MINOR PROJECT

Pyrrolidinyl peptide nucleic acid internally-labeled with cyanine-styryl dye as a fluorescence DNA probe

My research question is to find excellent the environment-sensitive fluorescence labels, alkyne-modified cyanine-styryl derivatives (STR), which were labeled onto pyrrolidinyl peptide nucleic acid with D-proline/(1S,2S)-2-aminocyclopentanecarboxylic acid backbone (acpcPNA) via sequential reductive alkylation-Click strategy with the aim to create self-reporting PNA probes that exhibit fluorescence change in response to the correct DNA target. The styryl dye family has received increasing attention as a photostable DNA label that revealed the excellent colorimetric response in difference microenvironments.

Researchers who have looked at this subject are Wen-hai Zhan and Rubner. Both of studies focused on the synthesis and evaluation of styryl dyes capable of exhibiting turn-on fluorescence through sequence-specific interaction with nucleic acid in fluorescence spectroscopy and diagnostic application.

Zhan et al. (2005) argue that the 2-propynyl substituted hemicyanine dyes were synthesized in a facile route and showed good optical properties that have a strong absorbance as well as a large strokes shift of the fluorescence emission maxima. The terminal alkynyl group of dyes was successfully employed for bioconjugation through a Click strategy with azide-modified cowpea mosaic virus (CPMV).

Rubner et al. (2012) argue that the alkyne-modified indole-quinoline styryl dye (CyIQ) was synthesized and incorporated it into DNA via click chemistry. The CyIQ dye exhibit good brightness and give excellent photostability.

Debate centers on this basic issue of the fluorescent properties of cyanine-styryl dyes which exhibit good brightness. Thus, we are interested in synthesizing new clickable styryl dyes for attachment onto acpcPNAs with the aim to use them as DNA sensing probes.

My work will be closer to Rubner’s in the focus on the synthesis of the alkyne-modified styryl family for fluorescent labeling and incorporated into nucleic acid as fluorescent probes.

Hopefully my contribution will be to synthesize and incorporate into acpcPNA which discriminate between the complementary DNA and single mismatch DNA and apply in SNP detection for diagnosis.

Reference list

Zhan, W-h., Barnhill, H. N., Sivakumar, K., Tian, H., Wang, Q. (2005). Synthesis of hemicyanine dyes for ‘click’ bioconjugation. Tetrahedron Letters, 46, 1691-1695.

Rubner, M. M., Holzhauser, C., Bohlander, P. R., Wagenknecht, H-A. (2012). A “Clickable” Styryl Dye for Fluorescent DNA labeling by Excitron and Energy Transfer Interactions. Chemistry - A European Journal, 18, 1299-1302. 

Assignment 2: Writing Introduction

Stage 1: DNA sequence analysis is widely used in several applications ranging from clinical diagnosis, food and agricultural sciences and forensic sciences. The development of rapid, sensitive and accurate method for DNA sequence determination is therefore a very important research area. There are two main approaches to investigate a DNA sequence, by direct sequencing and by the use of a hybridization probe. The latter method is more attractive for routine diagnosis because there is no requirement for expensive instruments.

Stage 2: The fluorescent hybridization probe is a short oligomer of DNA or its analogue that has a base sequence complementary to the region of interest in the DNA target. The conventional molecular beacon (MB) probe was designed and improved by Kramer et al.   (1996). There found that the MB probes are suited method for SNP detection. However, the MB probes was cannot performed well at room temperature. The limitation was avoided by using binary-probes for SNP analysis at room temperature. Therefore, the base-discriminating fluorescent (BDF) probes containing pyrenecaboxamide-labeled uracil (^PyU) and cytosine (^PyC) nucleobases were developed by Okamoto et al. (2004). Moreover, Seio et al. (2008) had synthesized and studied properties of N⁶-[N-(pyren-1-ylmethyl)carbamoyl]-deoxyadenosine (dA^pymcm) which was incorporated into internal oligonucleotides and showed fluorescence increasing in the presence of DNA target. Peptide nucleic acid (PNA) is one of DNA analogue in which the deoxyribose phosphate backbone is replaced by N-(2-aminoethylglycine) unit.  PNA has been first reported by Nielsene et al.  in 1991 (also known as aegPNA or Nielsen's PNA). Appella et al. (2005) synthesized fluorescent aegPNA probes as a sequence-free molecular beacon for DNA analysis. This PNA probe was increased fluorescent signal in the presence of the perfectly complementary DNA. Moreover, the thaizole orange modified FIT probes was described by Socher et al. (2008). The duplex of FIT probes with complementary DNA is exhibited increasing fluorescent signal.

Stage 3: Vilaivan et al. (2005) had reported a new version of pyrrolidinyl PNA called acpcPNA. This new PNA system contains a rigid backbone consisting of D-prolyl-2-aminocyclopentanecarboxylic acid (ACPC) subunits. Previously research in this area, the DNA and aegPNA was only developed for fluorescent probes. Nevertheless, the pyrrolidinyl PNA (acpcPNA) are no reported on the optical properties. Therefore, the fluorescent acpcPNA probes were developed.

Stage 4: The objective of this research is to develop fluorescent acpcPNA probes with styryl fluorescent dyes labeling via a sequential reductive alkylation/click chemistry. In addition, the optical properties of dyes-labeled acpcPNA in the absence and presence of target DNA are investigated.

Stage 5: The fluorescent acpcPNA probes can be discriminated the absence and presence of target DNA on ‘naked-eye’ detection and applied in SNP detection for diagnosis.