Part collection | Keystone

Cycle 1: SELEX-System Parts

Cycle 2：Biosensor-System Parts

Cycle 1: SELEX-System Parts

We describe the selection of nucleic acid aptamers with high specificity for Brain-Derived tau (BD-Tau) via the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) methodology. The selection regime involved positive screening against BD-Tau coupled with negative screening against generic Tau protein. The corresponding biological parts constituting our SELEX system are summarized in Table 1.

Table 1. The part collection

Parts Code	Parts Name	Type I	Type II	Description
BBa_252RIC0N	BD-tau(PHF6 domain)	Basic Part	Coding	BD-tau has been confirmed as a biomarker for Alzheimer's disease. The difference between BD-tau and peripheral tau lies in the fact that peripheral tau has an insertion of exon 4A between exons 4 and 5
BBa_25RK98WF	Tau	Basic Part	Coding	Total tau (T-tau) in cerebrospinal fluid has been proven to be a reliable biomarker for Alzheimer's disease specific neurodegeneration.
BBa_25R6CI56	Aptamer-1	Basic Part	ssDNA	Selection of BD-tau-binding aptamer by SELEX.
BBa_25QENYPB	Aptamer-2	Basic Part	ssDNA	Selection of BD-tau-binding aptamer by SELEX.
BBa_25XMPAOH	Aptamer-3	Basic Part	ssDNA	Selection of BD-tau-binding aptamer by SELEX.
BBa_25WL4D7V	Aptamer-08	Basic Part	ssDNA	Selection of BD-tau-binding aptamer by SELEX.
BBa_25ZYR9NO	Aptamer-14	Basic Part	ssDNA	Selection of BD-tau-binding aptamer by SELEX.
BBa_259EP3O5	Aptamer-16	Basic Part	ssDNA	Selection of BD-tau-binding aptamer by SELEX.
BBa_25MCN9SA	Selex_Fwd	Basic Part	Primer	PCR primers used in the SELEX process
BBa_257XM5WV	Selex_Rev	Basic Part	Primer	PCR primers used in the SELEX process
BBa_25B3VQE9	cDNA library	Basic Part	Oligo DNA	A single-stranded DNA (ssDNA) library with a 66-nucleotide random region flanked by fixed primer-binding sequences
BBa_259FM6T9	pET28a-PHF6 domain	Composite Part	Plasmid	Positive selection in SELEX
BBa_256M1I7W	pET28a-Tau	Composite Part	Plasmid	Negative selection in SELEX
BBa_25RGQIOH	pET28a	Basic Part	Plasmid_Backbone	Expression vector

Cycle 2：Biosensor-System Parts

In this study, the highest-affinity nucleic acid aptamer selected the SELEX system was used to construct a biosensor for BD-tau protein. This biosensor was coupled with the CRISPR-Cas12a signal amplification technology to quantify BD-tau protein levels in blood samples based on fluorescence detection. Furthermore, we used generic Tau protein as a positive control. All related components are listed in Table 2.

Table 2. The part collection

Parts Code	Parts Name	Type	Type II	Description
BBa_25M06Z8M	Tau aptamer	Basic part	ssDNA	Tau protein aptamer sequence (target: 2N4R-Tau)
BBa_25OUAJ0W	Tau aptamer-F	Basic part	ssDNA	This is a modified alpha-fetoprotein (AFP) aptamer sequence. When utilized in the construction of the biosensor, it forms an aptamer switch with the biotin-modified complementary ssDNA sequence (ComDNA).
BBa_253RQU0P	BD-tau aptamer 14-F	Basic part	ssDNA	This is a modified alpha-fetoprotein (AFP) aptamer sequence. When utilized in the construction of the biosensor, it forms an aptamer switch with the biotin-modified complementary ssDNA sequence (ComDNA).
BBa_2540VQLQ	comDNA 1	Basic part	Oligo DNA	ComDNA sequence that is complementary to a partial sequence of the Aptamer-F. It can bind to streptavidin on magnetic beads, thereby enabling the anchoring of the ComDNA to the magnetic beads.
BBa_25FNMXM6	comDNA 2	Basic part	Oligo DNA
BBa_25R1UJJB	comDNA 3	Basic part	Oligo DNA
BBa_254DI5CV	comDNA 4	Basic part	Oligo DNA
BBa_25AG7QMC	comDNA 5	Basic part	Oligo DNA
BBa_25HYDEQU	CrRNA T7 promoter-F	Basic part	Oligo DNA	The sequence of the forward primer used for preparing the crRNA transcription template.
BBa_257D5PXM	crRNA T7-R	Basic part	Oligo DNA	The sequence of the backward primer used for preparing the crRNA transcription template.
BBa_25G2G044	dsDNA-F	Basic part	Primer	The nucleotide sequence of the forward primer used for synthesizing the Cas12a protein-activating dsDNA.
BBa_2545L6R8	dsDNA-R	Basic part	Primer	The nucleotide sequence of the reverse primer used for synthesizing the Cas12a protein-activating dsDNA.
BBa_25083FD6	qPCR-F	Basic part	Primer	The forward primer for qPCR
BBa_25SMX4T3	qPCR-R	Basic part	Primer	The reverse primer for qPCR
BBa_25AJAYBU	Cas12a	Basic Part	Coding	Guided by CRISPR RNA (crRNA), Cas12a identifies and cleaves the target DNA sequence via its RuvC nuclease domain
BBa_25OZUK7L	pET28a-cas12a	Composite Part	Plasmid	Used for the expression of Cas12a