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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1034—Isolating an individual clone by screening libraries
  • C12N15/1048—SELEX
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6811—Selection methods for production or design of target specific oligonucleotides or binding molecules

Definitions

• the present disclosure describes novel aptamers, and methods to produce and use such aptamers.
• the disclosure describes slow off-rate (slow rate of dissociation) aptamers, slow off-rate aptamers containing C- 5 modified pyrimidines, and processes for the selection of slow off-rate aptamers by dilution, by the addition of a competitor, or by a combination of both approaches.
• slow off-rate aptamers to various targets such as proteins and peptides are described.
• Slow off-rate aptamers with unique structural features and melting temperatures are also described.
• the one or more slow off-rate aptamers contacted with the histological or cytological sample may serve as the nucleic acid target in a nucleic acid amplification method.
• the nucleic acid amplification method may include PCR, q-beta replicase, rolling circle amplification, strand displacement, helicase dependent amplification, loop mediated isothermal amplification, ligase chain reaction, and restriction and
• an aptamer or “nucleic acid ligand” is a set of copies of one type or species of nucleic acid molecule that has a particular nucleotide sequence.
• An aptamer can include any suitable number of nucleotides.
• “Aptamers” refer to more than one such set of molecules. Different aptamers may have either the same number or a different number of nucleotides. Aptamers may be DNA or RNA and may be single stranded, double stranded, or contain double stranded regions.
• each nucleic acid in a candidate mixture may have fixed sequences on either side of a randomized region, to facilitate the amplification process.
• the nucleic acids in the candidate mixture of nucleic acids can each further comprise fixed regions or "tail" sequences at their 5' and 3' termini to prevent the formation of high molecular weight parasites during the amplification process.
• photoreactive pyrimidines include 5-bromo-uracil (BrdU), 5-bromo-cytosine (BrdC), 5-iodouracil (IdU), and 5-iodo-cytosine (IdC).
• the photoreactive functional group will absorb wavelengths of light that are not absorbed by the target or the non-modified portions of the oligonucleotide.
• the target may be modified to different levels. Slow off -rate aptamers could be produced to differentiate the type or level of modification.
• a SELEX target does not include molecules that are known to bind nucleic acids, such as, for example, known nucleic acid binding proteins (e.g. transcription factors). Virtually any chemical or biological effector may be a suitable SELEX target. Molecules of any size can serve as SELEX targets.
• a target can also be modified in certain ways to enhance the likelihood or strength of an interaction between the target and the nucleic acid.
• slow off-rate enrichment process refers to a process of altering the relative concentrations of certain components of a candidate mixture such that the relative concentration of aptamer affinity complexes having slow dissociation rates is increased relative to the concentration of aptamer affinity complexes having faster, less desirable dissociation rates.
• the slow off-rate enrichment process is a solution-based slow off -rate enrichment process.
• a solution-based slow off -rate enrichment process takes place in solution, such that neither the target nor the nucleic acids forming the aptamer affinity complexes in the mixture are immobilized on a solid support during the slow off -rate enrichment process.
• the duration of the slow off -rate enrichment process is selected so as to retain a high proportion of aptamer affinity complexes having slow dissociation rates while substantially reducing the number of aptamer affinity complexes having fast dissociation rates.
• the slow off-rate enrichment process may be used in one or more cycles during the SELEX process. When dilution and the addition of a competitor are used in combination, they may be performed simultaneously or sequentially, in any order.
• the slow off-rate enrichment process can be used when the total target
• the nucleic acid target molecule mixture is irradiated causing some nucleic acids incorporated in nucleic acid-target molecule complexes to crosslink to the target molecule via the photoreactive functional groups, and the selection step is a selection for photocros slinking activity.
• the photoSELEX process is described in great detail in the PhotoSELEX Patents.
• photoaptamer and photoreactive aptamer are used interchangeably to refer to an aptamer that contains one or more photoreactive functional groups that can covalently bind to or "crosslink” with a target molecule.
• a method for identifying an aptamer having a slow rate of dissociation from its target comprising (a) preparing a candidate mixture of nucleic acid sequences; (b) contacting the candidate mixture with a tissue or cell sample wherein nucleic acids with the highest relative affinities to the target of interest in said tissue or cell sample preferentially bind the target, forming nucleic acid-target complexes; (c) applying a slow off-rate enrichment process to allow the dissociation of nucleic acid-target complexes with relatively fast dissociation rates; (d) partitioning the remaining nucleic acid-target complexes from both free nucleic acids and non-target molecules in the candidate mixture; and (e) identifying an aptamer to the target of interest.
• the process may further include the iterative step of amplifying the nucleic acids that bind to the target to yield a mixture of nucleic acids enriched in sequences that are able to bind to the target yet produce nucleic acid-target complexes having slow dissociation rates.
• the slow off -rate enrichment process can be selected from (a) diluting the candidate mixture containing the nucleic acid- target molecule complexes; (b) adding at least one competitor to the candidate mixture containing the nucleic acid- target molecule complexes, and diluting the candidate mixture containing the nucleic acid- target molecule complexes; (c) and adding at least one competitor to the candidate mixture containing the nucleic acid-target molecule complexes.
• the candidate mixture of nucleic acid molecules includes nucleic acids containing modified nucleotide bases that may aid in the formation of modified nucleic acid- target complexes with relatively slow dissociation rates.
• Aptamers identified according to the methods described herein are useful in a range of diagnostic and therapeutic methods.
• Slow off-rate aptamers will bind to the target for a longer duration. This is useful in diagnostic methods where the binding of an aptamer to the target may be used to detect the presence, absence, amount or quantity of the target molecule and a prolonged interaction of the aptamer and target facilitates such detection.
• a similar advantage may be afforded where slow off-rate aptamers are used in imaging methods, in vitro or in vivo.
• a prolonged interaction of aptamer and target also provides for improved therapeutic methods of treatment where the prolonged interaction may allow for an improved therapeutic effect, e.g. owing to the longer activation or inhibition of the target molecule or downstream signaling cascade.
• These slow off-rate aptamers and aptamers with high affinity may be used in cytological and histological molecular detection and
• slow off-rate aptamers obtained, identified or produced by the described methods can be used in a variety of methods of medical treatment or methods of diagnosis (in vitro or in vivo).
• slow off- rate aptamers can be used in a method of treatment of disease.
• slow off- rate aptamers can be used in a method for diagnosis of disease in vivo.
• slow off -rate aptamers can be used in vitro for the diagnosis of disease.
• a slow off -rate aptamer can be used in the manufacture of a therapeutic (e.g. pharmaceutical composition) or the manufacture of a diagnostic agent for use in a method of treatment or diagnosis of disease.
• Diagnostic or therapeutic applications of slow off -rate aptamers may involve a diagnostic or therapeutic outcome that depends on the specific and/or high affinity binding of the slow off-rate aptamer to its target.
• Slow off-rate aptamers may also be used in target validation and high throughput screening assays in the drug development process.
• Monoclonal antibodies often have high affinity and specificity, and may have slow dissociation rate constants; however, monoclonal antibodies have very slow clearance rates from the vasculature.
• Short peptides identified through, for example, phage display, may have fast clearance but poor affinity and specificity and fast dissociation rates from their intended targets.
• Affibodies a particular peptide version of an antibody mimetic, may have reasonable affinity and specificity and may have faster clearance than monoclonal antibodies, yet in order to achieve slow dissociation rates from their targets, affibodies are often made into dimers and higher order multimers, slowing their clearance at the same time that their dissociation rates are enhanced.
• the modified SELEX process disclosed herein includes the introduction of a slow off-rate enrichment process following equilibration of the candidate mixture of nucleic acids with the target or targets and a partitioning step prior to subsequent steps in the SELEX process.
• Introduction of a slow off-rate enrichment process to the basic SELEX process provides a means for enrichment of aptamer affinity complexes with slow dissociation rates from a set of nucleic acid-target complexes that includes a variety of dissociation rates.
• the modified SELEX process provides a method for identifying aptamers that bind target molecules and, once bound, have relatively slow rates of dissociation (also referred to herein as "off-rates”) from the target molecule.
• modifications are those wherein another chemical group is attached to the 5-position of a pyrimidine or the 2' position of a sugar.
• another chemical group is attached to the 5-position of a pyrimidine or the 2' position of a sugar.
• the type of other chemical group that can be incorporated on the individual nucleotides.
• the resulting modified nucleotide is amplifiable or can be modified subsequent to the amplification steps (see, e.g., U.S. Patent No. 6,300,074 entitled "Systematic evolution of ligands by exponential enrichment: Chemi- SELEX”.
• the present disclosure provides methods for identifying aptamers that bind one or more targets of interest within a cell or tissue sample and once bound have slow rates of dissociation or off -rates.
• the slow off-rates obtained with this method can exceed a half-life of about one hour and as much as about 240 minutes, that is, once a set of nucleic acid-target complexes is generated, half of the complexes in the set remain bound after one hour.
• a representative fixative would be 50% absolute ethanol, 2 mM polyethylene glycol (PEG), 1.85% formaldehyde. Variations on this formulation include ethanol (50% to 95%), methanol (20% - 50%), and formalin (formaldehyde) only. Another common fixative is 2% PEG 1500, 50% ethanol, and 3% methanol. Slides are place in the fixative for 10 to 15 minutes at room temperature and then removed and allowed to dry. Once slides are fixed they can be rinsed with a buffered solution like PBS. [00165] A wide range of dyes can be used to differentially highlight and contract or
• Primer beads were prepared by immobilizing biotinylated reverse PCR primer to streptavidin-coated paramagnetic beads (MyOne-Streptavidin Cl (SA beads), Invitrogen). 5 mL SA beads (10 mg/mL) were washed once with NaClT (5 M NaCl, 0.01% TWEEN-20), and resuspended in 5 mL biotinylated reverse PCR primer (5 ⁇ M in NaClT). The sample was incubated at 25 C for 15 minutes, washed twice with 5 rnL NaClT, resuspended in 12.5 rnL NaClT (4 mg/niL), and stored at 4 0 C.
• SA beads MyOne-Streptavidin Cl
• Selected aptamer DNA was amplified and quantified by QPCR.
• 48 ⁇ L DNA was added to 12 ⁇ L QPCR Mix (5X KOD DNA Polymerase Buffer, 25 mM MgCl 2 , 10 ⁇ M forward PCR primer, 10 ⁇ M biotinylated reverse PCR primer, 5X SYBR Green I, 0.125 U/ ⁇ L KOD XL DNA Polymerase, and 1 mM each dATP, dCTP, dGTP, and dTTP) and thermal cycled in an ABI5700 QPCR instrument with the following protocol: 1 cycle of 99.9 0 C, 15 seconds, 55 0 C, 10 seconds, 7O 0 C, 30 minutes; 30 cycles of 99.9 0 C, 15 seconds, 72 0 C, 1 minute. Quantification was done with the instrument software and the number of copies of DNA selected with target beads and (His) 6 beads were compared to determine
• DS Fluorescence images were obtained with a Nikon 80i upright microscope equipped with Digital Sight DS-RiI camera, mercury lamp illumination source, and neutral density and optical filters appropriate for DAPI or Cy3 imaging. Fast nuclear Aptamer dissociation was imaged at 5 Hz. For time course experiments, trials were taken without addition of reagents to confirm that photobleaching was not a significant source of fluorescence decay over the observed time period.

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• 2009
  • 2009-07-09 US US12/499,967 patent/US7964356B2/en active Active
• 2010
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