Sensors for human health and performance monitoring require biological recognition elements (BREs) at device interfaces for the detection of key molecular biomarkers that are measurable biological state indicators. BREs, including peptides, antibodies, and nucleic acids, bind to biomarkers in the vicinity of the sensor surface to create a signal proportional to the biomarker concentration. The discovery of BREs with the required sensitivity and selectivity to bind biomarkers at low concentrations remains a fundamental challenge. In this study, we describe an in-silico approach to evolve higher sensitivity peptide-based BREs for the detection of cardiac event marker protein troponin I (cTnI) from a previously identified BRE as the parental affinity peptide. The P2 affinity peptide, evolved using our in-silico method, was found to have ∼16-fold higher affinity compared to the parent BRE and ∼10 fM (0.23 pg/mL) limit of detection. The approach described here can be applied towards designing BREs for other biomarkers for human health monitoring.
We developed a search algorithm combining Monte Carlo (MC) and self-consistent mean field techniques to evolve a peptide sequence that has good binding capability to the anticodon stem and loop (ASL) of human lysine tRNA species, tRNA(Lys3), with the ultimate purpose of breaking the replication cycle of human immunodeficiency virus-1. The starting point is the 15-amino-acid sequence, RVTHHAFLGAHRTVG, found experimentally by Agris and co-workers to bind selectively to hypermodified tRNA(Lys3). The peptide backbone conformation is determined via atomistic simulation of the peptide-ASL(Lys3) complex and then held fixed throughout the search. The proportion of amino acids of various types (hydrophobic, polar, charged, etc.) is varied to mimic different peptide hydration properties. Three different sets of hydration properties were examined in the search algorithm to see how this affects evolution to the best-binding peptide sequences. Certain amino acids are commonly found at fixed sites for all three hydration states, some necessary for binding affinity and some necessary for binding specificity. Analysis of the binding structure and the various contributions to the binding energy shows that: 1) two hydrophilic residues (asparagine at site 11 and the cysteine at site 12) "recognize" the ASL(Lys3) due to the VDW energy, and thereby contribute to its binding specificity and 2) the positively charged arginines at sites 4 and 13 preferentially attract the negatively charged sugar rings and the phosphate linkages, and thereby contribute to the binding affinity.
Human tRNALys3UUU is the primer for HIV replication.
The HIV-1 nucleocapsid protein, NCp7, facilitates htRNALys3UUU recruitment from the host cell by binding to and remodeling
the tRNA structure. Human tRNALys3UUU is post-transcriptionally
modified, but until recently, the importance of those modifications
in tRNA recognition by NCp7 was unknown. Modifications such as the
5-methoxycarbonylmethyl-2-thiouridine at anticodon wobble position-34
and 2-methylthio-N6-threonylcarbamoyladenosine,
adjacent to the anticodon at position-37, are important to the recognition
of htRNALys3UUU by NCp7. Several short peptides
selected from phage display libraries were found to also preferentially
recognize these modifications. Evolutionary algorithms (Monte Carlo
and self-consistent mean field) and assisted model building with energy
refinement were used to optimize the peptide sequence in silico, while fluorescence assays were developed and conducted to verify
the in silico results and elucidate a 15-amino acid
signature sequence (R-W-Q/N-H-X2-F-Pho-X-G/A-W-R-X2-G, where X can be most amino acids, and Pho is hydrophobic)
that recognized the tRNA’s fully modified anticodon stem and
loop domain, hASLLys3UUU. Peptides of this sequence
specifically recognized and bound modified htRNALys3UUU with an affinity 10-fold higher than that of the starting
sequence. Thus, this approach provides an effective means of predicting
sequences of RNA binding peptides that have better binding properties.
Such peptides can be used in cell and molecular biology as well as
biochemistry to explore RNA binding proteins and to inhibit those
protein functions.
Our previously-developed peptide-design algorithm was improved by adding an energy minimization strategy which allows the amino acid sidechains to move in a broad configuration space during sequence evolution. In this work, the new algorithm was used to generate a library of 21-mer peptides which could substitute for λ N peptide in binding to boxB RNA. Six potential peptides were obtained from the algorithm, all of which exhibited good binding capability with boxB RNA. Atomistic molecular dynamics simulations were then conducted to examine the ability of the λ N peptide and three best evolved peptides, viz. Pept01, Pept26 and Pept28, to bind to boxB RNA. Simulation results demonstrated that our evolved peptides are better at binding to boxB RNA than the λ N peptide. Sequence searches using the old (without energy minimization strategy) and new (with energy minimization strategy) algorithms confirm that the new algorithm is more effective at finding good RNA-binding peptides than the old algorithm.
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