Compositions and methods for aptamer screening

• US 9,939,443 B2
• Filed: 12/19/2013
• Issued: 04/10/2018
• Est. Priority Date: 12/19/2012
• Status: Active Grant

• Current Assignee: Caris Science, Inc.
• Original Assignee: Caris Life Sciences Switzerland Holdings GmbH
• Inventors: Spetzler, David, Domenyuk, Valeriy, Hornung, Tassilo, Mayer, Gnter, Famulok, Michael
• Primary Examiner(s): ZARA, JANE J
• Application Number:
  US14/652,728
• Publication Number:
  US 20160069889A1
• Time in Patent Office: 1,573 Days
• Field of Search: 435 61, 435 611, 435 612, 435 613, 435 911, 435 9131, 536 231, 436501, 506 7, 506 9, 506 41
• CPC Class Codes: C12N 15/1048 SELEX
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2525/205 Aptamer
• CPC Class Codes: G01N 33/5076 involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: G01N 33/57488 involving compounds identifable in body fluids

Abstract

Methods are provided for selecting aptamers that are specific to a target of interest from amongst a library of potential aptamer sequences. Aptamers disclosed can be used to detect and/or characterize biological entities of interest, e.g. microvesicles and/or surface antigens. Further disclosed are biomarkers that can be used for diagnosing different disorders including different types of cancer.

Claims

1. A method of identifying at least one aptamer comprising:

(a) providing a pool of candidate aptamers;

(b) contacting a first at least one biological sample with the pool provided in step (a), wherein the first at least one biological sample comprises a microvesicle population;

(c) identifying a first subpool of candidate aptamers that bound at least one microvesicle in the first at least one biological sample in step (b);

(d) contacting a second at least one biological sample with the pool provided in step (a), wherein the second at least one biological sample comprises a microvesicle population;

(e) identifying a second subpool of candidate aptamers that bound at least one microvesicle in the second at least one biological sample in step (d); and

(f) comparing the members of the first and second subpools of candidate aptamers to identify at least one member of a pool that is overrepresented in one of the subpools versus the other subpool, thereby identifying the at least one aptamer.

2. The method of claim 1, wherein each of the first at least one biological sample is separately contacted with aliquots of the pool in step (b).

3. The method of claim 1, wherein each of the second at least one biological sample is separately contacted with aliquots of the pool in step (d).

4. The method of claim 1, further comprising repeating steps (b)-(c) at least 1 time prior to step (f), wherein the first subpool of candidate aptamers from step (c) is used as the starting pool of candidate aptamers when repeating step (b).

5. The method of claim 1, further comprising repeating steps (d)-(e) at least 1 time prior to step (f), wherein the second subpool of candidate aptamers from step (e) is used as the starting pool of candidate aptamers when repeating step (d).

6. The method of claim 1, further comprising repeating steps (a)-(f) at least 1 time, wherein the identified one aptamer from step (f) is used as the starting pool of candidate aptamers when repeating step (a).

7. The method of claim 1, wherein the first at least one biological sample comprises a diseased sample.

8. The method of claim 7, wherein each of the first at least one biological sample comprises a sample representative of a same disease.

9. The method of claim 7, wherein the second at least one biological sample comprises a non-diseased sample.

10. The method of claim 7, wherein the first at least one biological sample comprises a same disease as the second at least one biological sample, and wherein the disease in the first and second samples differs in at least one of clinical stage, pathologic stage, aggressiveness, growth rate, receptor status, prior treatment, response to a same to treatment, and response to different treatments.

11. The method of claim 7, wherein the disease comprises a cancer.

12. The method of claim 11, wherein the cancer comprises a prostate cancer, a lung cancer, a breast cancer, a brain cancer, an ovarian cancer, a colorectal cancer, or a melanoma.

13. The method of claim 1, wherein the first at least one biological sample and the second at least one biological sample comprise bodily fluids.

14. The method of claim 13, wherein the bodily fluid comprises peripheral blood, sera, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper'"'"'s fluid or pre-ejaculatory fluid, female ejaculate, sweat, fecal matter, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, umbilical cord blood, or a derivative of any thereof.

15. The method of claim 13, wherein the bodily fluid comprises peripheral blood, plasma, serum or a derivative of any thereof.

16. The method of claim 1, wherein the biological sample comprises a tissue sample or cell culture sample.

17. The method of claim 1, wherein the identifying in step (c) and/or step (e) comprises sequencing the candidate aptamers that bound the at least one microvesicle.

18. The method of claim 17, wherein comparing the members of the first and second subpools in step (f) comprises comparing the sequences of the aptamers identified in step (c) and/or step (e).

19. The method of claim 1, further comprising filtering the identified at least one aptamer.

20. The method of claim 19, wherein filtering the identified at least one aptamer comprises calculating a stability of the at least one aptamer, identifying a secondary structure of the at least one aptamer, and/or separately contacting each of the at least one aptamer with a biological sample.