Structural properties and binding mechanism of DNA aptamers sensing saliva melatonin for diagnosis and monitoring of circadian clock and sleep disorders

Meenakshi Pundir; Maria C De Rosa; Liubov Lobanova; Shahad Abdulmawjood; Xiongbiao Chen; Silvana Papagerakis; Petros Papagerakis

doi:10.1016/j.aca.2023.340971

Structural properties and binding mechanism of DNA aptamers sensing saliva melatonin for diagnosis and monitoring of circadian clock and sleep disorders

Anal Chim Acta. 2023 Apr 22:1251:340971. doi: 10.1016/j.aca.2023.340971. Epub 2023 Feb 15.

Authors

Meenakshi Pundir¹, Maria C De Rosa², Liubov Lobanova³, Shahad Abdulmawjood⁴, Xiongbiao Chen⁵, Silvana Papagerakis⁶, Petros Papagerakis⁷

Affiliations

¹ Laboratory of Precision Oral Health and Chronobiology, College of Dentistry, University of Saskatchewan, 107 Wiggins Rd, Saskatoon, S7N 5E4, Canada; Division of Biomedical Engineering, University of Saskatchewan, 57 Campus Dr, Saskatoon, S7K 5A9, Canada; Laboratory of Oral, Head and Neck Cancer - Personalized Diagnostics and Therapeutics, Department of Surgery, College of Medicine, University of Saskatchewan, 107 Wiggins Rd, Saskatoon, S7N 5E4, Canada.
² Department of Chemistry, Faculty of Science, Carleton University, 1125 Colonel by Drive, Ottawa, Ontario, K1S 5B6, Canada. Electronic address: mariaderosa@cunet.carleton.ca.
³ Laboratory of Precision Oral Health and Chronobiology, College of Dentistry, University of Saskatchewan, 107 Wiggins Rd, Saskatoon, S7N 5E4, Canada.
⁴ Department of Chemistry, Faculty of Science, Carleton University, 1125 Colonel by Drive, Ottawa, Ontario, K1S 5B6, Canada.
⁵ Division of Biomedical Engineering, University of Saskatchewan, 57 Campus Dr, Saskatoon, S7K 5A9, Canada; Department of Mechanical Engineering, School of Engineering, University of Saskatchewan, 57 Campus Dr, S7K 5A9, Saskatoon, Canada. Electronic address: xbc719@mail.usask.ca.
⁶ Laboratory of Precision Oral Health and Chronobiology, College of Dentistry, University of Saskatchewan, 107 Wiggins Rd, Saskatoon, S7N 5E4, Canada; Laboratory of Oral, Head and Neck Cancer - Personalized Diagnostics and Therapeutics, Department of Surgery, College of Medicine, University of Saskatchewan, 107 Wiggins Rd, Saskatoon, S7N 5E4, Canada; Department of Otolaryngology-Head and Neck Surgery, School of Medicine, University of Michigan, 1500 E Medical Center Dr, Ann Arbor, MI, 48109, United States. Electronic address: silvana.papagerakis@usask.ca.
⁷ Laboratory of Precision Oral Health and Chronobiology, College of Dentistry, University of Saskatchewan, 107 Wiggins Rd, Saskatoon, S7N 5E4, Canada; Division of Biomedical Engineering, University of Saskatchewan, 57 Campus Dr, Saskatoon, S7K 5A9, Canada. Electronic address: petros.papagerakis@usask.ca.

PMID: 36925277
DOI: 10.1016/j.aca.2023.340971

Abstract

Circadian desynchrony with the external light-dark cycle influences the rhythmic secretion of melatonin which is among the first signs of circadian rhythm sleep disorders. An accurate dim light melatonin onset (established indicator of circadian rhythm sleep disorders) measurement requires lengthy assays, and antibody affinities alterations, especially in patients with circadian rhythm disorders whose melatonin salivary levels vary significantly, making antibodies detection mostly inadequate. In contrast, aptamers with their numerous advantages (e.g., target selectivity, structural flexibility in tuning binding affinities, small size, etc.) can become preferable biorecognition molecules for salivary melatonin detection with high sensitivity and specificity. This study thoroughly characterizes the structural property and binding mechanism of a single-stranded DNA aptamer full sequence (MLT-C-1) and its truncated versions (MLT-A-2, MLT-A-4) to decipher its optimal characteristics for saliva melatonin detection. We use circular dichroism spectroscopy to determine aptamers' conformational changes under different ionic strengths and showed that aptamers display a hairpin loop structure where few base pairs in the stem play a significant role in melatonin binding and formation of aptamer stabilized structure. Through microscale thermophoresis, aptamers demonstrated a high binding affinity in saliva samples (MLT-C-1F K_d = 12.5 ± 1.7 nM; MLT-A-4F K_d = 11.2 ± 1.6 nM; MLT-A-2F K_d = 2.4 ± 2.8 nM; limit-of-detection achieved in pM, highest sensitivity attained for MLT-A-2F aptamer with the lowest detection limit of 1.35 pM). Our data suggest that aptamers are promising as biorecognition molecules and provide the baseline parameters for the development of an aptamer-based point-of-care diagnostic system for melatonin detection and accurate profiling of its fluctuations in saliva.

Keywords: Aptamer; Circadian rhythm sleep disorders; Circular dichroism; Melatonin; Microscale thermophoresis.

MeSH terms

Aptamers, Nucleotide* / analysis
Circadian Clocks*
Circadian Rhythm
Humans
Melatonin*
Saliva / chemistry
Sleep Disorders, Circadian Rhythm* / diagnosis
Sleep Wake Disorders*

Substances

Melatonin
Aptamers, Nucleotide