a body portion and two coatings disposed thereon, in accordance with some embodiments;

[0088] FIG. 5 shows one non-limiting example of a probe comprising a body portion and three coatings disposed thereon, in accordance with some embodiments;

[0089] FIG. 6 shows one non-limiting example of a probe comprising a body portion and four coatings disposed thereon, in accordance with some embodiments;

[0090] FIG. 7 depicts schematically one example of a process by which light comprising both an amount of the light that has been reflected from an interface internal to a probe and an amount of light that has been reflected from the end of a probe can be generated, in accordance with some embodiments;

[0091] FIG. 8 depicts schematically the binding of oligo(dT) to a probe, the binding of mRNA to the oligo(dT), and the anticipated wavelength shift during these processes, in accordance with some embodiments;

[0092] FIG. 9 shows wavelength shifts as a function of time during processes described in Example 2;

[0093] FIG. 10 shows wavelength shifts as a function of time during processes described in Example 3;

[0094] FIG. 11 shows the key associated with FIG. 10 and calculated binding rates between the mRNA and a probe;

[0095] FIGs. 12 and 13 show wavelength shifts as a function of time during processes described in Example 4;

[0096] FIG. 14 shows the wavelength shifts as a function of time during processes described in Example 5;

[0097] FIG. 15 shows calculated binding rates and a curve that fits the calculated binding rates as a function of known mRNA concentrations, in accordance with some embodiments;

[0098] 7

[0099] #14820240vlFIGs. 16, 18, 20, and 22 show wavelength shifts as a function of time during processes described in Example 5 during processes described in Example 6;

[0100] FIGs. 17, 19, 21, and 23 show calculated binding rates and associated curve fits for the wavelength shifts shown in FIGs. 16, 18, 20, and 22, respectively; and

[0101] FIGs. 24 and 25 further summarize the data shown in FIGs. 16-23.

[0102] DETAILED DESCRIPTION

[0103] Kits and methods for detecting messenger RNA (mRNA) are generally provided. The kits and methods described herein may make use of probes that, upon exposure to mRNA, bind the mRNA thereto. When the mRNA is present in a liquid with which the probe is contacted, the probe may bind the mRNA thereto in an amount and / or at a rate that depends on the concentration of the mRNA in the liquid. In some embodiments, contacting a probe with a liquid comprising mRNA may result in the generation of a signal that is indicative of the concentration of the mRNA in the liquid.

[0104] Some methods described herein comprise performing mRNA detection in the presence of a divalent cation. Some kits described herein include a probe and reagents for performing such methods. Without wishing to be bound by any particular theory, it is believed that the presence of certain divalent cations, such as Mg2+, in a liquid comprising mRNA may cause the mRNA to have a size that is more condensed than it would have in an otherwise-equivalent liquid lacking the divalent cation. It is also believed that this condensed structure can cause certain signals generated upon binding between the mRNA and the probe to have larger magnitudes than would be generated upon such binding if the mRNA were less condensed. Moreover, it is believed that many buffers comprising divalent cations are understood by the scientific community to undesirably cause RNA degradation, and so their suitability for use in a technique for determining mRNA concentration is believed to be unexpected.

[0105] As noted above, certain embodiments relate to methods. FIG. 1 shows one nonlimiting example of such a method. In FIG. 1, the method 100 comprises the steps 102-114. It should be noted that all of the steps shown in the method 100 are optional. Therefore, the methods contemplated herein may include some, all, or none of the steps shown in the method 100. Similarly, although the steps shown in the method 100 are shown in a particular order, it is possible for them to be performed in a different order and / or for two or more steps to be performed for periods of time that overlap (e.g., partially or fully).

[0106] 8

[0107] #14820240vlThe first, optional step of the method 100 is the step 102, which comprises contacting a probe with a liquid comprising mRNA at an mRNA concentration. As noted above, contacting a probe with a liquid comprising mRNA may result in binding of the mRNA to the probe. The liquid comprising the mRNA may be a liquid for which it would be desirable to determine the concentration of mRNA therein, such as a sample obtained from a transcription reaction (e.g., an in vitro transcription reaction), a sample comprising cellular lysates, and / or a sample comprising tissue homogenates. This liquid may be a crude sample (e.g., a liquid obtained as a sample directly from a particular bioprocessing instrument and / or reaction vessel, a liquid obtained directly from a tissue and / or cell lysis process) or may be a liquid that has undergone one or more steps (e.g., one or more heating steps, one or more enzyme digestion steps) between being obtained and having the probe contacted therewith. As one example, in some embodiments, the liquid may be a crude sample that has been mixed with a buffer and / or another component comprising one or more species that are believed to enhance signal generation.

[0108] Contact between a probe and a liquid may be effectuated in a variety of suitable manners. In some embodiments, a probe is translated such that it contacts the surface of a liquid and / or is partially immersed in a liquid. It is also possible for a container containing a liquid to be translated such that the surface of the liquid contained therein contacts the probe and / or such that the probe becomes immersed in the liquid.

[0109] The second, optional step of the method 100 is the step 104, which comprises contacting the probe with a liquid comprising a divalent cation (e.g., a liquid in which the divalent cation is dissolved). A variety of suitable liquids comprising a divalent cation may be employed for this purpose, such as aqueous liquids comprising divalent cation-containing salts and / or buffers comprising divalent cation-containing salts (e.g., aqueous liquids and / or buffers in which such salts are dissolved).

[0110] The third, optional step of the method 100 is the step 106, which comprises detecting a signal. The signal may be a signal that is indicative of a rate of binding between mRNA and the probe. Such a signal may be indicative of a concentration of mRNA in the liquid (e.g., when mRNA is present in the liquid in an amount such that the rate of binding between mRNA and the probe can be readily correlated to the amount of mRNA in the liquid). As discussed in further detail below, the signal may be an optical signal.

[0111] Signals may be detected during a variety of suitable points in time. As one example, some methods comprise detecting a signal while and / or after the probe is contacted with a liquid comprising mRNA. As another example, some methods comprise detecting a signal 9

[0112] #14820240vlwhile and / or after the probe is contacted with a liquid comprising a divalent cation. Further examples of periods of time during which signals may be detected may be prior to contacting the probe with the liquid comprising the mRNA and during and / or after steps in which mRNA bound to a probe is intended to be removed therefrom. In some embodiments, a method to comprise detecting two or more signals at different points in time (e.g., during two or more of the periods of time described above).

[0113] The detection of a signal may comprise detecting the signal at a single point in time and / or may comprise detecting the signal over a period of time. As two examples of the former, detecting a signal may comprise detecting the signal a point in time at which the amount of binding between the mRNA in a liquid and a probe has plateaued or a point in time at which the baseline is stable. As one example of the latter, detecting a signal may comprise detecting the signal while mRNA in the liquid is binding to a probe.

[0114] As described in further detail below, signals that are detected may be signals from which the concentration of mRNA in the liquid comprising the mRNA can be readily determined. Such signals may include signals during and / or after contact of the probe with the liquid comprising the mRNA. It is also possible for a variety of other types of signals to be detected, such as signals that indicate a baseline measurement (e.g., signals detected prior to the contact of the probe with the liquid comprising the mRNA) and / or signals that can be employed to assess the performance of one or more processes performed by the instrument (e.g., signals detected during steps intended to remove bound mRNA from the probe and / or during preparation of the probe for contact with a liquid comprising mRNA).

[0115] The fourth, optional step of the method 100 is the step 108, which comprises determining the mRNA concentration (i.e., the concentration of the mRNA in the liquid comprising the mRNA) based on the signal. Such a step may be suitably performed when the signal on which the determination of the mRNA concentration is based is a signal indicative of an amount of binding between the mRNA and the probe and / or a rate of binding between the mRNA and the probe. The amount of binding between the mRNA and the probe may be determined from a signal obtained at a single point in time (e.g., a point in time at which the amount of binding between the mRNA in a liquid and a probe has plateaued). The rate of binding between the mRNA and the probe may be determined from a signal obtained over a period of time (e.g., a period of time during which the mRNA is binding to the probe).

[0116] In some embodiments, determining the mRNA concentration in a liquid may comprise curve fitting a signal obtained over a period of time. The signal may be curve fit to an expected binding rate curve, which may be employed to determine the rate of binding 10

[0117] #14820240vlbetween the mRNA and the probe. The rate of binding between the mRNA and the probe may then be employed to determine the concentration of the mRNA in the liquid based on a known relationship between this concentration and the rate of binding to the probe. It is also possible for the signal to be fit to a curve that directly correlates measured signal to known concentrations of mRNA in the liquid with which the probe is contacted.

[0118] The fifth, optional step of the method 100 is the step 110, which comprises contacting the probe with a regeneration liquid. The regeneration liquid may be a liquid that has a composition that removes mRNA bound to the probe from the probe. Contacting the probe with a regeneration liquid may therefore allow for a probe to which mRNA is bound to be returned to the state it was in prior to the mRNA binding thereto and / or to be used in one or more subsequent processes comprising some or all of the steps of the method 100 (e.g., a subsequent process in which the probe is contacted with a different liquid comprising mRNA and then a signal is generated that allows for the determination of the concentration of mRNA in the different liquid). For this reason, it may be performed upon the conclusion of the detection of a signal associated with mRNA binding to the probe (e.g., after contacting the probe with the liquid comprising mRNA, after contacting the probe with a liquid comprising a divalent cation, and / or after detecting a signal).

[0119] This sixth, optional step of the method 100 is the step 112, which comprising contacting the probe with a neutralization liquid (e.g., after contacting the probe with a regeneration liquid). Some regeneration liquids may remove bound mRNA from the probe and also leave the probe in a state that is less suitable for binding further mRNA thereto. For instance, some regeneration liquids may, during contact with the probe, deposit one or more species on the probe and / or cause the probe surface to have one or more species that hinders the binding of mRNA thereto (e.g., a charge undesirable and / or unsuitable for binding mRNA thereto). In such instances, it can be desirable to contact the probe with a liquid that compensates for these effects (e.g., by removing such deposited species and / or altering the surface charge of the probe). This may be accomplished in the sixth, optional step of the method 100.

[0120] The seventh, optional step of the method 100 is the step 114, which comprises contacting the probe with a standard. The standard may be any of a variety of suitable liquids. In some embodiments, a probe is contacted with a standard that is a liquid lacking mRNA (i.e., a negative standard). Additionally or alternatively, it is also possible for a probe to be contacted with a standard that includes a known mRNA that is present in a known amount (i.e., a positive standard). Although shown as the seventh step in the method 100, as 11

[0121] #14820240vlnoted above, the steps of the method 100 may be performed in other orders. For instance, a probe may be contacted with one or more standards prior to be contacted with a liquid comprising mRNA at an mRNA concentration.

[0122] Further features of the method steps 102-114 of the method 100, and other features of exemplary methods, are also described in further detail below.

[0123] As noted above, certain embodiments relate to kits. Such kits may be capable of and / or configured to be employed to perform one or more methods described herein.

[0124] Similarly, some methods described herein may comprise using one or more components of a kit described herein.

[0125] FIG. 2 shows one non-limiting example of a kit. The kit 216 shown in FIG. 2 comprises a probe 218, a salt 220 comprising a divalent cation, and a salt 222 comprising a monovalent cation. Salts provided as part of a kit may be provided as the salt (e.g., in solid and / or crystalline form) or may be provided dissolved in a liquid (e.g., dissolved in an aqueous liquid, such as water or an aqueous buffer). In some embodiments, a kit comprises a single liquid (e.g., a single aqueous liquid, such as water or a single aqueous buffer) comprising both the salt comprising the divalent cation and the salt comprising the monovalent cation. It is also possible for a kit to provide two different liquids, one of which comprises the salt comprising the divalent cation (e.g., a first aqueous liquid, such as a first portion of water and / or a first aqueous buffer) and the other of which comprises the salt comprising the monovalent cation (e.g., a second aqueous liquid, such as a second portion of water and / or a second aqueous buffer).

[0126] Further features of the kit 216, and other features of exemplary kits, are described in further detail below.

[0127] As noted above, some methods described herein may comprise contacting a probe with one or more liquids and some systems described herein may comprise a probe. In some embodiments, a probe comprises a body portion and one or more coatings disposed on a body portion. FIG. 3 shows one non-limiting example of a probe 318 comprising a body portion 324 and a coating 326 disposed on the body portion 324. Further examples of suitable coatings and features of the body portion are provided in further detail below.

[0128] As used herein, when a component is referred to as being “disposed on” another component, it can be directly disposed on that component layer, or an intervening component also may be present. A component that is “directly disposed on” or “in contact with” another component means that no intervening component is present.

[0129] 12

[0130] #14820240vlIn some embodiments, a probe comprises a coating (e.g., a coating disposed on the probe’s body portion) that is particularly suitable for binding mRNA thereto, such as a coating comprising deoxythymidine and / or a thymidine analog. Without wishing to be bound by any particular theory, it is believed that mRNA typically comprises a plurality of adenine repeat units, which form a structure referred to as a “poly(A) tail.” It is also believed that adenine may bind particularly strongly to deoxythymidine and thymidine analogs. For this reason, it is believed that probes comprising coatings comprising deoxythymidine and / or thymidine analogs may be particularly suitable for binding mRNA to the probe (e.g., via binding between the mRNA’s poly(A) tail and the deoxythymidine and / or thymidine analog). In some embodiments, a method comprises binding mRNA to a probe by binding a poly(A) tail present in the mRNA to deoxythymidine and / or a thymidine analog present in a coating present in a probe.

[0131] The deoxythymidine and / or thymidine analog present in a coating of a probe may itself be present in a variety of suitable manners. In some embodiments, a coating present in a probe comprises a molecule comprising a plurality of repeat units, and the plurality of repeat units comprise deoxythymidine and / or a thymidine analog. The molecule may be an oligomer or a polymer. In such embodiments, it may be a homo-oligomer or a homopolymer, or may be a co-oligomer or a copolymer (e.g., an oligomer or a copolymer that comprises more than one type of repeat unit, some of which comprise deoxythymidine and / or a thymidine analog, and some or none of which may be repeat units other than deoxythymidine and thymidine analogs). It is also possible for a molecule to be a polymer that comprises a block of repeat units of a single deoxythymidine and / or thymidine analog and further comprise one or more end groups and / or linkers (e.g., an end group that facilitates binding of the molecule to portion of the probe on which the coating is disposed, a linker chemically linking the end group to the block of repeat units of the single deoxythymidine and / or thymidine analog).

[0132] A variety of suitable thymidine analogs may be included in the molecules described herein. In some embodiments, a molecule comprises an engineered thymidine analog, one non-limiting example of which is locked nucleic acid (LNA) thymidine.

[0133] In some embodiments, a coating comprises oligomers formed from deoxythymidine repeat units, which can also be referred to as “oligo(dT).”

[0134] In some embodiments, a coating comprises an oligomer comprising a plurality of repeat units comprising deoxythymidine and / or a thymidine analog (e.g., an oligo(dT)) that comprises greater than or equal to 15, greater than or equal to 18, greater than or equal to 20,

[0135] 13

[0136] #14820240vlgreater than or equal to 22, greater than or equal to 25, or greater than or equal to 28 deoxythymidine repeat units. In some embodiments, a coating comprises an oligomer comprising a plurality of repeat units comprising deoxythymidine and / or a thymidine analog (e.g., an oligo(dT)) that comprises less than or equal to 30, less than or equal to 28, less than or equal to 25, less than or equal to 22, less than or equal to 20, or less than or equal to 18 deoxythymidine repeat units. Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 15 and less than or equal to 30). Other ranges are also possible.

[0137] In some embodiments, a coating comprises oligo(dT)i5-3o and / or oligo(dT)is-i8. In some embodiments, a coating is an oligo(dT)is.

[0138] In some embodiments, a coating comprises an oligo(dT) that, upon exposure to a poly (A) tail at 25 °C, forms a stable complex therewith.

[0139] Non-limiting examples of suitable linkers include alkyl linkers (e.g., C6 linkers, C12 linkers) and oligomeric glycols (e.g., triethylene glycol).

[0140] One non-limiting example of a suitable end group is biotin.

[0141] In some embodiments, a molecule present in a coating present in a probe (e.g., an outermost coating) comprises a molecule comprising a biotin end group and oligo(dT), and the oligo(dT) is directly bonded to the biotin end group or directly bonded to a linker that is directly bonded to a biotin end group. Such molecules may be referred to as “biotin-oligo(dT).”

[0142] In some embodiments, a probe comprises a body portion, a coating comprising deoxythymidine and / or a thymidine analog (e.g., as repeat units in a molecule comprising a plurality of repeat units), and further comprises one or more further coatings. These further coatings may be positioned between the body portion and the coating comprising the deoxythymidine and / or the thymidine analog (e.g., they may be disposed on the body portion, and the coating comprising the deoxythymidine and / or the thymidine analog may be disposed on them).

[0143] FIG. 4 shows one non-limiting example of a probe 418 including one further coating, FIG. 5 shows one non-limiting example of a probe 518 including two further coatings, and FIG. 6 shows one non-limiting example of a probe 618 including three further coatings. In FIG. 4, the probe 418 comprises a body portion 424, a coating 428 disposed on the body portion 424, and a coating 426 disposed on the coating 428. In FIG. 5, the probe 518 comprises a body portion 524, a coating 528 disposed on the body portion 524, a coating 530 disposed on the coating 528, and a coating 526 disposed on the coating 530. In FIG. 6, the probe 618 comprises a body portion 624, a coating 628 disposed on the body portion 624, a 14

[0144] #14820240vlcoating 630 disposed on the coating 628, a coating 632 disposed on the coating 630, and a coating 626 disposed on the coating 633. The coating 426 shown in FIG. 4, the coating 526 shown in FIG. 5, and the coating 626 in FIG. 6 each comprise a molecule comprising a plurality of repeat units comprising deoxythymidine and / or a thymidine analog. In some embodiments, a coating comprising a molecule comprising a plurality of repeat units comprising deoxythymidine and / or a thymidine analog is the outermost coating present in a probe.

[0145] Coatings positioned between a body portion and a coating comprising deoxythymidine and / or a thymidine analog (e.g., as repeat units in a molecule comprising a plurality of repeat units) may serve a variety of purposes. In some embodiments, such a coating assists with bonding the coating comprising deoxythymidine and / or the thymidine analog to the remainder of the probe. In such embodiments, the coating comprising the deoxythymidine and / or the thymidine analog may be directly disposed on the coating that assists with bonding that coating to the remainder of the probe. As one non-limiting example of a coating that assists with bonding a coating to a remainder of a probe, in some embodiments, a probe comprises a coating comprising streptavidin positioned between a body portion and a coating comprising a molecule comprising repeat units comprising deoxythymidine and / or a thymidine analog. In some such embodiments, this molecule comprises biotin (e.g., as an end group). In such embodiments, biotin- streptavidin binding may assist with bonding the coating comprising deoxythymidine and / or the thymidine analog to the probe.

[0146] Streptavidin present in a coating may be present in a variety of suitable forms. In some embodiments, a coating comprises cross-linked streptavidin (e.g., via EDC (l-ethyl-3-(3-dimethylamino) propyl carbodiimide) / NHS (N-hydroxysuccinimide) coupling) and / or a streptavidin multimer.

[0147] In some embodiments, a coating positioned between a body portion and a coating comprising deoxythymidine and / or a thymidine analog may comprise one or more features that assist with the generation of a signal, such as an optical signal. For instance, a coating may have a composition and an arrangement such that light impinging on the first coating after traveling through the body portion is partially reflected at an interface between the first coating and the body portion and partially transmitted through the first coating. This may be accomplished by selecting a composition for the coating having an appropriate refractive index, transparency, and thickness. It may also be accomplished by positioning the coating with respect to the body portion such that light traveling through the body portion would be 15

[0148] #14820240vlincident thereon (e.g., it may be positioned at a location such that light could be supplied to an opposing portion of the body portion).

[0149] It is also possible for a body portion of a probe to have one or more features that assist with the generation of a signal, such as an optical signal.

[0150] As will be described in further detail below, in some embodiments, a method comprises detecting an optical signal, such as an optical signal generated by interference between light reflected at two different locations associated with a probe. In some embodiments, a coating present in a probe assists with reflecting light to generate such interference.

[0151] In some embodiments, a probe comprises one or more coatings that assist with the generation of an optical signal that comprise a material having the same composition as a body portion of the probe.

[0152] In some embodiments, one or more coatings that assist with the generation of an optical signal comprises a glass and / or a body portion of a probe comprises a glass. Nonlimiting examples of suitable glasses include SiCh and Ta20s. In some embodiments, a probe comprises a body portion comprising SiCh, a coating disposed (e.g., directly) on the body portion comprising Ta20s, a coating comprising SiCb disposed (e.g., directly) on the coating comprising Ta20s, and a coating comprising deoxythymidine and / or a thymidine analog disposed (directly or indirectly) on the coating comprising SiCb.

[0153] In some embodiments, one or more coatings that assist with the generation of an optical signal and / or a body portion of a probe comprises a polymer. Non-limiting examples of suitable polymers include polystyrene and polyethylene.

[0154] The coatings described herein may have a variety of suitable thicknesses. In some embodiments, one or more coatings (and / or all coatings together) have a thickness of greater than or equal to 50 nm, greater than or equal to 100 nm, greater than or equal to 200 nm, greater than or equal to 500 nm, greater than or equal to 750 nm, greater than or equal to 1 micron, greater than or equal to 2 microns, greater than or equal to 3 microns, or greater than or equal to 4 microns. In some embodiments, one or more coatings (and / or all coatings together) have a thickness of less than or equal to 5 microns, less than or equal to 4 microns, less than or equal to 3 microns, less than or equal to 2 microns, less than or equal to 1 micron, less than or equal to 750 nm, less than or equal to 500 nm, less than or equal to 200 nm, or less than or equal to 100 nm. Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 50 nm and less than or equal to 5 microns, greater than or equal

[0155] 16

[0156] #14820240vlto 100 nm and less than or equal to 5 microns, or greater than or equal to 500 nm and less than or equal to 1 micron). Other ranges are also possible.

[0157] Some probes described herein are optical probes. Such probes may be particularly well-suited for generating and / or transmitting optical signals described herein. In some embodiments, a probe is transparent to and / or may transmit light at a plurality of wavelengths (e.g., visible wavelengths, infrared wavelengths, near infrared wavelengths). In some embodiments, a probe is a fiber-optic probe and / or has a body portion that comprises one or more fibers, such as one or more optical fibers.

[0158] The probes described herein may be contacted with a variety of suitable liquids comprising mRNA at an mRNA concentration. It should be noted that the mRNA concentration may be 0 wt% (i.e., the liquid may lack mRNA) or the mRNA concentration may be greater than 0 wt% (i.e., the liquid may comprise mRNA). In some embodiments, the mRNA concentration is unknown. Some methods described herein may comprise determining an unknown mRNA concentration (e.g., in a one-step assay).

[0159] In some embodiments, a liquid comprises mRNA at an mRNA concentration of greater than or equal to 0.025 micrograms / mL, greater than or equal to 0.05 micrograms / mL, greater than or equal to 0.075 micrograms / mL, greater than or equal to 0.1 micrograms / mL, greater than or equal to 0.2 micrograms / mL, greater than or equal to 0.5 micrograms / mL, greater than or equal to 0.75 micrograms / mL, greater than or equal to 1 microgram / mL, greater than or equal to 2 micrograms / mL, greater than or equal to 5 micrograms / mL, greater than or equal to 7.5 micrograms / mL, greater than or equal to 10 micrograms / mL, greater than or equal to 20 micrograms / mL, greater than or equal to 50 micrograms / mL, greater than or equal to 75 micrograms / mL, greater than or equal to 100 micrograms / mL, greater than or equal to 150 micrograms / mL, greater than or equal to 200 micrograms / mL, greater than or equal to 500 micrograms / mL, greater than or equal to 850 micrograms / mL, greater than or equal to 1000 micrograms / mL, or greater than or equal to 1500 micrograms / mL. In some embodiments, a liquid comprises mRNA at an mRNA concentration of less than or equal to 2000 micrograms / mL, less than or equal to 1000 micrograms / mL, less than or equal to 850 micrograms / mL, less than or equal to 500 micrograms / mL, less than or equal to 200 micrograms / mL, less than or equal to 150 micrograms / mL, less than or equal to 100 micrograms / mL, less than or equal to 75 micrograms / mL, less than or equal to 50 micrograms / mL, less than or equal to 20 micrograms / mL, less than or equal to 10 micrograms / mL, less than or equal to 7.5 micrograms / mL, less than or equal to 5 micrograms / mL, less than or equal to 2 micrograms / mL, less than or equal to 1

[0160] 17

[0161] #14820240vlmicrogram / mL, less than or equal to 0.75 micrograms / mL, less than or equal to 0.5 micrograms / mL, less than or equal to 0.2 micrograms / mL, less than or equal to 0.1 micrograms / mL, less than or equal to 0.075 micrograms / mL, or less than or equal to 0.05 micrograms / mL. Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 0.025 micrograms / mL and less than or equal to 850 microgram / mL, greater than or equal to 0.025 micrograms / mL and less than or equal to 1 microgram / mL, or greater than or equal to 0.025 micrograms / mL and less than or equal to 2000 microgram / mL,). Other ranges are also possible.

[0162] As noted above, in some embodiments, a probe may be contacted with a liquid comprising mRNA at an mRNA concentration that further comprises one or more components in addition to the mRNA. As one example, a probe may be contacted with a liquid that is a crude sample and / or comprises a crude sample. Such liquids may further comprise other components typically present in crude samples. Non-limiting examples of such species include RNA polymerase, dithiothreitol, DNA templates, spermidine, nucleotides, buffering salts, ethylenediaminetetraacetic acid (EDTA), cellular lysates, tissue homogenates, surfactants (e.g., nonyl phenoxypolyethoxylethanol (NP-40), 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol (Triton X-100), sodium deoxycholate, sodium dodecyl sulfate (SDS)), DNase inhibitors, RNase inhibitors, and protease inhibitors.

[0163] When a surfactant is present in a liquid comprising mRNA at an mRNA concentration, it may be present in a variety of suitable amounts. In some embodiments, a surfactant makes up greater than or equal to 0.01 wt%, greater than or equal to 0.02 wt%, greater than or equal to 0.05 wt%, greater than or equal to 0.075 wt%, greater than or equal to 0.1 wt%, greater than or equal to 0.2 wt%, greater than or equal to 0.5 wt%, greater than or equal to 0.75 wt%, greater than or equal to 1 wt%, greater than or equal to 1.25 wt%, greater than or equal to 1.5 wt%, or greater than or equal to 1.75 wt% of a liquid comprising mRNA at an mRNA concentration. In some embodiments, a surfactant makes up less than or equal to 2 wt%, less than or equal to 1.75 wt%, less than or equal to 1.5 wt%, less than or equal to 1.25 wt%, less than or equal to 1 wt%, less than or equal to 0.75 wt%, less than or equal to 0.5 wt%, less than or equal to 0.2 wt%, less than or equal to 0.1 wt%, less than or equal to 0.075 wt%, less than or equal to 0.05 wt%, or less than or equal to 0.02 wt% of a liquid comprising mRNA at an mRNA concentration. Combinations of the above -referenced ranges are also possible (e.g., greater than or equal to 0.01 wt% and less than or equal to 2 wt%, or greater than or equal to 0.5 wt% and less than or equal to 2 wt%). Other ranges are also possible.

[0164] 18

[0165] #14820240vlWhen a liquid comprising mRNA at an mRNA concentration comprises two or more surfactants, each surfactant may individually be present in one or more of the abovedescribed ranges and / or all of the surfactants together may make up an amount of the liquid comprising mRNA at the mRNA concentration in one or more of the above-described ranges.

[0166] In some embodiments, a liquid comprising mRNA at an mRNA concentration is an aqueous liquid and / or a buffer, such as an aqueous buffer. Such liquids may further comprise salts and / or other buffering components. Additionally, it is possible for a buffer comprising mRNA at an mRNA concentration to also be crude samples (i.e., when the crude sample is a buffer), to be obtained from a crude sample (e.g., crude samples to which a buffer has been added, possibly after undergoing one or more purification steps), and / or to be obtained by dissolving mRNA obtained from a crude sample in a buffer. In some embodiments, a buffer comprising mRNA at an mRNA concentration is a Tris buffer, a HEPES buffer, and / or a citrate buffer. It is also possible for a buffer comprising mRNA at an mRNA concentration to include a monovalent cation (e.g., in the form of a cation of a salt dissolved therein) and / or a divalent cation (e.g., in the form of a cation of a salt dissolved therein).

[0167] Without wishing to be bound by any particular theory, it is believed that the presence of both a monovalent cation and a divalent cation in a liquid (e.g., a buffer) comprising mRNA may facilitate the binding of binding of the mRNA to a probe and may enhance the magnitude of a signal generated upon such binding. It is believed that the monovalent cation may facilitate binding between the mRNA and deoxythymidine and / or thymidine analogs present in a coating present in the probe. It is also believed that the divalent cation may cause the mRNA to have a structure that is more condensed than it would have in the absence of the divalent cation, which is believed to facilitate the generation of signals having larger magnitudes upon such binding.

[0168] Divalent cations present in liquids comprising mRNA at an mRNA concentration may be selected as desired. In some embodiments, such a liquid comprises an alkaline earth metal cation, such as Mg2+and / or Ca2+.

[0169] The concentration of a divalent cation in a liquid (e.g., a buffer) comprising mRNA at an mRNA concentration may be particularly advantageous for causing the generation of a signal with a relatively large value upon contact between the buffer and a probe. In some embodiments, the concentration of the divalent cation in a liquid comprising mRNA at an mRNA concentration is greater than or equal to 1 mM, greater than or equal to 2 mM, greater than or equal to 5 mM, greater than or equal to 7.5 mM, greater than or equal to 10 mM, greater than or equal to 15 mM, greater than or equal to 20 mM, greater than or equal to 30

[0170] 19

[0171] #14820240vlmM, greater than or equal to 50 mM, greater than or equal to 60 mM, or greater than or equal to 75 mM. In some embodiments, the concentration of the divalent cation in a liquid comprising mRNA at an mRNA concentration is less than or equal to 100 mM, less than or equal to 75 mM, less than or equal to 50 mM, less than or equal to 30 mM, less than or equal to 20 mM, less than or equal to 15 mM, less than or equal to 10 mM, less than or equal to 7.5 mM, less than or equal to 5 mM, or less than or equal to 2 mM. Combinations of the abovereferenced ranges are also possible (e.g., greater than or equal to 1 mM and less than or equal to 100 mM). Other ranges are also possible.

[0172] When a liquid comprising mRNA at an mRNA concentration comprises two or more divalent cations, each divalent cation may individually be present in one or more of the above-described ranges and / or all of the divalent cations together may make up an amount of the liquid comprising mRNA at the mRNA concentration in one or more of the abovedescribed ranges.

[0173] Monovalent cations present in liquids comprising mRNA at an mRNA concentration may be selected as desired. In some embodiments, such a liquid comprises an alkali metal cation, such as Na+and / or K+.

[0174] The concentration of a monovalent cation in a liquid (e.g., a buffer) comprising mRNA at an mRNA concentration may be selected as desired. In some embodiments, the concentration of the monovalent cation in a liquid comprising mRNA at an mRNA concentration is greater than or equal to 50 mM, greater than or equal to 75 mM, greater than or equal to 100 mM, greater than or equal to 125 mM, greater than or equal to 150 mM, or greater than or equal to 175 mM, greater than or equal to 200 mM, greater than or equal to 500 mM, greater than or equal to 750 mM, greater than or equal to 1000 mM, greater than or equal to 1250 mM, greater than or equal to 1500 mM, or greater than or equal to 1750 mM. In some embodiments, the concentration of a monovalent cation in a liquid comprising mRNA at an mRNA concentration is less than or equal to 2000 mM, less than or equal to 1750 mM, less than or equal to 1500 mM, less than or equal to 1250 mM, less than or equal to 1000 mM, less than or equal to 750 mM, less than or equal to 500 mM, less than or equal to 200 mM, less than or equal to 175 mM, less than or equal to 150 mM, less than or equal to 125 mM, less than or equal to 100 mM, or less than or equal to 75 mM. Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 50 mM and less than or equal to 2000 mM, or greater than or equal to 50 mM and less than or equal to 200 mM). Other ranges are also possible.

[0175] 20

[0176] #14820240vlWhen a liquid comprising mRNA at an mRNA concentration comprises two or more monovalent cations, each monovalent cation may individually be present in one or more of the above-described ranges and / or all of the monovalent cations together may make up an amount of the liquid comprising mRNA at the mRNA concentration in one or more of the above-described ranges.

[0177] The probes described herein may be contacted with a variety of suitable liquids comprising a divalent cation (e.g., subsequent to being contacted with a liquid comprising mRNA at an mRNA concentration). A variety of suitable divalent cations may be employed, such as alkaline earth metal cations (e.g., Mg2+and / or Ca2+).

[0178] The concentration of the divalent cation in a liquid comprising a divalent cation (e.g., a liquid contacted with a probe subsequent to being contacted with a liquid comprising mRNA at an mRNA concentration) may be particularly advantageous for causing the generation of a signal with a relatively large value upon contact between the liquid and a probe. In some embodiments, the concentration of the divalent cation in such a liquid is greater than or equal to 1 mM, greater than or equal to 2 mM, greater than or equal to 5 mM, greater than or equal to 7.5 mM, greater than or equal to 10 mM, greater than or equal to 15 mM, greater than or equal to 20 mM, greater than or equal to 30 mM, greater than or equal to 50 mM, greater than or equal to 60 mM, or greater than or equal to 75 mM. In some embodiments, the concentration of the divalent cation in such a liquid is less than or equal to 100 mM, less than or equal to 75 mM, less than or equal to 50 mM, less than or equal to 30 mM, less than or equal to 20 mM, less than or equal to 15 mM, less than or equal to 10 mM, less than or equal to 7.5 mM, less than or equal to 5 mM, or less than or equal to 2 mM. Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 1 mM and less than or equal to 100 mM). Other ranges are also possible.

[0179] When a liquid comprising a divalent cation comprises two or more divalent cations, each divalent cation may individually be present in one or more of the above-described ranges and / or all of the divalent cations together may make up an amount of the liquid comprising the divalent cation in one or more of the above-described ranges.

[0180] In some embodiments, a liquid comprising a divalent cation (e.g., a liquid with which a probe is contacted subsequent to being contacted with a liquid comprising mRNA at an mRNA concentration) is an aqueous liquid and / or a buffer, such as an aqueous buffer. In some embodiments, such a liquid is a Tris buffer, a HEPES buffer, and / or a citrate buffer. It is also possible for such a liquid to include a monovalent cation (e.g., in the form of a cation of a salt dissolved therein).

[0181] 21

[0182] #14820240vlMonovalent cations present in liquids comprising a divalent cation may be selected as desired. In some embodiments, such a liquid comprises an alkali metal cation, such as Na+and / or K+.

[0183] The concentration of a monovalent cation in a liquid comprising a divalent cation (e.g., a liquid with which a probe is contacted subsequent to being contacted with a liquid comprising mRNA at an mRNA concentration) may be selected as desired. In some embodiments, the concentration of the monovalent cation in such a liquid is greater than or equal to 50 mM, greater than or equal to 75 mM, greater than or equal to 100 mM, greater than or equal to 125 mM, greater than or equal to 150 mM, greater than or equal to 175 mM, greater than or equal to 200 mM, greater than or equal to 500 mM, greater than or equal to 750 mM, greater than or equal to 1000 mM, greater than or equal to 1250 mM, greater than or equal to 1500 mM, or greater than or equal to 1750 mM. In some embodiments, the concentration of the monovalent cation in such a liquid is less than or equal to less than or equal to 2000 mM, less than or equal to 1750 mM, less than or equal to 1500 mM, less than or equal to 1250 mM, less than or equal to 1000 mM, less than or equal to 750 mM, less than or equal to 500 mM, 200 mM, less than or equal to 175 mM, less than or equal to 150 mM, less than or equal to 125 mM, less than or equal to 100 mM, or less than or equal to 75 mM. Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 50 mM and less than or equal to 2000 mM, or greater than or equal to 50 mM and less than or equal to 200 mM). Other ranges are also possible.

[0184] When a liquid comprising a divalent cation comprises two or more monovalent cations, each monovalent cation may individually be present in one or more of the abovedescribed ranges and / or all of the monovalent cations together may make up an amount of the liquid comprising the divalent cation in one or more of the above-described ranges.

[0185] As noted above, some methods may comprise a step in which a probe is contacted with a regeneration liquid. The regeneration liquid may remove bound mRNA from the probe. The regeneration liquid may remove bound mRNA from the probe by dissolving some or all of the bound mRNA therein. It is also possible for a regeneration liquid to remove bound mRNA from the probe by causing a chemical reaction to occur that severs a bond between the mRNA and the probe (e.g., a bond between a poly(A) tail present in the mRNA and a deoxythymidine and / or thymidine analog in a coating). As a third example, in some embodiments, a regeneration liquid decomposes mRNA in a manner such that the decomposition products are soluble in the regeneration liquid. As a fourth example, in some embodiments, a regeneration liquid comprises a species that has a higher affinity for the 22

[0186] #14820240vlprobe (and / or a coating therein) than the mRNA and / or a species that is configured to displace the mRNA from the probe.

[0187] The regeneration liquid may perform one or more of the above-described processes by causing a conformational change in the mRNA and / or a coating to which the mRNA is bound (e.g., a coating comprising deoxythymidine and / or a thymidine analog). The regeneration liquid may cause this change by changing a chemical and / or physical parameter of the liquid with which the probe is contacted, such as its pH, conductivity, and / or temperature.

[0188] In some embodiments, a regeneration liquid is and / or comprises water. In some embodiments, a regeneration liquid is and / or comprises a buffer. It is also possible for the regeneration liquid to comprise a salt (e.g., an acetate salt, such as sodium acetate; a citrate salt; a phosphate salt; a Tris buffer salt; NaOH) and / or an organic molecule (e.g., glycine, biotin, histidin). The salt may assist with buffering the regeneration liquid. In some embodiments, a regeneration liquid comprises a species, such as a small molecule or a salt, that is capable of and / or configured to bind to mRNA, such as mRNA bound to the probe. It is also possible for a species present in a regeneration liquid to bind to a coating present in a probe (e.g., a coating comprising deoxythymidine and / or a thymidine analog), thereby displacing mRNA bound thereto (e.g., a species in a regeneration liquid may bind to deoxythymidine and / or a thymidine analog present in a coating disposed on a body portion of a probe, thereby displacing mRNA therefrom).

[0189] As noted above, some methods may comprise a step in which a probe is contacted with a neutralization liquid. In some embodiments, a probe is contacted with a neutralization liquid after being contacted with a regeneration liquid, before being contacted with a second liquid comprising mRNA at an mRNA concentration, and / or before being contacted with a standard. The neutralization liquid may remove any undesirable species deposited on a probe from a regeneration liquid. It is also possible for a regeneration liquid to adjust one or more physical and / or chemical properties of the probe (e.g., pH, conductivity, and / or temperature). In some embodiments, a neutralization liquid comprises a buffer. Non-limiting examples of suitable liquids include phosphate-buffered saline and Tris buffers.

[0190] In some embodiments, a neutralization liquid has a pH that assists with returning the pH and / or charge at a probe surface to neutral. For instance, when a probe is contacted with a regeneration liquid that has a basic pH, it may subsequently be contacted with a neutralization liquid having an acidic pH. As another example, when a probe is contacted with a regeneration liquid that has an acidic pH, it may subsequently be contacted with a neutralization liquid having a basic pH.

[0191] 23

[0192] #14820240vlThe methods and kits described herein may be employed to generate and detect a variety of suitable signals. In some embodiments, a method comprises detecting a signal that is an optical signal (e.g., a signal comprising light) and / or kit comprises one or more components suitable for the use in a method of detecting a signal that is an optical signal. In some embodiments, such an optical signal is a signal that was generated via an interferometry technique, such as via biolayer interferometry. As noted above, some methods comprise employing a probe suitable for performing an interferometry technique (e.g., biolayer interferometry) and / or a kit comprises such a probe.

[0193] As also noted above, in some embodiments, a probe has a design that facilitates the reflection of light at two or more locations associated therewith. Such light may interfere with each other to form the optical signal (i.e., the optical signal may comprise light reflected from two different locations). In some embodiments, light is transmitted through the probe, to a first location at which it is reflected, and then to a second location at which it is reflected. The reflected light may travel back through the probe to form an optical signal comprising both types of reflected light, and both types of reflected light may interfere with each other.

[0194] In some embodiments, a probe comprises one or more interfaces internal thereto. Such probes may be particularly suitable for generating optical signals comprising light generated via an interferometry technique, such as biolayer interferometry. In some embodiments, a probe comprises one or more internal interfaces at which light is reflected. The amount of light reflected at any particular internal interface may depend on the differences in the refractive indices of the components on either side of the internal interface. It is also possible for light to reflect from the end of a probe.

[0195] When present, an internal interface may extend across a probe cross-section (e.g., the cross-section perpendicular to the axis along which light is transmitted through the probe, the cross-section perpendicular to the long axis of the probe). In some embodiments, a probe comprises an internal interface positioned between a body portion and a coating disposed directly on the body portion. It is also possible for a probe to comprise an internal interface positioned between two coatings that are directly adjacent to each other. For instance, with respect to FIG. 6, the probe 620 comprises the internal interface 634 between the body portion 624 and the coating 628, the internal interface 636 between the coating 628 and the coating 630, the internal interface 638 between the coating 630 and the coating 632, and the internal interface 640 between the coating 626 and the coating 632.

[0196] As noted above, optical signals may comprise reflected from two locations associated with a probe, such as one or more interfaces internal to the probe and / or from the end of the 24

[0197] #14820240vlprobe. Such light may comprise two or more such types of light, light interference between these two or more such types of light (e.g., interference between light traveling through optical pathways having different optical path lengths due to the reflection at different locations), the absence of one or more such types of light, and / or the absence of such interference. As two examples, light supplied by a probe may comprise interference between light that is reflected from two different interfaces associated with a probe and / or mRNA bound to the probe (e.g., an interface between a body portion of a probe and a coating disposed on the body portion of the probe, an interface between two coatings present in the probe, an interface between a coating present in the probe and an environment external to the probe) or the absence of such interference.

[0198] Light that is reflected from an interface associated with a probe may be supplied to a probe from a light source. Such a light source may be optically coupled to a probe such that light is transmitted from the light source and across the probe (e.g., parallel to an optical axis of the probe). Upon reaching an end of the probe, the light may be transmitted out of the probe and / or may reflect from an interface between the probe and an environment external to the probe (and / or from the end of the probe). If mRNA is bound to the probe, some light may reflect from the interface between the probe and the mRNA and / or some light may be transmitted through the mRNA. The mRNA may also change the effective refractive index at the end of the probe and / or change the effective optical path length of the light transmitted through the probe. Light transmitted through the mRNA will then encounter the environment with which the mRNA is in contact. Some light encountering this environment may be transmitted into the environment with which the mRNA is in contact (e.g., an environment external to the probe) and / or may reflect from the interface between the environment and the mRNA.

[0199] Light reflected from one or more of the above-described locations (and / or any further locations) may travel back through the probe. If light is reflected from multiple locations (e.g., at an interface between the probe and mRNA bound to the probe, at an interface between mRNA bound to the probe and an environment external to the probe, at an interface between a coating present in a probe and mRNA bound the probe, at an interface between two coatings present in a probe, at an interface between a body portion of the probe and a coating disposed thereon, from the end of the probe), such light may interfere which each other. Light interference may cause the intensity of the interfered light to be higher or lower depending on whether the interference is positive or negative, which may depend on the phase shift between the multiple sources of interfering light. The phase shift may depend on 25

[0200] #14820240vlthe differences in the path lengths traveled by the light prior to interfering, the refractive index of the material(s) through which the light passes prior to interfering, and / or on the wavelength of light. Thus, in some embodiments, light supplied by a probe and / or detected by an optical detector comprises light having a variety of wavelengths. Additionally, obtaining information about the intensity of interfered light across a variety of wavelengths may provide information about the presence or absence of a layer comprising mRNA bound to the probe, the thickness of such a layer, and / or the refractive index of such a layer. This information may be employed to determine the presence, absence, and / or amount of mRNA bound to the probe.

[0201] Optical signals comprising light reflected from two or more locations associated with the probe, such as from an interface internal to the probe and from the end of the probe, may therefore be indicative of an amount of mRNA bound to the probe. For instance, as more mRNA binds to the probe, the effective path length traveled by light before being reflected at the end of the probe may change and / or the refractive index at the end of the probe may change. The magnitude of such changes may be indicative of the thickness of a layer of mRNA that is bound to the probe (e.g., to a molecule comprising deoxythymidine and / or a thymidine analog present in a coating therein) and / or the coverage of the surface of the probe by mRNA bound thereto. Such changes may also affect the wavelengths of light at which constructive and destructive interference occurs, and thereby affect the magnitude of the wavelength shift of an interference pattern of such reflected light (i.e., a pattern of the variation in magnitude of intensity of the light present in the optical signal as a function of wavelength). The magnitude of the wavelength shift of an interference pattern may therefore be employed to determine the amount and / or thickness of mRNA bound to the probe. Either or both of such parameters may be employed to determine the concentration of mRNA in a liquid comprising mRNA at an mRNA concentration by correlating the amount of binding to the amount of binding expected for a particular concentration of mRNA in such a liquid when contacted with a probe over a particular period of time.

[0202] FIG. 7 depicts schematically one example of a process by which light comprising both an amount of the light that has been reflected from an interface internal to a probe and an amount of light that has been reflected from the end of a probe can be generated. As shown in FIG. 7, light that travels down a probe may reflect from an interface between a coating disposed on a body portion of a probe and from an interface between an outermost coating present in the probe and the environment external to the probe. The phase shift between these two sources of reflected light may depend on the amount of a species bound to the 26

[0203] #14820240vlprobe (FIG. 7 depicts binding between an antigen and a coating comprising an antibody, but the same principle would apply for the coatings described herein and mRNA) and on the wavelength of the reflected light, which may affect the intensity of the reflected light measured. Analysis of the intensity of the reflected light as a function of wavelength may therefore be employed to determine an amount of mRNA bound to the probe. As shown in FIG. 7, the intensity of the reflected light as a function of wavelength may form an interference pattern, and a change in the magnitude of the phase shift between light reflecting from two or more interfaces described herein may cause a wavelength shift of such an interference pattern.

[0204] As noted above, detection of a signal may be performed at one or more discrete points in time or over a period of time. Additionally, such detection may be performed in a manner that yields a single data point (e.g., an endpoint, the average intensity of light at a particular wavelength as measured over a period of time, the average intensity of light at a particular wavelength as computed by averaging a plurality of measurements of light intensity, the intensity of light at a particular wavelength as determined from a single measurement, the magnitude of a wavelength shift in the interference pattern determined over a period of time, the average magnitude in the wavelength shift of an interference pattern as computed by averaging a plurality of measurements of the wavelength shift of the interference pattern, the magnitude of the wavelength shift of an interference pattern as determined from a single measurement) and / or a plurality of data points. The plurality of data points may describe the variation of the signal over time (e.g., in a kinetic measurement). The plurality of data points may be obtained from different measurements that take place over different (overlapping or non-overlapping) periods of time.

[0205] In some embodiments, detecting a signal over time comprises detecting its variation over time. The variation may comprise an increase, a decrease, or a lack of variation. In some embodiments, the variation comprises the first derivative of the intensity of the light at one or more wavelengths and / or the first derivative of the magnitude of a wavelength shift in an interference pattern. The variation in the intensity of the light (and / or one or more wavelengths thereof) and / or the magnitude of the wavelength shift of the interference pattern over a period of time may be determined from multiple measurements made over the period of time that yield multiple values of the light intensities and / or the magnitude of the wavelength shift of the interference pattern over the period of time.

[0206] In some embodiments, light present in an optical signal and / or from which an interference pattern is generated comprises visible light. It is also possible for the light to 27

[0207] #14820240vlcomprise infrared light. Additionally, the light may be and / or comprise polarized light or unpolarized light.

[0208] In some embodiments, a method described herein is performed by an instrument, is capable of being performed by an instrument, and / or is configured to be performed by an instrument. Similarly, some kits may be capable of being employed in conjunction with an instrument and / or configured to be employed in conjunction with an instrument. One nonlimiting example of such a suitable instrument is a biolayer interferometer.

[0209] In some embodiments, an instrument suitable for performing a method described herein and / or for being used in conjunction with a kit includes a light source (e.g., that supplies light at a plurality of wavelengths) and an optical detector (e.g., that detects the intensity of light impinging thereon as a function of wavelength). Such instruments may further comprise one or more components that assist with the transmission of light from a light source to a probe and / or from a probe to a detector (e.g., one or more optical cables).

[0210] It is also possible for an instrument described herein to comprise a housing that can be configured to mechanically support and / or translate one or more components described herein and / or can be capable of doing so. For instance, in some embodiments, an instrument comprises a housing in which a probe is positioned, that is configured to receive a probe, and / or that is capable of receiving a probe. Such a housing may mechanically support the probe, may fix the position of the probe (e.g., via mating, a pressurized fit, clamps, a holder, an adhesive, etc.). In some embodiments, a housing is configured such that a probe may be reversibly inserted into and / or removed therefrom and / or may have a design into which a probe can be reversibly inserted and / or from which a probe can be reversibly removed. In some embodiments, a housing is configured to translate and / or is capable of translating the probe. This may be achieved in an automated manner (e.g., via instructions provided by software, via a motor, via an electrical controller) and / or by manual operation (e.g., via an operator repositioning the probe as desired, such as in an instrument when the housing comprises two or more parts that are movable with respect to each other, such as parts joined by a hinge).

[0211] In some embodiments, an instrument comprises a housing that is configured to support and / or capable of supporting one or more liquids (e.g., one or more liquids to be contacted with a probe by a method described herein). As one example, in some embodiments, one or more liquids described herein (e.g., a liquid comprising mRNA at an mRNA concentration, a liquid comprising a divalent cation, a standard, a regeneration liquid, a neutralization liquid) are contained in containers and the housing may be configured to 28

[0212] #14820240vlsupport and / or capable of supporting such containers. For instance, the housing may comprise a platform on which the container(s) can be disposed and / or may comprise one or more recesses into which the container(s) can be inserted. In some embodiments, a housing comprises one or more components that fix the position of the container(s) (e.g., via mating, a pressurized fit, clamps, a holder, an adhesive, etc.). In some embodiments, a housing is configured such that one or more containers may be reversibly inserted into and / or removed therefrom and / or may have a design into which one or more containers can be reversibly inserted and / or from which a probe can be reversibly removed. In some embodiments, a housing is configured to translate and / or is capable of translating one or more containers. This may be achieved in an automated manner (e.g., via instructions provided by software, via a motor, via an electrical controller) and / or by manual operation (e.g., via an operator repositioning the container(s) as desired, such as in an instrument when the housing comprises two or more parts that are movable with respect to each other, such as parts joined by a hinge).

[0213] In some embodiments, liquids in a plurality of liquids are contained in containers that are each wells in a single multiwell plate.

[0214] In some embodiments, a method comprises translating a probe to contact a plurality of liquids sequentially (e.g., comprising two or more of the liquids contacted with the probe as described in the method 100) and / or translating a plurality of liquids (e.g., comprising two or more of the liquids contacted with the probe as described in the method 100) such that they contact a probe sequentially.

[0215] In some embodiments, a probe and / or a container containing a liquid contacted with a probe may undergo shaking during contact between the probe and the liquid.

[0216] EXAMPLE 1

[0217] This Example describes the expected morphology at the end of a probe during the performance of a process in which the following steps are performed: (1) Biotin-oligo(dT) is bound to a coating present in the probe that comprises streptavidin to form a coating comprising biotin-oligo(dT) disposed on the coating comprising streptavidin; and (2) A liquid comprising mRNA is contacted with the probe, causing binding between the mRNA and the biotin-oligo(dT). It also describes the expected wavelength shift in an interference pattern generated during biolayer interferometry using the above-described probe as a function of time.

[0218] 29

[0219] #14820240vlFIG. 8 shows the above-described process schematically, the associated morphology at the end of the probe schematically, and the expected wavelength shift in the interference pattern prior to the above-described steps and during the above-described steps. In the chart shown in FIG. 8, the x-axis is time in seconds, and the y-axis is the magnitude of the wavelength shift of the interference pattern in nm.

[0220] EXAMPLE 2

[0221] This Example shows that biotin-oligo(dT) binds more strongly to a coating comprising streptavidin than unbiotinylated oligo(dT). It also shows that mRNA does not appreciably bind to probes comprising streptavidin in the absence of a coating disposed thereon comprising a molecule comprising deoxythymidine and / or a thymidine analog.

[0222] A probe having an outermost coating comprising streptavidin was contacted with the following sequence of liquids: (1) A liquid comprising biotin-C12-oligo(dT)i8 or a control; (2) A buffer; and (3) A liquid comprising mRNA. The buffer lacked the biotin-C12-oligo(dT)i8 and the mRNA. The liquid comprising the mRNA further included 50 mM Tris and 0.5 M NaCl.

[0223] During these steps, the wavelength shift of an interference pattern generated via biolayer interferometry employing the above-described probe was measured. These wavelength shifts are shown in FIG. 9. In FIG. 9, the x-axis is time in seconds, the y-axis is the magnitude of the wavelength shift of the interference pattern in nm, and the identity of the liquid contacted with the probe in step 1 for each trace is shown by the key. As can be seen from FIG. 9, biotin-oligo(dT) binds much more strongly to the coating comprising streptavidin than unbiotinylated oligo(dT).

[0224] It can be seen from FIG. 9 that mRNA did not exhibit detectable binding to the probe in the absence of a coating comprising oligo(dT) disposed thereon, and exhibited the largest wavelength shift (and, therefore, the highest degree of binding) to the probe including the coating comprising purified biotin-oligo(dT).

[0225] EXAMPLE 3

[0226] This Example shows that magnitude of the wavelength shift of an interference pattern obtained during biolayer interferometry upon contact between a liquid comprising mRNA at an mRNA concentration and a probe can be increased by subsequently contacting the probe with a liquid comprising Mg2+.

[0227] 30

[0228] #14820240vlProbes comprising a coating comprising streptavidin were contacted with a liquid comprising biotin-oligo(dT) to form a coating comprising oligo(dT). These probes were then contacted with liquids comprising mRNA at mRNA concentrations of various values. These liquids further comprised 50 mM Tris and 0.5 M NaCl. Subsequently, these probes were contacted with a buffer comprising 50 mM Tris, 0.5 M NaCl, and 50 mM MgCh. Finally, these probes were contacted with a regeneration liquid. During these steps, biolayer interferometry was performed to determine the wavelength shift of the interference pattern associated with light traveling through and reflecting at various locations associated with the probe.

[0229] FIG. 10 shows the above-described wavelength shift of the interference patterns. In FIG. 10, the x-axis is time in seconds, the y-axis is the magnitude of the wavelength shift of the interference pattern in nm, and the various traces correspond to liquids having different mRNA concentrations.

[0230] FIG. 11 shows the key associated with FIG. 10 and the calculated binding rates between the mRNA and the probe. The calculation was performed by fitting the measured wavelength shift of the interference patterns to binding rate models programmed into the Octet Analysis Studio Software.

[0231] Table 1 shows the wavelength shift of the interference patterns at various points in time and a calculation of the wavelength shift enhancement observed after exposure to the buffer comprising Tris, NaCl, and MgCh.

[0232] Table 1.

[0233]

[0234] 31

[0235] #14820240vlAs can be seen from FIG. 11 and Table 1, contacting the probes with the liquid comprising Mg2+increased the magnitude of the wavelength shift measured during biolayer interferometry.

[0236] EXAMPLE 4

[0237] This Example shows that various molecules comprising oligo(dT) can be suitable for use in coatings for binding mRNA.

[0238] Biotin-C12-oligo(dT)i8 (B-C12-OdT18), biotin-C6-oligo(dT)i8 (B-C6-OdT18), and biotin-triethylene glycol-oligo(dT)i8 (B-TEG-OdT18) were contacted with a probe comprising a coating comprising streptavidin. During such contact, biolayer interferometry was performed to determine the wavelength shift of the interference pattern associated with light traveling through and reflecting at various locations associated with the probe. FIG. 12 shows these wavelength shifts as a function of time. In FIG. 12, the x-axis is time in seconds, the y-axis is the magnitude of the wavelength shift of the interference pattern in nm, and the identity of the liquid contacted with the probe is shown by the key. As can be seen from FIG.

[0239] 12, these molecules bound to the streptavidin-containing coating to form coatings comprising oligo(dT).

[0240] Subsequently, the oligo(dT)-coated probes were contacted with liquids comprising mRNA at various mRNA concentrations. These liquids further comprised 50 mM Tris, 0.5 M NaCl, and 50 mM MgCh. During such contact, biolayer interferometry was performed to determine the wavelength shift of the interference pattern associated with light traveling through and reflecting at various locations associated with the probe. FIG. 13 shows these wavelength shifts as a function of time. In FIG. 13, the x-axis is time in seconds, the y-axis is the magnitude of the wavelength shift of the interference pattern in nm, and the identity of the liquid contacted with the probe is shown by the key. As can be seen from FIG. 13, each coating was suitable for binding with mRNA and for generating signals that differed with mRNA concentration.

[0241] EXAMPLE 5

[0242] This Example shows that binding between a probe comprising a coating comprising oligo(dT) and mRNA present in a liquid comprising both mRNA and Mg2+can be detected via biolayer interferometry.

[0243] Liquids comprising mRNA at known mRNA concentrations of various values were contacted with probes comprising a coating comprising oligo(dT). These liquids further 32

[0244] #14820240vlcomprised 50 mM Tris, 0.5 M NaCl, and 0.5 M MgCh. During each such contact, the magnitude of the wavelength shift of the interference pattern associated with the probe was measured as a function of time via biolayer interferometry. FIG. 14 shows these measurements and FIG. 15 shows the calculated binding rates (i.e., the initial slope of the magnitude of the wavelength shift of the interference pattern as a function of time) and a curve that fits the calculated binding rates as a function of known mRNA concentrations. In FIG. 14, the x-axis is time, the y-axis is the magnitude of the wavelength shift of the interference pattern, and the mRNA concentration is labeled by each trace. In FIG. 15, the x-axis is mRNA concentration in micrograms / mL, and the y-axis is the binding rate in nm / s. As can be seen from FIG. 14, the magnitude of the wavelength shift of the interference pattern increased with both time and the mRNA concentration. As can be seen from FIG. 15, the binding rate also increased with the mRNA concentration.

[0245] After establishing the curve that fits the calculated binding rates shown in FIG. 15, a calculated mRNA concentration was determined for each liquid based on this curve and the calculated binding rates. Table 2 shows the recovery average (the percentage of the calculated mRNA concentration with respect to the known mRNA concentration) and the coefficient of variation of the recovery average for each liquid comprising mRNA. Each calculated mRNA concentration was calculated by fitting the measured wavelength shift of the interference patterns to binding rate models programmed into the Octet Analysis Studio Software. These binding rate models correlate measured wavelength shifts of interference patterns as a function of time to known concentrations of mRNA in liquids contacted with probes of the type employed. As can be seen from Table 2, various concentrations of mRNA were able to be accurately and reproducibly determined.

[0246] Table 2.

[0247]

[0248] 33

[0249] #14820240vlEXAMPLE 6

[0250] This Example shows that, for any given mRNA concentration in a liquid comprising mRNA, the magnitude of the wavelength shift detected upon binding of the mRNA therein to a probe comprising a coating comprising oligo(dT) is higher in the presence of Mg2+.

[0251] Probes comprising a coating comprising oligo(dT) were contacted with either a liquid of either Type A or Type B. Liquids of Type A comprised mRNA at an mRNA concentration and further comprised 50 mM of a base salt and 0.5 M NaCl. Liquids of Type B comprised mRNA at an mRNA concentration and further comprised 50 mM of a base salt, 0.5 M NaCl, and 50 mM MgCh. The base salts were either Tris or 4-(2-hydroxyethyl)-l-piperazine ethanesulfonic acid (HEPES). During contact, biolayer interferometry was employed to detect wavelength shifts of interference patterns.

[0252] FIGs. 16 and 18 show these wavelength shifts for probes contacted with liquids of Type A, and FIGs. 17 and 19 show the binding rates and associated curve fits calculated from FIG. 16 and FIG. 18, respectively. FIGs. 20 and 22 shows these wavelength shifts for probes contacted with liquids of Type B, and FIGs. 21 and 23 show the binding rates and associated curve fits calculated from FIGs. 20 and 22, respectively. The base salt for the liquids shown in FIGs. 16 and 20 was Tris. The base salt for the liquids shown in FIGs. 18 and 22 was HEPES.

[0253] In FIGs. 16, 18, 20, and 22, the x-axis is time in seconds, the y-axis is the magnitude of the wavelength shift of the interference pattern in nm, and the mRNA concentration is labeled by each trace. In FIGs. 17, 19, 21, and 23, the x-axis is mRNA concentration in micrograms / mL, and the y-axis is the calculated binding rate in nm / s.

[0254] The data shown in FIGs. 16-23 is further summarized in FIGs. 24 and 25 and Tables 3-4. FIG. 24 and Table 3 show the binding rate as a function of mRNA concentration for liquids of Types A and B including a Tris base salt. FIG. 25 and Table 4 show the binding rate as a function of mRNA concentration for liquids of Types A and B including a HEPES base salt.

[0255] Table 3.

[0256]

[0257] 34

[0258] #14820240vl

[0259]

[0260] Table 4.

[0261]

[0262] As can be seen from the above-described data, the presence of Mg2+during binding of mRNA to oligo(dT) can enhance the signal obtained during biolayer interferometry without appreciably affecting the measured binding rate.

[0263] While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the teachings of the present invention is / are used. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, kit, and / or method described

[0264] 35

[0265] #14820240vlherein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the scope of the present invention.

[0266] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0267] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0268] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0269] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements 36

[0270] #14820240vland not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0271] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0272] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0273] 37

[0274] #14820240vl

Claims

CLAIMSWhat is claimed is:

1. A kit, comprising:a probe;a salt comprising a divalent cation; anda salt comprising a monovalent cation, wherein:the probe comprises a body portion, a first coating disposed on the body portion, and a second coating disposed on the first coating,the first coating has a composition and an arrangement such that light impinging on the first coating after traveling through the body portion is partially reflected at an interface between the first coating and the body portion and partially transmitted through the first coating,the second coating comprises a molecule,the molecule comprises a plurality of repeat units, andthe plurality of repeat units comprises deoxythymidine and / or a thymidine analog.

2. A method, comprising:contacting a probe with a liquid comprising mRNA at an mRNA concentration; after contacting the probe with the liquid comprising mRNA at the mRNA concentration, contacting the probe with a liquid comprising a divalent cation;while and / or after contacting the probe with the liquid comprising the divalent cation, detecting a signal; anddetermining the mRNA concentration based on the signal, wherein:the probe comprises a body portion and a coating disposed on the body portion,the coating disposed on the body portion comprises a molecule, the molecule comprises a plurality of repeat units,the plurality of repeat units comprises deoxythymidine and / or a thymidine analog, andthe signal is indicative of an amount and / or a rate of binding between the mRNA and the molecule.38#14820240vl3. A method, comprising:contacting a probe with a liquid comprising mRNA at an mRNA concentration; while and / or after contacting the probe with the liquid comprising mRNA at the mRNA concentration, detecting a signal; anddetermining the mRNA concentration based on the signal, wherein:the liquid comprising mRNA further comprises a divalent cation and a monovalent cation;the probe comprises a body portion and a coating disposed on the body portion,the coating disposed on the body portion comprises a molecule, the molecule comprises a plurality of repeat units,the plurality of repeat units comprises deoxythymidine and / or a thymidine analog, andthe signal is indicative of an amount and / or a rate of binding between the mRNA and the molecule.

4. A kit as in claim 1, wherein the divalent cation is Mg2+.

5. A kit as in claim 1, wherein the divalent cation is Ca2+.

6. A kit as in claim 1, wherein the monovalent cation is Na+.

7. A kit as in claim 1, wherein the monovalent cation is K+.

8. A method as in claim 2 or claim 3, wherein the coating is a second coating, and wherein the probe further comprises a first coating.

9. A kit as in claim 1, wherein the probe further comprises a third coating positioned between the first coating and the second coating.

10. A kit as in claim 9, wherein the third coating comprises streptavidin.

11. A kit as in claim 1, wherein the molecule is chemically bound to biotin.39#14820240vl12. A kit as in claim 1, wherein the plurality of repeat units comprises deoxythymidine.

13. A kit as in claim 1, wherein the plurality of repeat units comprises the thymidine analog.

14. A kit as in claim 13, wherein the thymidine analog is an engineered thymidine analog.

15. A kit as in claim 14, wherein the engineered thymidine analog is LNA thymidine.

16. A method as in claim 2 or claim 3, wherein the liquid comprising the mRNA comprises a crude sample.

17. A method as in claim 2 or claim 3, wherein the liquid comprising the mRNA comprises RNA polymerase.

18. A method as in claim 2 or claim 3, wherein the liquid comprising the mRNA comprises dithiothreitol.

19. A method as in claim 2 or claim 3, wherein the liquid comprising the mRNA comprises a DNA template.

20. A method as in claim 2 or claim 3, wherein the liquid comprising the mRNA comprises cellular lysates.

21. A method as in claim 2 or claim 3, wherein the liquid comprising the mRNA comprises tissue homogenates.

22. A method as in claim 2 or claim 3, wherein the liquid comprising the mRNA comprises a surfactant.

23. A method as in claim 22, wherein the surfactant is NP-40 or Triton X-100.40#14820240vl24. A method as in claim 22, wherein the surfactant makes up greater than or equal to 0.01 wt% and less than or equal to 2 wt% of the liquid.

25. A method as in claim 2 or claim 3, wherein the liquid comprising the mRNA comprises a DNase inhibitor.

26. A method as in claim 2 or claim 3, wherein the liquid comprising the mRNA comprises an RNase inhibitor.

27. A method as in claim 2 or claim 3, wherein the liquid comprising the mRNA comprises a protease inhibitor.

28. A kit as in claim 1, wherein the salt comprising the divalent cation is dissolved in water.

29. A kit as in claim 1, wherein the salt comprising the monovalent cation is dissolved in water.

30. A kit as in claim 1, wherein the kit comprises a buffer in which the salt comprising the divalent cation and the salt comprising the monovalent cation are dissolved.

31. A kit as in claim 1, wherein the kit comprises a first buffer in which the salt comprising the divalent cation is dissolved and a second buffer in which the salt comprising the monovalent cation is dissolved.

32. A method as in claim 2 or claim 3, wherein the liquid comprising the mRNA is a buffer.

33. A method as in claim 3, wherein the liquid comprising the mRNA is a buffer comprising the monovalent cation.

34. A method as in claim 2 or claim 3, wherein the liquid comprising the mRNA is a buffer comprising the divalent cation.41#14820240vl35. A method as in claim 2 or claim 3, wherein the liquid comprising the mRNA is a Tris buffer.

36. A method as in claim 2 or claim 3, wherein the liquid comprising the mRNA is a HEPES buffer.

37. A method as in claim 2 or claim 3, wherein the liquid comprising the mRNA is a citrate buffer.

38. A method as in claim 34, wherein a concentration of the divalent cation in the buffer is greater than or equal to 1 mM and less than or equal to 100 mM.

39. A method as in claim 33, wherein a concentration of the monovalent cation in the buffer is greater than or equal to 50 mM and less than or equal to 200 mM.

40. A method as in claim 3, further comprising contacting the probe with a liquid comprising the divalent cation after contacting the probe with the liquid comprising the mRNA at the mRNA concentration.

41. A method as in claim 2 or claim 3, further comprising contacting the probe with a regeneration liquid after contacting the probe with the liquid comprising the mRNA at the mRNA concentration.

42. A method as in claim 41, wherein the regeneration liquid is a buffer.

43. A method as in claim 42, wherein the buffer comprises NaOH.

44. A method as in claim 41, further comprising contacting the probe with a neutralization liquid after contacting the probe with the regeneration liquid.

45. A method as in claim 44, wherein the neutralization liquid is a buffer.

46. A method as in claim 2 or claim 3, wherein the mRNA comprises a poly(A) tail.42#14820240vl47. A method as in claim 46, wherein the binding between the mRNA and the molecule occurs via binding between the poly(A) tail and the deoxythymidine and / or between the poly(A) tail and the thymidine analog.

48. A method as in claim 2, further comprising, while and / or after contacting the probe with the liquid comprising mRNA at the mRNA concentration, detecting a signal.

49. A method as in claim 2 or claim 3, wherein the signal is an optical signal.

50. A method as in claim 49, wherein the optical signal comprises light reflected from two different locations associated with the probe.

51. A method as in claim 49, wherein the optical signal comprises light reflected from an interface between the body portion and the coating and light reflected from the end of the probe.

52. A method as in claim 49, wherein the optical signal comprises light interference generated by interferometry.

53. A method as in claim 52, wherein the interferometry is biolayer interferometry.

54. A method as in claim 2 or claim 3, wherein the method is performed by an instrument.

55. A method as in claim 2 or claim 3, wherein the method further comprises contacting the probe with a standard.

56. A method as in claim 2 or claim 3, wherein the liquid comprising mRNA is contained by a container.

57. A method as in claim 2 or claim 3, wherein the liquid comprising the divalent cation is contained by a container.

58. A method as in claim 55, wherein the standard is contained by a container.43#14820240vl59. A method as in claim 41, wherein the regeneration liquid is contained by a container.

60. A method as in claim 44, wherein the neutralization liquid is contained by a container.

61. A method as in claim 56, wherein the container is a well in a multiwell plate.

62. A method as in claim 61, wherein the multiwell plate further comprises a well containing the liquid comprising the divalent cation, a well containing a standard, a well containing a regeneration liquid, and / or a well containing a neutralization liquid.

63. A method as in claim 62, further comprising translating the probe to contact the liquids sequentially.44#14820240vl

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