The confirmation of the presence of sperm cells in a sexual assault case is vital. Despite the routine use of microscopic examination facilitated by histological staining techniques, the method is prone to a lack of specificity and sensitivity.
In new research, scientists at Kings College London and the University of Warwick are testing out low-cost, effective alternatives. Their method is based on the development of aptamers, single-stranded DNA molecules capable of selectively binding to a given target.
Recently published in the journal Analytical and Bioanalytical Chemistry, the team has identified several promising aptamer candidates and demonstrated that they were able to selectively bind to sperm cells over other cell types.
“Aptamers possess several advantages over their protein counterparts, including enhanced stability, greater speed of production, and ease of chemical modification,” reads the study. “Importantly, aptamers can also be mass-produced at low cost with no batch-to-batch variation.”
For their study, the scientists modified a previously published Cell-SELEX protocol to incorporate massively parallel sequencing (MPS), or next generation sequencing, to elucidate the sequences of potential binding ligands.
Three separate instances of high-throughput sequencing were performed to determine the sequence and relative abundance of ssDNAs contained within 14 rounds of SELEX cycle aptamer pools. While the 10 rounds of selection did not yield any sequences that were repeated more than once, the latter rounds were more successful.
In round 14, a total of 13,138 sequences were found to be repeated multiple times in the MPS dataset. In fact, 15 sequences were found to possess read counts greater than 25—meaning they show potential for the recognition of human sperm cells.
The researchers then took the three sequencing candidates that displayed the highest read counts from round 14 and put them through additional binding experiments to confirm the aptamer sequences could bind to sperm cells from different donors.
“[This] is crucially important for any potential future application to forensic casework,” say the researchers.
The sequences were incubated with dilution series of sperm cells obtained from two donors in enzyme-linked oligonucleotide assays (ELONA). For each donor/aptamer combination, the researchers noted consistent increases in absorbance with greater sperm cell concentrations, indicating dose-dependent binding responses and demonstrating the ability of each aptamer to interact with sperm cells from different individuals.
Additionally, while there is no “typical” volume of semen recovered during investigations, it’s important to note this research relied on 5 uL of semen during the microscale thermophoresis portion of the experiment—what forensic analysts would refer to as a trace amount.
“[This] technique would likely result in a drastic reduction in the cost and amount of time needed to screen items of evidence for semen in cases of sexual assault, as well as prevent the possibility of missed evidence—and therefore potential miscarriages of justice,” said the study’s lead author, James Gooch, a research fellow within the forensic group at Kings College London.
Now, Gooch and his research team are working to further optimize the aptamers to make them even more specific—before the optimized sequences are ready to undergo validation procedures to allow them to be used within forensic casework.
Beyond forensics, the team said they believe the ligands can be applied to other areas of biomedical testing, including fertility evaluation.
Photo: Human sperm cells stained for routine microscopy-based imaging. Credit: User: Bob J Galindo